JPH0463680B2 - - Google Patents

Description

The scope of the claims 1. Gage by association with one or more controls
A method for characterizing a test sample of DNA.
The test sample is then probed with a polynucleotide probe.
and detect hybridized fragments of DNA.
and the hybridized fragments were subjected to step 1 above.
or to multiple controls;
Here, the polynucleotide probe is
Hybridization between the probe and the minisatellite region of
miniaturization of the genome to the extent that it allows for dissection.
Few sequences are homologous to the satellite region.
At least 3 tandem repeats (must be exact repeats)
comprising a polynucleotide containing (not necessary)
(labeled or marker components if necessary)
), and the DNA of the sample is
does not cleave sequences corresponding to program repeats to a significant extent
fragmented by one or more restriction enzymes,
and (a) each of said repeats contains a core;
- multiple minisates from different genomic loci
Consensus of similar lengths present in lights
At least 70% homologous to the core region
the law of nature; (b) said core has a length of 6 to 16 nucleotides;
death; (c) a null in said repeating unit that does not contribute to said core;
the total number of cleotides is less than or equal to 15; and (d) Multiple polymorphic minisatellite regions or hypervariability
said hybrid by associating it with a genetic locus.
The soy fragments distinguish the DNA of the sample.
Ru; A method characterized by: 2. The said genome means a human or animal genome.
and the sample is a human or animal genome.
2. The method according to claim 1, wherein the method is a sample of DNA. 3. The provider of the test sample and one or more control samples.
The same control sample was used to confirm the identity with the donor.
fragment and hybridize from each sample.
The claim is characterized in that the fragments are compared.
The method described in 1. 4. The provider of the test sample and one or more control samples.
to establish the familial relationship between the donor and the donor.
The control sample was similarly fragmented, and the fragments from each sample were
It is characterized by comparing hybridized fragments.
2. The method according to claim 1. 5 Obtained using at least two types of the above probes.
Claim 1, wherein the patterns of positive fragments are compared.
Method described. 6 At least the repeating unit of the probe is single-stranded.
The method according to claim 1. 7. The claim that the probe is entirely single-stranded.
The method described in Section 1. 8. The polynucleotide is read as 5'→3'.
The following general formula is: H. (J.Core.K)_o・L (1) [In the formula, "core" is read with the same meaning as above.
The following array: GGAGGTGGGCAGGAXG (2) AGAGGTGGGCAGGTGG (3) GGAGGYGGGCAGGAGG (4) T(C)_nGGAGGAXGG(G)_pC (5A) T(C)_nGGAGGA(A)_qGGGC (5B) At least 6 sequences selected from
indicates the sequence with the following nucleotides, where () X is A or G, Y is C or T
, T is T or U, and m is 0, 1 or 2.
, p is 0 or 1, and q is 0 or 1.
and n is at least 3; () J and K together represent 0 within said repeating unit.
~15 additional nucleotides; ( ) H and L each sandwich the repeating unit.
0 or at least 1 additional nucleotide
represents; () The repeating unit (J, core, K) is
the polynucleotide is recognizably repeated;
As long as it has a consensus sequence that
exact repetition in both number and type of codes.
There doesn't have to be; and () "Core" also refers to the actual core in all n repeating units.
The core array in question is defined above in terms of equations (2) to (5).
on average more than 70% compared to the “true” core sequence
As long as there is homology, it can be considered a modified core sequence.
Can also be used] or complementary to the above sequence
Claim 1, wherein the polypeptide has a sex sequence.
The method described in. 9 Present in each (J, core, K) repeat sequence
Claim 8, wherein the total number of nucleotides does not exceed 25.
The method described in. 10 core has up to 16 nucleotides,
The method according to claim 8. 11 Core is 7 or more consecutive nucleotides
9. The method of claim 8, wherein the method represents an array of . 12 The continuous nucleo having 12 or more cores
9. The method of claim 8, wherein the method represents a sequence of tido. 13 The core has the configuration shown in formula (2), (3) or (4).
14 to 16 said consecutive nuclei selected from the column
9. The method of claim 8, wherein the method represents an octide sequence. 14 The actual core arrangement is different from the true core arrangement.
according to claim 8, which has a homology of 80% or more.
Method. 15 J is 0 or 1, and K is 0 or 1
15. The method according to claim 14. 16 Present in each (J/Core/K) repeating unit
The total number of nucleotides in the claim does not exceed 20.
10. 17 The polynucleotide has the following formula: (5′)GPGGGCWGGWXG(3′) (6) (In the formula, P is not G, and W is A, T, or U.
, and X is A or G) Consecutive (5′→3′) core sequences selected from within
has three or more repeats of a 6-31 nt sequence containing
polynucleotide, or a variant thereof (in all repeats)
The actual total core array of is defined for equation (6).
on average more than 70% of the total “true” core arrays
(provided that it has homology to the above), or
are polynucleotides with sequences homologous to these.
2. The method according to claim 1, wherein the method is otide. 18 Each iteration or variant iteration contains the array of equation (6).
18. The method of claim 17. 19 Consecutive (5′→3′) array: PGGGCWG (7) is present in all repeating units of said polynucleotide.
and P and W in claim 17.
The person according to claim 1, which has the given meaning.
Law. 20 Consecutive (5′→3′) array: TGGGCA (8) is present in all repeating units of said polynucleotide.
and T is T or U.
The method described in claim 1. 21. Claims 17 to 20, wherein P is T or U.
The method described in any one of the above. 22. Any of claims 17 to 20, wherein W is A.
or the method described in paragraph 1. 23 The continuous core sequence or conserved sequence
23. The sequence of claims 17-22, wherein the sequence is identical to the repeat sequence.
The method described in any one of the above. 24 The polynucleotide is one of the following consecutive
5′→3′ core arrangement: GGPGGGCWGGWXG (9) (In the formula, P is not G, and W is A, T, or U.
, and X is A or G) has 3 or more repeats of a sequence not exceeding 31 nt containing
polynucleotide, or a variant thereof (in all repeats)
The actual total core array of is defined for equation (9)
>70% homology to the “true” core sequence
) or for these
A polynucleotide with a complementary sequence
2. The method according to claim 1. 25 W is A at the 5′ end and
according to claim 24, which is T or U at the end.
Method. 26 The probe has the following sequence (complementary sequence)
Including): Name (Core) GGAGGTGGGCAGGA^A _GG (33.6) (A) AGGGCTGGAGG (33.15) AGAGGTGGGCAGGTGG (33.5) GGGAGG^C _TGGGCAGGAGG (M13.Core B) GTGGGCAGGAAG Name (M13.Core C) TGGGCAG (M13. Core D) GGTGGGCAGGTGG (In the formula, T is T or U) from at least three repeats of a sequence selected from
A method according to claim 1, consisting essentially of: 27 The probe has the following sequence (complementary sequence)
Including): (33.6) (A) AGGGCTGGAGG (33.15) AGAGGTGGGCAGGTGG from at least three repeats of a sequence selected from
A method according to claim 1, consisting essentially of: Background of the invention 1 Field of invention This invention searches the human or animal genome.
(probing)
Polynucleotides that can be labeled as
and use this probe to extract genomic DNA.
Concerning how to identify. This identification method is based on an example
Examples include paternal and maternal testing, forensics, and genetic diseases.
It is useful in the diagnosis of diseases and cancer. 2 Description of prior art To identify genetic differences in genomic DNA
The main conventional method is restriction fragment length polymorphism.
(restriction fragment length
by detecting polymorphisms (RFLP).
It is something that For example, J.F. Gusella et al., Nature
Huntington Dance by 306, 234-238 (1983)
Identification of the locus of the disease-causing DNA defect, and W.
K. Cavanee et al., Nature305, 779-784 (1984)
See analysis of predisposition to retinoblastoma. Most RFLPs are caused by small-scale changes in the DNA,
Usually creates a restriction endonuclease cleavage site
or arise from destructive base substitutions. human DNA
The mean heterozygosity is
The limiting error is low (approximately 0.001 per base pair).
nuclease at a given locus
RFLP will be detected only rarely. detected
Most RFLPs never have 50%
Allele frequencies that do not exceed and are usually very low
heterozygosity, determined by the degree of
dimorphic (restriction endonuclea)
cleavage site). Conclusion
As a result, all such RFLPs
Whenever mozygous, information in pedigree analysis
It lacks sex. Genetic analysis is a highly likely
Due to the availability of variable region probes and
correspondingly high heterozygosity
could be considerably simplified. This kind of
Area 1 is A.R.Wyman et al., Proc.Nat.Acad.
Sci.USA,77, 6754−6758 (1980), by coincidence
A library of random segments of human DNA
isolated from Lee. Multiple alleles at this locus
Structural basis of multiallelic variation
The foundation is still unknown. Then, again by chance
Several other hypervariable regions are found in the human insulin gene.
Denshi [G.J.Brll et al., Nature295 31-35 (1982),
Zeta globin gene [N.J. Proudfoot et al.
Call31, 553−563 (1982), and S.E.Y.
Goodbourn et al., Prou.Nato AcAd.Sci.USA.80,
5002-5026 (1983)], and c-Ha-ras-1 Carcinogenesis
Genes [D.J.Capon et al., Nature302, 33−37
(1983)]. In each case,
Variable regions consist of short sequences (“minisatellites”) of proteins.
Consists of dem repeats and is polymorphic
(polymorphism) is mitotic or meiotic
Due to divisive unequal exchange,
or produced by slippage of DNA during replication.
Based on allelic differences in the number of shear repeats. arising
Differences in the length of the nisatellites cleave the repeat unit
can be detected using no restriction endonucleases
Ru. The inventor and his co-researchers have already
The 33bp sequence in the intron of the oglobin gene
A short mini-satellite consisting of four tandem repeats
[P. Weller et al., EMBO J.3,
439-446 (1984)]. This 33bp repeat has already been characterized
The other human minisatellites mentioned above
However, it is noted that the sequence shows weak similarity.
I was noticed. This report shows that the minisatellite region is translocated.
It is assumed that this occurs due to (transposition).
Human - 33bp repeats of myoglobin gene are transposable
(transposable), then this one is the number of iterations
often associated with multiple allelic polymorphisms based on differences in
for tandem repeat regions of the human genome that are associated with
Could you please provide the probe? 3 Additional unpublished background information Human - Genomic DNA from myoglobin gene
A DNA protein comprising tandem repeats of a 33 bp sequence of
probing by lobes. polymorphism
Genomes of three individuals (father, mother, and daughter) with differences
observed in several different regions of DNA,
This difference is due to the size of larger fragments (2-6kb)
occurs in Data is more than 1 mini
Stable inheritance based on differences in the length of satellite regions
The results were consistent with the polymorphism. Summary of the invention Further research has shown that the myoglobin gene 33bp -
Much more effective than front view probe
shows the variability in minisatellites or hypervariable regions.
It is possible to explore genomic SNA using the method shown below.
It was shown that This invention is directed to
Between many minisatellites, different minisatellites
It is said to contain DNA regions with a high degree of homology.
It is based on the discovery of This common core region is short
Thus, it is approximately 16 base pairs. Now at least 3 times
A short core sequence of tandemly repeated nucleotides
A probe having as an essential component the column
Many different minisatellite regions in genomic DNA
, and their DNA phase in these regions.
An individual can be identified or identified by mentioning the difference.
It is useful for detecting
It was found that there is one. These traditional
lobe is better than that given by RFLP.
It leads to the discrimination of degrees, but is essentially unique for individuals.
It doesn't work with fingerprints. note
Of note, more than one mini-satellite
This can be defined by reference to a target region or a hypervariable locus.
The core probe of the invention is capable of fingerprinting DNA.
It has been found that it is possible to
It is this discovery that adds a degree of non-obviousness to inventiveness.
and this is of fundamental scientific novelty and importance.
It is an invention that has. Core sequence knowledge?
et al., minisatellites from various loci in the genome
has the property of hybridizing with the light region
DNA probes can be produced. but
However, mere recognition or identification of a specific core sequence
In itself, it is useful for the manufacture of probes
is insufficient. Tandem repeats in the core region
Producing a polynucleotide or its derivative containing
It is also necessary to build probe like this
is a minisatellite from human or animal DNA
can be isolated as or
It can be created using construction techniques. good results
Achieve additional constraints for hybridization
It is also important to These are acceptable
Consensus - degree and overall homology with the core
of non-core DNA within and outside the repeat unit as
Includes knowledge of acceptable lengths. Thus, this invention utilizes the following recognition and discovery.
Ru: (1) Knowledge of any one core sequence can be applied to this method.
that it can be used in This is further confirmed by
Including knowledge: (2) studying different hypervariable loci in the genome;
have the necessary degree of consensus depending on the
that different core sequences can be found; (3) would recognize a different spectrum of polymorphisms;
that a collection of DNA probes can accumulate; (4) By probes with specific gene classification,
Better results can be achieved than with other probes.
thing; (5) More than one program in any one classification
The use of lobes amplifies the probability of identification
thing; (6) Further research on the first generation of successful probes
Easier and more successful probes
can be produced, for example, synthetically. Thus, according to the first aspect of the invention, a large number of
typic minisatellites – with length-specific binding properties
A method for producing a polynucleotide is provided, and
Here's how: () Restricted activity to other polymorphic DNA regions
can perform hybridization,
Identifying natural tandem repeat sequences in DNA, () is presumed to be responsible for such binding.
The natural consensus-core sequence of the repeat sequence
identify the column, and () Lower genome than natural repeat sequences - genes
with locus-specificity and high polymorphic fragment susceptibility
natural constructs with minisatellite binding properties.
complete or incomplete sequence derived from the core sequence.
Isolate or artificially create tandem repeat sequences
to accomplish, It consists of The core components of the probe are based on the same basic principles
It can be defined in a variety of ways. the most basic group
The basic principle is that the repeat sequence of the probe is
Common core common to Nomu DNA minisatellites
-consisting of a nucleotide sequence from a region of
This should be included. This common core array
is a high level between one minisatellite and another.
For example, if the consensus is 80% or more,
In this sense, it is “common”. these mini
Satellites are, for example, the myoglobin gene
This was discovered by searching the genomic DNA for 33bp repeat sequences.
referred to as “λ33-positive” fragment in the specification of
Detected by obtaining hybridized fragments
It will be done. These fragments and myoglobin gene
The 33 bp repeat contains approximately 16 bp of common core sequence. child
The λ33-positive fragment of
Plasma of genomic DNA to form other fragments
Can be used as a robe itself
However, it can also be accompanied by some small change.
Ru. Another principle is that the core nucleotide sequence
effectively hack into the minisatellite region of sample DNA.
It is not so short that it cannot be hybridized, and it is
Not so long that type characteristics cannot be detected successfully, e.g.
A 33bp tandem repeat in the miniglobin gene
It is very similar. In general, the core
6 Nuks Found in the Common Core of Nisatellites
should have up to about 16 nucleotides
It is. Is the repeating sequence of the probe exclusively a core?
at either end of the core array.
Contain a small number of flanking nucleotides
I can do it. The repeating unit can be any number or species of nucleotides.
for any of the classes, and for the core or repeating unit.
is an exact iteration for any of the non-core components.
It doesn't have to be. However, this is approximately
Even if the term
It is convenient to describe and define it as a position.
A block of n repeating units can have any
It may have a nucleotide sequence and its extent
and type is usually unimportant. The polynucleotide of this invention can be obtained by any of the following methods.
specifically includes those defined in any of the following. First definition The following general formula read in the meaning of 5′→3′ H. (J.Core.K)n.L (1) [During the ceremony, A “core” consists of at least six consecutive nuclei.
represents a sequence with an otide, this nucleotide
The following array is read equivalently: GGAGGTGGGCAGGAXG (2) AGAGGTGGGCAGGTGG (3) GGAGGYGGGCAGGAGG (4) T(C)_nGGAGGAXGG(G)_pc (5A) T(C)_nGGAGGA(A)_qGGGC (5B) is selected from one of the following, where: X is A or G, Y is C or T, m
is 0, 1 or 2, p is 0 or 1, and q is
is 0 or 1, and n is 3 or more; J and K together are 0 to 15 in the repeating unit
represents an additional nucleotide of; and H and L are 0 or 1 or more sandwiching the repeat sequence, respectively.
the additional nucleotides above; and the
and i.e.: () “Core” and J and K are each (J. Core
-． K) Require same sequence or length in repeat units
It doesn't have sushi either; () “Core” represents a further different core arrangement.
You can also forget; () Actual total core sequence in all n repeat units
is for formulas 2 to 5 in the same number n of repeating units.
The “true” total core array defined above and 70
% or more homology; [provided that] a polynucleotide having; and A polynucleotide with a complementary sequence to this
Otide. Second definition The following general formula: H. (J. Core. K.) n.L (1) [During the ceremony, “Core” has the same meaning as 5′ → 3′ 6 to 16
represents a sequence of consecutive nucleotides, (1)
Myoglobin tandem repeat arrangement of about 33 nt per repeat unit
human or animal using probe DNA containing sequences
obtained by searching the genomic DNA of
5′→3′ of the first human or animal minisatellite
(2) common core area of the first mini-satellite;
Contains a tandem repeat sequence comprising an arch region.
human or animal DNA using probe DNA
Second person or movement obtained by searching
5′→3′ common core region of minisatellites of objects, and
and (3) including the common core region of the second minisatellite.
A probe containing a tandem repeat sequence consisting of
Search for human or animal genomic DNA using DNA
A third human or animal species obtained by
Selected from the 5′→3′ common core region of Nisatellite.
Each of the above tandem repeat sequences has 3 or more units of repeats.
vengeance] a polynucleotide having;
A polynucleotide with a complementary sequence. Third definition The following general formula: H. (J.Core.K)n.L (1) [During the ceremony, “Core” means 75% or more, preferably 80% core.
of human or animal genomic DNA showing a
6 or more from within the common core area of the minisatellite
Any of the sequences with consecutive nucleotides above
represents; The “core” is not necessarily the same sequence in each repeat unit.
and all other symbols
is as defined above] a polynucleotide having; and for this
A polynucleotide of complementary sequence. Fourth definition The following formula: (5′)GPGGGCWGGWXG(3′) (6) (In the formula, P is not G, and W is A or T.
and X is A or G) selected from the array represented by or its variants
6 to 35 nt containing a contiguous (5′→3′) core sequence
A polypeptide having three or more repeats of the sequence (all
The actual total core array during the iteration is the same number of iterations
The “true” core configuration defined for equation (6) in
conditional on having 70% or more homology to the column
); and a complementary array point to this.
Renucleotide. In the above expressions, and throughout, the array is a regular
It is indicated by the notation 5′→3′. This invention relates to DNA, RNA, and
Any other type of polynuc that can hybridize
Includes leotide. Polynucleus defined above
Otides are unlabeled and double-stranded
(ds) form or single chain (ss) form. This invention is a ss for use as a probe.
shaped labeled polynucleotides, and these
labeled precursor and then ss-pro
This includes those capable of producing a tube. These are preferably in any conventional manner³³P-
radioactively labeled, but this method is
well-known in hybridization technology.
may be radioactively labeled by other means.
Proceedings of the 1981 ICN−
UCLA Symposium on Developmental
Biology using Purified Genes, Keystone;
Colorado, March 15-20, 1981, Vol XX
1981, pp. 647-658, Academic Press,
D.C. described in Donald D. Brown et al., eds.
biotin or similar species by the method of Ward et al.
or A.D.B.
Malcolm et al., Abstracts of the 604th
Biochemical Society Meeting, Cambridge,
Enzyme treatment using the method of the United Kingdom (Meeting of July 1, 1983)
You can even be belled. According to another aspect of the invention, human or
is the genetic source of samples containing animal DNA.
Polynucleotide probes useful for determining the future of
This product contains labeled ingredients or markers.
Contains a car component and restricts sample DNA.
obtained by fragmentation with endonuclease.
Hybridization of the probe to the corresponding DNA fragment
human or animal to the extent that it allows for daization.
Sequences homologous to minisatellite regions of the genome of
3 or more tandem repetitions of (including variants)
comprising a polynucleotide comprising
and do the following, i.e.: a Each repeat consists of multiple repeats from different genomic loci.
Containers of similar length present in minisatellites
Census - core region with 70% or more homology
Contains ar; b the core has a length of 6 to 16 nucleotides; c Nucleos within the repeat unit that do not contribute to the core
The total number of chidos is 15 or less; It is characterized by This invention further provides human or animal genomes.
This method encompasses DNA sample identification methods.
searches the DNA with the probe of this invention,
and detect hybridized fragments of DNA.
It consists of This aspect of the invention: Total DNA from a sample of cellular material is restricted and encoded.
Fragmented using donuclease, Highly variable DNA fragments can be combined with labeled components or
Contains repeating core components in addition to marker components
hybridize with the probe defined above;
and into DNA fragments of different lengths, or more generally
are labels attached to bands of different molecular sizes or
determine the concentration of the marker, Including. Usually, fragmented DNA is processed by hybridization
Before electrophoresis, e.g.
and then detect the marker concentration.
We know that its individual elements are specific to genetic origin.
Obtain a characteristic pattern. Definition The following used in this invention and the description so far:
A definition may help. hypervariable human at one recognized locus or site
A region of animal or animal DNA that is different in length or sequence.
If it exists in many different forms, it is hypervariable.
It is said to be a target. Restriction fragment length polymorphism (RFLP) This allows even separation and probes after electrophoresis.
The pattern of human or animal DNA fragments detected by
Genetic diversity in turn. mini satellite It is composed of tandem repeats of short DNA sequences.
This is the region of human or animal DNA that is produced. vinegar
It is not always the case that all repeating units exhibit exact identity.
is not necessary either. (The probe of this invention is polymorphic
It consists of a mini-satellite. ) polymorphism Genes or DNA that show differences between individuals
Segments of are said to be polymorphic. core (array) Originally used to mean consensus-core sequence.
However, any iterations derived from it
It is extended to arrays or modified arrays. Consensus-core (sequence) Two or more minisates with different origins or genetic loci
Substantial or complete match between repeating units of light
A sequence that can be identified as iteration (array) a given core sequence or a cell containing a core sequence.
complete or incomplete tandem repetition of a segment
It is an array. defined core (array) completely identical to one of equations (2) to (8) within its own length.
This is the core arrangement that matches the Deformed body (core arrangement) differs by a small degree from the defined core sequence
(50% or more homology) Actual core sequence. Complete iteration (array) of or containing a given core sequence
An array that is an exact tandem copy of the segment.
Ru. incomplete repeat (array) at least one unit has a base pair substitution and/or
differs in length from at least one other unit
It is an array. (Normally, there are only a few in one probe sequence.)
At least 3 tandem arrays are present and the probe
at least one core array defined within the array
and there will be at least one variant
cormorant. ) Homology % In comparing two sequences of the same length, base pairs
The number of bp obtained by subtracting the number of bp substitutions from the number of (bp)
% of Nucleotides (nt) and base pairs (bp) are synonymous
used for. Both can refer to DNA or RNA.
I can do it. Abbreviations C, A, G, T are as usual (De
(oxy)cytidine, (deoxy)adenosine, (sepoxy)cytidine, (deoxy)adenosine,
oxy)guanosine, and deoxythymidine or
It means either uridine. tandem repeats (artificial or naturally isolated)
body) can be completely iterative, but
More preferably, it is an incomplete repeat. Preferably less
At least two repeats are consensus-core sequence
is an incomplete repetition of Preferably, three or more
repeats, and more preferably seven or more repeats.
Present in lobe array. Probe production is based on natural clones by cloning.
Nisatellite isolation and DNA sequencing
can include specifics. This is also required
Cutting out the core and its subsequent tandem
Conversion to repeats or non-identical fragments with different origins
This may include facilitating an exchange. This is also
May include cloning of polynucleotides
Wear. On the other hand, the construction stage is based on the identified consensus code.
may include the synthesis of a sequence or a fragment thereof.
Ru. Consensus - core sequence preferably 6bp
and preferably contains 16 bp or less
do. Next, create tandem repeats of the synthetic core.
Ru. Needless to say, polynucleotides have shown good results.
cloning of or partially successful intermediates of
be the result of a succession of operations at different subsequent times
and the final polypeptide is the parent minisatellite
of natural origin as having a slight resemblance to
or fragments of synthetic origin. The probe of this invention is useful in the following fields:
be. 1. Paternal and maternal studies in humans. 2. For example, family groups in settlement disputes and inheritance disputes.
proof of. 3 Zygasity test in twins
Experiment. 4. Studies on consanguineous marriage in humans. 5 General pedigree analysis in humans. 6. Identification of genetic disease loci in humans.
This allows for specific
It becomes possible to create a probe. 7 Forensic medicine (a) Identification of semen from rape victims; (b) blood, hair and semen, for example from soiled clothing;
identification of, (c) Identification of human remains 8 Cell Chimaerism
studies, e.g. donor cells versus recipient cells after bone marrow transplantation.
follicle tracking. 9 Livestock breeding and pedigree analysis/certification. (This is an example
routine control and inspection of purebred animals;
For example, in litigation cases related to race horse and dog breeding.
This could include a pedigree check. )Sara
shows relationships with economically important genetic traits.
To provide genetic markers that may be useful. 10 Contamination of pure cell lines and routine identification procedures
Routine inspection of cultured animal cell lines
quality control. 11 Information on tumor cells and tumor molecules
Analysis. 12 Polynucleotide or probe derived therefrom
has potential uses in plant breeding
It is expected that [Brief explanation of drawings] Figure 1 shows the 33bp repeat sequence of the myoglobin gene.
Insert the column into the plasmid and clone it
This is a schematic explanation of its manufacturing method. Second
Diagram shows hybridization to various DNA probes
Photograph of an autoradiograph of genomic DNA fragments
is a photocopy of, and furthermore, its DNA.
was identified in two autoradiograms.
Shows the pedigree of relatives. Figure 3 shows this invention.
relatives explored with three different probes of
DNA samples from three unrelated human individuals
The autoradiogram of the sample is shown. Figure 4 shows this
9 human cases probed with the inventive probe
Autoradiograph of a DNA sample from an individual
, several of them are related, and two
are identical twins. Figure 5 shows the prototype of this invention.
2 humans and 17 humans explored by Loeb
Autoradiation of DNA samples from animals other than animals
Show ogram. FIG. 6 shows the search according to the invention.
Ghanaian family caught in settlement conflict investigated
A series of autotra of DNA samples from members of
Showing a geogram. Figure 7 shows neurofibromatosis.
Autoradiation of DNA samples from blood relatives who have
Indicates grams. Figure 8 shows the minisatellite fragment.
Possible co-inheritance and fetal hemoglobin
Created for Genetic Persistence (HPFH) Testing
Autoradiogram of DNA from Gujerati Pedigree
Indicates the time. Figure 9 A and B are related to large kinship relationships.
reception of HPFH and various minisatellites in
Genetic diagram showing splicing. Figure 10
is the preparation of cloned artificial minisatellites.
This is a diagram showing. Figure 11 shows the new
Unrelated, explored with synthetic probes
One series of DNA samples from the placentas of two individuals.
A traradiogram is shown. Figure 12 shows large kinship relationships.
A series of audits of the DNA samples searched from
A traradiogram is shown. Figure 13 shows two different programs.
Various results obtained for twins using lobes
Autoradiogram showing DNA banding pattern
It is. Figure 14 shows single-stranded probe and double-stranded probe.
Comparing the band patterns formed using the
This is an autoradiogram. Figure 15 and 1
Figure 5A shows DNA fingerprints obtained from forensic samples.
This is an autoradiogram showing the crest. Figure 16 is
Auto showing DNA fingerprints obtained from a family of dogs
It is a radiogram. Figure 17 shows a short-haired domestic cat.
Autoradiograph showing DNA fingerprints from a family of
It is mu. Figure 18 uses two different probes.
An image showing DNA fingerprints obtained from various sheep.
This is a radiogram. Figure 19 shows three different animals.
Autoradiogram showing DNA fingerprints from a pig
It is. Figure 20 shows a family of cows and additional cows.
In an autoradiogram showing the DNA fingerprints of
Yes; and Figure 21 shows a diagram using a different probe.
Figure 20 is a similar autoradiogram. Description of preferred embodiments Human DNA cloned into Phage λL47.1
10-20kb ofSau3A part human genome library
Lee, 33bp myoglobin repeat probe
“Hybridization with pAV33.7
Cleaned. 3×10^FiveRecombinant library
– 40 or more strongly or weakly hybridized
A plaque was identified. These positive plaques
(λ33.1−15) and purified 8 random selections of
using Southern blot analysis of phage DNA.
DNA that hybridizes in each recombinant
is located within the unit short (0.2-2kh) region of the recombinant
It was shown that Sequence analysis revealed that eight recombinants
This region of each of the 3 to 29 tandem cos of the repeat
Contains minisatellites comprising P.
The length of the repeat sequence is 16bp in λ33.15 to 64bp in λ33.4.
It was shown that the range is within the range. most mini satella
The site contained an integral number of repeats. next,
In λ33.6, the 37bp repeat is a basic 12bp unit.
It consisted of two forked trimmers. λ33 pairs each
Converting is clone-specific between each minisatellite.
Differences in the human genome as determined by DNA sequence
He represented the area. Eight cloned minisatellite regions are human
DNAHinfDetected by pAV33.7 in the I digest.
Possible polymorphic 2-6kb DNA fragments smaller than 0.5~
2.2kbHinfIt was located within the IDNA fragment. claw
Polymorphism in any of the minisatellite regions
In order to determine whether the³²P-labeling
Place a single-stranded DNA probe at the appropriate location for each minisatellite.
Prepared from M13 subclone, and high stringency
in,HinfNot related to the 14 people killed in I
Hybridized to a panel of British white DNA
I met. Typical hybridization pattern
each under these hybridization conditions.
Probe detects unique region of human genome
and that three of these regions are highly polymorphic.
Show that something is true. Further details of the above steps are provided in the example.
Ru. Now, regarding the above definition of polynucleotide,
The definition is the following general formula H. (J.C.K.) n.L (8) (In the formula, C indicates the core sequence, and other symbols
is as above) matches. One unit in all definitions
The core array of can be the same or different from
good. For example, this applies to the repeating unit as a whole.
1 or 2 extra sequences compared to the consensus sequence
Contains nucleotides, one or two nucleotides
Lacking an octide or (for example) 1 to 4 nuclei
The punch line may be different. The core can be defined in a variety of ways. general definition
The definition refers to a procedure by which the core sequence can be identified.
It can be obtained by applying genome
For example, the satellite region of DNA is GGAGGTGGGCAGGAXG (2) It is compared with an array such as . Everything included in equation (2)
selected from all possible X-nucleotide length sequences.
The minisa that shows the greatest homology with the X-nucleotide
X-nucleotide long sequence taken from tellite
is considered to be a core array. Needless to say
This is a very narrow way to define the core.
and one or a few core 6 nucleotides
long, one or a few seven nucleotides long, and up to 16
should yield a nucleotide length of (maximum
, for example, there is more than one of 100% homology
Therefore, it is a “few”). defined like this
An average of 70% of these cores relative to
Diverse to the extent that all n repeat units have the above homology
It is assumed that gender can exist. in a repeating array
Flanking sequences J and K are for homology
Not included in calculations, only compared between cores.
The defined core can have various lengths.
and can be “mixed”. That is, some
For example, in the repeating unit of formula (2)
homologous to GGGCAGGAXG and elsewhere
is homologous to, for example, GGGCAGGTGG in formula (3).
Therefore, the deformed body is necessarily connected to the “core” itself.
Considered as homology with (core) n rather than homology.
should be considered. The “first” definition defined earlier takes into account the above considerations.
Equations (2) to (5) derived from but shown
Medium, depending on circumstances, any length from 6 to maximum 12 to 16
It is simple in that the core is defined as an array of
Purified. Similar regulations exist for deformed bodies.
Ru. The second definition is that each additional mini-satellite
Successive hybridizations that can generate fragments
Define the core as a yon stage. human genome
Sufficient numbers for extensive libraries of DNA
Perform hybridization to obtain a sufficient number of hybrids.
Test the hybridized fragments and probe from these.
make a new library, explore the genomic DNA again, and make sense of it.
Logically, by doing this an infinite number of times, you can
Consensus code widely symbolized in light DNA
It is possible to reach the range of the array
will be easily understood. In reality, the space is wide enough.
very large numbers to reach the sensor score area.
These operations must be performed several times and on a large scale.
It is not expected that the
Only three search operations (in hand) are included in the definition. In the “third” definition, W represents the core.
and 25% (e.g. 4 out of 16 nucleotides)
leotide) is widely shared with more and less different diversity.
Relying on the possibility of a consensus core area
Ru. Approximately 16 nuku with possible variability of less than 25%
Define the core region above leotide in this way.
For example, core W consists of all consecutive 6 or more from within the area.
It is defined as the sequence of nucleotides. The “fourth” definition relates to synthetic polynucleotides.
The redefined consensus code obtained from the
Contains formula (6). These studies support other definitions of shorter core sequences.
Lead righteousness. Therefore, preferably a contiguous (5′→3′) sequence: PGGGCWG (7) is conserved in all repeat units, where P and
W has the meaning given in the fourth definition above.
do. P is preferably T and W is preferably
It is A. Preferably also a contiguous (5′→3′) sequence: TGGGCA (8) is conserved in all repeat units. According to another point of view, consecutive 5′→3′ Core array: GGPGGGCWGGWXG (7) (In the formula, P is not G, and W is A or T.
and X is A or G) a polynucleotide having three or more repeats comprising;
or its variants (actual total core configuration during all iterations)
“True” defined with respect to formula (7) in iterations with the same number of columns
“has more than 70% homology with the total core sequence of
), and complementary to the above.
Polynucleotide sequences are provided. In all the above definitions, the core is less
Both have a length of 6 nucleotides, and more preferably
7 or 8 nucleotides and most preferably
or greater than 12, e.g. 14-16. formula
(2) sequence GGGCAGGAXG (terminal 10 nucleotides at the 3′ end)
) has a high consensus and is particularly
It seems hopeful. The deformed body core preferably has 70% or more, and
More preferably have a homology of 80 or 85% or more
Ru. Flanking sequences J and K within each repeat unit are preferred.
Preferably omitted or shortened at each end, e.g.
for example 0.1 or 2 nucleotides and preferably
Or J and K together should exceed 20.
should not exceed 20, and more preferably should not exceed 20.
stomach. All nulls in the sum of J + core + K in the repeating unit
The cleotide number is preferably 36, and even more preferably
preferably greater than 31 and most preferably greater than 25
Shouldn't. The number n of repeating units is preferably at least 10, conveniently
The profit range is 10 to 40, but in principle any
can be a number, even a number up to 10000
It is. Flanking sequences H and L are unrelated. child
These can be omitted or any number of
Exists as up to 20,000 nucleotides, e.g.
However, with such long probes
It is not very wise to do so. Repeated array is ss−
Even if DNA, these contain ds-DNA
can do. Any known search technique may be used as the identification method.
The most common method is to collect sample DNA,
Do not cleave tandem sequences or search for them.
cleave to a meaningless degree that does not interfere with the ability to be searched.
Restriction enzyme (one type if appropriate)
or multiple types). Example 1 Long tag from human myoglobin gene
Construction of a probe containing a double repeat sequence The construction of this probe is shown in 5 stages (a) to (e).
It is explained in the mock-up in Figure 1 which shows the floor. Out
Myoglobin gene (a) is derived from P. Weller et al., EMBO
J.3, 439-446 (1984).
As shown there, the gene is mostly
33bp sandwiched between two identical 9bp sequences (r in Figure 1)
In the first intron, consisting of four repeats of the sequence
It has a region where it is located. This region is 169bpHint
isolated in f I fragment (b) and end-repaired it.
and plasmid pUC13SmaClaw on the part
[J. Vieira et al.,
Gene19, 259-268 (1982)]. limit
endonucleaseAvaBy (A), the third and
The monomer is isolated by cleaving the fourth repeat.
Released (c). (Single substitutions in repeats 1 and 2 are
remove the part and replace it withDde(D) Part
Form. ) non-identicalAve.33bp via sticky ends
Head:tail port with unknown number of reactive units due to monomer linkage
Rimmer (d) was produced. at least 10 iterations
Agarose gel electrophoresis for preparative separation of polymers containing
isolated and end-repaired pUC13Sma
ligated into the E. coli JM83 site and cloned into E. coli JM83
(see J. Vieiva et al., supra).
The port in the resulting plasmid (e), designated pAV33.7.
The structure of the remer DNA insertion site isBamH+EcoR
Cut out the insertion part in the polylinker by
, α−³²By P-dCTPBamH site odor
to create a file-in label, andAvaby
Confirmed by partial digestion. labeled
The partial digestion products were electrolyzed on a 2% agarose gel.
Separated by electrophoresis. pAV33.7 is shown in (e).
Sea urchin 767bpBamH-EcoContained in R fragment
Found to contain 23 repeats of 33bp monomer
It was. (2) Human with myoglobin 33bp repeat probe
- Sequencing a selection of minisatellite regions of the genome
fixed Cloned into bacteriophage λL47.1
Library of 10-20 kb human DNA fragments [P.
Weller et al., supra, and W.A.M. Lenen et al., Gene
20, 249-259 (1980)], P. Weller et al., supra.
by the method of³²P-labeled in step (1) above.
Hybridization with the 767bp pAV33.7 insert described in
Screening was carried out by daization. 8 pieces
A selection of positive plaques was purified and labeled as λ33.1−15.
A recombinant named . These Phage DNA
eachHintf orHaeDigested by 1.5
electrophoresed through a % agarose gel and
The 33-repeat-related sequences in
By way of hybridization
The position has been determined. Each recombinant has one “λ33-positive”
Hinf andHaegave a fragment, but λ33.4 and
11 is detectableHaeDidn't give any fragments (this
in the repeat region in the recombinant of et al.Haecleavage site exists
). λ33-positiveHintf andHaepiece
isolated by preparative gel electrophoresis and optionally
End repair and double-stranded DNA M133mp8
blunt-end ligated to the Sma site (J. Messing et al.
Gene19, 269-276 (1982)]. E. coli
After transformation into JM101, positive ss-M13 recombinants were isolated.
release, and dideoxynucleotide chain
-Sequenced by termination method. All
All λ33-positive fragments contain tandem repeat regions.
, which in some cases requires direct sequencing.
I was able to Repeated regions are sequencing primers
In other cases, if it is too far from the site, restrict the M13 insertion site.
Shortened by restriction endonuclease cleavage
and sequenced again. The structure of the λ33-positive fragment is λ33.1, 33.3, 33.4,
8 pieces called 33.5, 33.6, 33.10, 33.11 and 33.15
shown later in map form. Actual restriction enzyme used
The length of the elementary and nucleotide fragments is λ33.1:
Hae, 2000; 33.0:Hintf, 465; 33.4:Hint
f, 2000; 33.5:Hintf, 1600; 33.6:Hae
, 720; 33.10:Hae, 720; 33.11:Hinf,
1020;λ33.15:HintIt was f, 1220. Each map
repeats the bases in uppercase script on a rectangular box
Indicates an array. “Repeat” is always complete in number of bases.
Not all, and due to base substitution.
It's somewhat different. Boxes indicate iterations that differ from consensus.
vinegar. Blank space within the box indicates a match was found.
The base symbols A, C, G, and T in the box are consensus
What is shown above it in the array is
Indicates that it has been replaced. X is A or C
be. Y is C or T. − is a consensus
It is the missing nucleotide in comparison. >>>
The <<< (arrowhead) symbol is used as is in the sequence.
Not autoradio glasses for sequencing gels
, the sequence is a consensus “repeat”
(It goes without saying that the word “repetition” can be used in the same way as above.
use). piece
λ33.3, 33.5, 33.10, 33.11 and 33.15 are fully aligned
others have been partially sequenced.
Ru. The number below “con” on the left side of the box is the repetition of the array.
It is a number. The number at the bottom of this column indicates the number of iterations.
vinegar. i.e. λ33.1 is the uppercase script above the box
26 repeats of the 62 nucleotide (nt) sequence shown in
including. Lowercase script at the top and bottom of each map
The sequences shown in
These are flanking sequences present on the 5′ and 3′ sides.
and these flanking sequences are iterated
do not have. In other words, this structure
That of the random copolymers indicated by the columns
similar, where the block represented by tandem repeats
Units of the copolymer appear. 【table】 【table】 【table】 【table】 (3) located within the repeat sequence of each λ33-positive fragment;
Discovery of common short “core” sequences shared by
and identification Repeated sequences in each region were analyzed using dot matrix analysis.
Myoglobin 33bp repeat sequence in pAV33.7
, and its reverse complement. very obvious
In particular, myoglobin 33bp repeat sequence and λ33-positive
There is no sequence similarity between the consensus repeats of the fragments.
One small clear area was found. same area
is shared by all eight λ33 fragment repeats.
and may be referred to as the "common core." under
The chart below shows the comparison. In this chart, the decision made in step (2) above
The entirety of each consensus sequence determined is shown.
These are in myoglobin 33bp probe and human
λ33-positive fragment isolated from genomic DNA
This is the part that repeats in tandem. This cheer
The common core area is divided into 16 blocks of uppercase script.
Shown as leotide length. 【table】 * This sequence is a trimer and therefore a furan
The king area contributes to the core.
Again, X is A or G, and Y is C or T.
Yes, - is the missing nucleotide. these 16
Substantial degree of identity exists for nucleotides
8 of them showed 100% agreement, and number 9
The “X” of the eye is either A or G.
I will. These 9 nucleotides have an nucleotide in the bottom row.
A dashed line is attached to indicate the null area of the common core area.
Indicates cleotide. indicated in lowercase script
Residues of tandem repeats are located at the edges of the common core region.
exist. The start/end of each iteration consensus is a symbol
Identified by ▼; non-standard in the case of λ33.4 and 33.15.
There is one integer iteration, and therefore separate
The start and end of the repetition are indicated by different symbols ▽.
Common core regions can only be identified through complex analyses.
Let's recognize what could have been done. Because this is
One consensus sequence of frequently λ33-positive fragments
does not belong to the whole, and the myoglobin gene
Because it spans two consecutive sequences of 33bp repeats
It is. defined as being within the scope of this invention
To illustrate the degree of match to the polynucleotide,
The various determining factors are shown in Table 1 below. 【table】 【table】 Underlined numbers are not allowed
Indicates length.
(4) Polymorphic human-genomic DNA fragments contain individual λ33
- Hybridization of positive fragments with probes
discovery that it can be detected by Regarding Figure 2, a random sample of British Whites (1-6)
Selection of small and large Anglo-Asian ancestry (7-18)
DNA was extracted from white blood cells collected from members of the
Manufactured. For convenience, the pedigree is a square that indicates males and a square that indicates females.
By the circles shown and the marriage by the lines between them
shown. Two close marriages between cousins and parents
Indicated by two lines. 10μg sample of DNAHinf
and passed through a 20 cm long 2% agarose gel.
electrophoresed in situ, denatured in situ, and blotted.
Sartorius Nitro by Ing
Transferred to cellulose filter. single chain³²P-ra
Convert the hybridization probe into a mini
M13 containing satellite (tandem repeat) regions
Prepared from recombinant. The exact probe used was
I will write about it later. The procedure was as follows. Approximately 0.4μg
4 ng of single-stranded DNA in Nocoumer sequencing primer
[M.L.Dockworth et al., Nucleic Acids Res.
9, 1691-1706 (1981)] and 10 μl of 10 mM
MgCl₂, 60℃ in 10mM Tris-HCl (pH 8.0)
Annealed for 30 minutes. Primer extension,
16μl 80μM dATP, 80μM dTTP, 10mM
Tris-HCl (pH 8.0), 0.1mM EDTA and 3μl
(30 μCi) α−³²P-dCTP (3000 Ci mmole^-1)
and 1 μl of 5 units μl^-1Klenow Fragment (Baelin
) and incubate for 15 minutes at 37°C.
This was done by baiting. Extension is 2.5μl
Add 0.5mM dCTP and further 15 min at 37°C
Completed by chasing
did. (What is “Chasing”?
To complete the transfer of dsDMA onto the M13ssDNA template.
means adding the dNTP mixture.
DNA in the insert or distal to the insert.
Appropriate restriction endonucleases in M13 polylinters
1/10 volume of 1.5M
Modified by adding NaOH, 0.1M DKTA.
and extend from the primer.³²P-label
Convert single-stranded DNA fragments into a 1.5% low melting point agarose gel
Recovered by electrophoresis using (Sea Plaque)
Ta. Excised band (specific activity >10⁹cpm／
μgDNA) was sheareed with 1mg of alkali.
Carrier human placenta DNA (0.3MNaOH, 20mM
Taken for 5 minutes at 100°C in EDTA and then in HCl.
re-neutralized) at 100°C, and
directly into the hybridization chamber.
Ta. Carrier DNA also serves as a link to repetitive DNA sequences.
to suppress hybridization after
It was also helpful. The exact hybridization procedure used
(A) 33.1, minisatellite (62nt sequence)
26 repeats = 1612nt) + approx. 360nt frantenghito
Approximately 2000nt subcloning containing DNAHaeCut off
Piece; (B) 33.4, 2015ntHintContained in f fragment
695nt non-− on the proximal side of the minisatellite primer
mini satelliteEcoR fragment; and (C, D,
E) 33.15, λ33.15 minisatellite array (shown above)
An average of 2 nucleotide differences from the common core region
29 repeats of 16nt sequence) + 128nt flanking human
A 592nt subcloned fragment containing DNA,
Ta. A.J. Jeffreys et al., Celltwenty one, 555-564 (1980)
Hybridize as described above.
I went to Shion. However, the background label
Dextran sulfate is added to lower the
% (W/V) by polyethylene glycol 6000
Replaced. Filters A and B in 0.5×SSC
Hybridize overnight at 65°, then 65°
and washed in 0.2×SSC. Filter C~E
was hybridized and 65% in 1×SSC.
Washed at ℃. Tangs with fixed filter
1 to 3 at -80℃ using a thenic acid reinforced screen
Autoradiography was performed for days. Iterative core probe as shown in Figure 2.
33.15 isHinIn human DNA digested with f
Extremely complex profiles of hived fragments
detected. Maximum (4~20knt)Hintf fragment
only the hives can be well separated, and these
Redization profile is solid-specific
Shows extreme polymorphism to the extent that it provides a DNA “fingerprint”
did. Pedigree analysis is based on a single blood relationship (first cousin marriage).
Even between 16 and 18 in Figure 2, all individuals were identified.
confirmed the extreme polymorphic diversity that can be
Ta. The family (D, E) in Figure 2 isHintf-cut
Most of the fragments are passed down from each parent to only some of the offspring.
This shows that these discontinuities
Most of the pieces exist in a heterozygous state,
and the heterozygosity of these large hypervariable fragments is
It is shown that it must reach 100%.
In contrast, all fragments in the descendants are either one or
on the other hand (the only exception being
), and hence a set of stable heredity
Provides genetic markers that can be used. specifically from my father
transmitted to son or from mother to daughterband
(Filter D, see Figure 2)
and). This excludes Y and X linkages respectively
and these minisatellite fragments are
meaning that it is derived from autosomes. these
Where in the set of autosomes does the DNA fragment come from?
Although it is not yet known how many
It does not originate from a single region of the chromosome. yes
pairs of parent fragments that segregate independently in their progeny rather than
can be identified (filter D, see Figure 2).
thing). Precisely at least one AB or --
If there are descendants, the
The pair of bands AB (missing from one side) is an allelic gene.
cannot be a descendant; existence of A- or -3 recombinant progeny
Furthermore, the lack of close linkage between A and B
Probability. The origin of the family shown in Figure 2D
Careful examination of the autoradiographs of those
At least 10 separations in the mother by the criteria of
Possible bands, 8 of which are mutually exclusive.
are genetic and not closely linked.
stomach. The other two bands each have 8 unlinked
may be one allele of the fragment, here
Restricting the progeny so that only A- and -B progeny are analyzed in
observed only in the number of
A small number of samples show that such fragment pairs are monogenic.
Enough to prove that it is an allele at the child locus
Not. In conclusion, the core probe
Simultaneously useful on a few unlinked hypervariable loci
can give information. This conclusion is shown in Example 8.
will be considered in more detail. On the other hand, the other two probes (filter A and
B, FIG.
and many different bands.
Polymorphic regions cannot be detected simultaneously, and
Therefore, it cannot be used for general diagnosis. Example 2 Detecting a new set of hypervariable regions in human DNA
Additional deformed body (core
-) use Cloned λ33 - derived from positive fragments λ33.5 and λ33.6
The other two probes coming in are similar to Example 1, step (4).
It was prepared as follows. 33.5 probe is clicked during M13mp8
Consists of a loaned 308nt DNA fragment and on
It consists of 14 repeats of the consensus sequence shown below.
This is the common core along with the 70nt Franteng human DNA.
-This is a 17nt long variant of the sequence. 33.6 probe is
Contains a 37nt sequence of 18 repeats, this sequence is common
3 repeats of a shortened sequence of approximately 12 nt derived from the core region +
comprising an additional TC at the 5'-end of the TC. 18
×17nt repeat block is sandwiched between 97nt human DNA
there was. The structure of the 37nt sequence can be expressed as follows.
can. TGG AGG AGG GGC TGG AGG A-G GGC (or TGG
AGG AGG GC) TCCGG AGG AGG GGC This probe was similarly cloned into M13mp8.
was made into In the later description, the 11nt consensus sequence
AGGGCTGGAGG given for this probe
It will be done. both probes³²Label by P and Example 1.
Similar to step (4), 14 unrelated Caucasian pa
It was hybridized with human DNA from flannel.
Both probes perform a complex set of hybridizations.
detected fragments, many of which have extremely polymorphic changes.
showed that. Some fragments detected by 33.5 are
New and with 33.15 core probe
This has not been detected until now. 33.6p
The robe is almost a completely new set of mini satays.
Detecting lights and correct inheritance of these
was proven by pedigree analysis. (See Example 8)
and). The following examples further explain and illustrate this invention.
Ru. Digestion of sample DNA preferably involves
Restriction endonuclea that recognizes the 4 base pairs of
This is done using ze. For the longest hypervariable fragment
4bp recognition system using all DNA fingerprint patterns
It is largely independent of restriction endonucleases.
was found. This means that these large fragments
Rather than being derived from longer minisatellites,
and each is subjected to restriction endonuclease cleavage
site lacking and normal high density of 4 bp cleavage sites
full-length homogeneous DNA flanked by human DNA containing
Strongly suggests that it contains minisatellites.
This is because most of these large minisatellite fragments
which are unlinked and separated into lineages independently.
The examples above and in Example 8 show that
matches the results given. In a preferred embodiment, the sample of DNA is
Separate hybridizations resulting in two different fingerprints
In the process, two different probes, e.g.
Using probes from lambda 33.15 and 33.6 fragments
and double fingerprints will be taken. Two unrelated individuals are the same
have the same fingerprint. The probability is already low, but this method
This will further decrease. For example, Example 4 preferably
is an incompletely segregated hybrid with a terminal length of 4 kb.
10 even when is DNA fragments are ignored^-19Toi
indicates a low probability. The invention includes a method of paternity testing. in child
Approximately half of the polymorphic minisatellite fragments originate from the father.
and these paternal fragments are the DNA of the mother and child.
It can be identified by comparing fingerprints. these
all of the paternal fragments are normally present in the father's DNA.
There will be. Probe length 33.15 and over 4kb
Using DNA fragments, a hypothetical father can be identified as typical in a child.
All six paternal-specific DNA segments identified
The probability of having a piece by chance is 10^-FiveIt is an order of
And the use of both probes 33.6 and 33.15 makes it
Ten^-8is expected to decrease to the order of
Ru. Of course, the exact probability is obtained by the exact separation and
Depends on the complexity of the DNA pattern and less than 4kb
Additional paternal fragments of full length are analyzed, or
This could be improved if more probes were used.
cormorant. Due to DNA fingerprinting, one drop of human blood can produce enough
Rapidly isolate DNA (0.5-5 micrograms)
can be done. And that DNA has already been
Individuals including 2 people + 2 sisters whose crests have been collected
DNA fingerprints were created from a random panel. 2 people
A pre-characterized individual has a DNA fingerprint ratio
can be easily and unambiguously identified based on comparison.
have a substantial number of minisatellite fragments in common.
The same was true for the two sisters. DNA is taken from different cells of a given individual
can all give the same fingerprint. Therefore, the essence
DNA fingerprints are identified on children and blood DNA.
The pattern is similar for identical twins. Sara
The DNA fingerprint of the blood DNA and the
transformed with Epstein-Barr virus.
isolated from a lymphoblastoid cell line
pattern, as shown by comparison with DNA
appears to be maintained in cultured cells. Non-human movements to which this invention can be applied
This includes most mammals, birds, amphibians, and fish.
be caught. For example, chickens, hamsters, rabbits, mice.
Sparrow, Sparrow, Magusodaka, Frog, Newt and Fish
It is. In the case of chickens, the hybridizing DNA fragment
A very complex blurred part of the fragment was produced. Hae
Digestion leaves behind “clear” fingerprints and is unclear
part was removed. Therefore, the chicken's DNA also
one or more repeating units ofHaeContains cleavage site
contains long core-containing satellites and thus
It is believed that cleavage by Hae reduces this satellite to very small DNA fragments that migrate to the bottom of the gel during DNA electrophoresis. Occasionally, other animals produce smeared bands, and these are thought to be separable if the DNA is digested with a suitable enzyme that cleaves the longer fragments. In the following additional examples, the temperature is in °C. Example 3 From fresh human placenta as described by AJ Jaffreys, Cell 18 , 1-10 (1979).
DNA was isolated. Using three individual placentas,
Labeled 1-3. An 8 microgram sample of DNA was digested with Hinf and/or Sau 3A in the presence of 4mM spermidine trichloride to aid complete digestion, recovered by ethanol precipitation after phenol extraction, and 20 cm 1.5kb through a long 0.6% agarose gel at 30V for approximately 24 hours.
Electrophoresis was performed until all DNA fragments of less than 100 mL were removed from the gel. The DNA was then transferred to a sartrium-nitrocellulose filter by blotting. High specific activity (10 ⁹ cpm ³² P/
larger than microgram DNA) single-stranded M13DNA. The probe was prepared as described in Example 1, Step 1. The exact probe used was: (a)
Comprising most of the λ33.5 minisatellite (17nt x 14 repeats) + approximately 60nt flanking human DNA,
33.5 probe consisting of a 220nt Hae DNA fragment subcloned into the Sma site of M13mp8; (b) consisting of a 720nt Hae fragment subcloned into the Sma site of M18m8, consisting of a minisatellite + approximately 50nt flanked human DNA;
33.6 probe; and (c) same as Example 1, step (4)
33.15 probe (this is a minisatellite + 128nt
Pst+ Sma containing flanking human DNA
It was a 599 nt Pst - Aha fragment subcloned into M13mp19 DNA digested with Southern blot hybridization and washing were carried out in 1×SSC at 65° C. as previously described for filters C-E in Example 1, step (4). Filters were autoradiographed for 4 days at room temperature without an intensifying screen. Each probe produces a different fragment pattern, the complexity of which is largely independent of the tetranucleotide restriction endonuclease used. Figure 3 shows the pattern obtained. Separation of polymorphic fragments less than 4 kb in length was improved upon double digestion with Hin f+ Sau 3A, which produced relatively separate and unaltered fragments with Sau 3A cleavage sites accumulated within one or more repeat units. It is based on the removal of background hybridization probably caused by Hin f minisatellite fragments. In double digests, the number of separable polymorphic fragments detected by probe 33.15 is approximately equal to that of the long monodigested minisatellite fragment, which likely contains Sau 3A cleavage sites in most or all single repeat units. It can increase from about 15 to about 23 per individual at the cost of losing 20%. Example 4 A sample of 8 micrograms of human blood DNA from a random sample of 20 unrelated British Caucasians was digested with Hin f and analyzed using minisatellite probes 33.6 or 33.15 as described in Example 3. A hybrid was formed by the method. Compare each DNA fingerprint (individual A) with the pattern in an adjacent gel track (individual B), with a co-migrating counterpart of approximately the same number of bands in A and approximately the same autoradiographic intensity in B that is clearly absent in B. The number of bands was recorded. Results shown in the table below are all mean values for pairwise comparisons. A small amount (approximately 6%) of additional weak hybridizing fragments in A matched the strong hybridizing fragments in B. In such a case,
Such fragments were ignored as it is not possible to determine whether the bands in A are also present in B. If the co-migrating bands in A and B are always the same allele of the same minisatellite locus, then the average probability x that the allele in A is also present in B is = 2q - q ² , so q = 1-(1-
x) Average allele frequency (homozygosity) by ^1/2
Related to q. In fact, the (unknown) proportion of co-migrating bands in A and B are derived by chance from different minisatellite loci, and therefore the average allele frequency and homozygosity estimates are maximum, and the electrophoresis of the minisatellite fragments Depends on separation. The probabilities shown in Table 1 are for the individual size fragments of DNA indicated. The three numbers must be combined to obtain an overall probability for the most readable part of the fingerprint, i.e. all DNA larger than 4kb in size. For example, the average probability that all fragments detected by probe 33.15 in individual A are also present in B is 0.08 ^2.9 ×
0.20 ^5.1 ×0.27 ^6.7 = 3 × 10 ^-11 . 2 in A
The probability that all fragments detected by species probes 33.15 and 33.6 are also present in B is 5×
10 ^-19 . [Table] Example 5 This example illustrates the somatic stability of DNA fingerprints and their use in paternity testing. Lymphoblastoid cell lines transformed by EB virus and stored in liquid nitrogen were reestablished in liquid culture after 2 years. These cultured lymphocytes were washed twice with physiological saline, and DNA from the lymphocyte pellet and white blood cells was collected by AJ Jeffreys.
Cell, Vol. 18, 1-10 (1979). Sperm DNA was produced in the same manner, except that spermatozoa collected from semen were treated with 1M2-mercaptoethanol for 5 minutes at room temperature before being dissolved with SDS. 5 of DNA digested with Hin f and hybridized with probe 33.6 (a) or 33.15 (b).
DNA fingerprints were prepared as described in Example 3 using microgram samples. Example 6 This example illustrates the use of probes of the invention to detect highly polymorphic regions in the DNA of various vertebrates. DNA samples were prepared from blood taken from chickens, sparrows and baby birds, rabbit and mouse livers, human placentas, and gutted carcasses of frogs and fish. An 8 μg DNA sample was digested with Hin f, whereas chicken DNA was digested with Hin f.
Restriction digests were electrophoresed through a 0.6% agarose gel, denatured, transferred to Sartorius nitrocellulose filters, and hybridized as described above using human minisatellite probe 33.15. The stringency of hybridization was 65° C. in 1×SSC. The results,
The following samples are shown in FIG. 1, 2: Unrelated human placental DNA 3: Rabbit DNA from F ₁ hybrids of Alaskan and Vienna White breeds 4: Rabbit DNA from Alaskan breeds 5,6 Mouse from two wild-caught Greek mice ( Mus musculus ) DNA 7 Mouse from the inbred strain DBA-2 ( Mus
musculus) DNA 8 Mouse from inbred strain C57/BL10 ( Mus
musculus) DMA 9,10 Chicken DNA: Note: DNA was digested with Hae . 11, 12: Sparrow DNA 13, 14, 15: Little bird DNA 16, 17: Frog (Xenopus tropicalis) DNA 18, 19: Small fish DNA A variety of valid DNA fingerprints have been obtained from almost all tested vertebrates. It can be seen that it is clearly as informative as a human DNA fingerprint. Chicken DNA cleaved with Hinf also produced a less strong but highly complex obscurity of hybridizing DNA (not shown). However, upon digestion with Hae, this stain was removed, resulting in a clean polymorphic fingerprint pattern. The two inbred strains of mice have simpler fingerprints than wild mice. This is because in the wild, most hypervariable minisatellite loci are heterozygous;
This is thought to be because inbreeding results in homozygosity, which reduces the number of hybridizing DNA fragments by half in inbred lines. The following examples further illustrate the application of the particular probes described above. Example 7 This example describes the use of DNA fingerprint analysis in an immigration case that would have been extremely difficult, if not impossible, to solve using traditional genetic methods. This case concerns a Ghanaian boy who was born in the UK, immigrated to Ghana to be reunited with his father, and then returned alone to the UK to be reunited with his mother, brother and two sisters. But there,
There was evidence to suggest that a swap had taken place with an unrelated boy or with the son of one of the mother's sisters, all of whom were living in Ghana. As a result, the returning boy was not allowed to reside in the UK. At the request of the family's attorney, an analysis was conducted to determine the boy's mother. Neither the father nor the mother's sisters were used in the analysis, as this would complicate matters. Furthermore, although the mother was certain that the boy was her son, she was not sure about the boy's father. Therefore, DNA fingerprints from blood DNA samples taken from available members of the family (mother M, brothers B, sisters S1 and S2, and the boy in question Detect. The above two minisatellite probes 33.6 and 33.15
It was produced by hybrid formation by the Southern method. The mother (M), the boy in question (X), his brother (B),
An 8 μg sample of blood DNA from sisters (S1, S2) and an unrelated individual (U) was digested with Hin f;
Electrophoresis was performed on a 0.7% agarose gel, and a hybrid was formed with the probe using the Southern method. The autoradiograph is shown in Figure 6. Fragments present in the mother's (M) DNA fingerprint are indicated by short horizontal lines. Paternal fragments that are absent in M but present in at least one of the non-disputed siblings (B, S1, S2) are indicated by long lines. The "maternal" and paternal fragments transmitted to X are indicated by dots. The DNA fingerprint of X contains no additional isolated fragments. All fragments were recorded from the original autoradiographs taken at different exposures. Partially resolved, relatively weak bands that could not be reliably recorded, especially towards the bottom of the gel, were ignored. The first step was to establish the paternity of X from the pattern of hypervariable fragments. Although the father was not available, most of his DNA fingerprints are paternal specificities present in at least one of the three non-disputed siblings (B, S1, S2) but not in M.
They were able to reconstruct it from DNA fragments. Approximately half of the 39 paternal fragments thus identified are
It was present in the DNA fingerprint. DNA fragments are DNA fingerprints of unrelated individuals (see individual U in Figure 6).
Since it is rarely shared between
very strongly suggests that B, S1 and S2 have the same father. After excluding these father-specific DNA fragments, 40 fragments remained in X, all of which were present in M. This strongly proves that M is the mother of X and therefore X, B, S1 and S2 are true siblings. It was already mentioned above that the average probability that a fragment in one person's DNA fingerprint will be present in a randomly selected second individual is about 0.2 for Northern Europeans. The corresponding values for Father and M are 0.26, establishing that the DNA fingerprint variability in these Ghanaians is not significantly different from that of Northern Europeans. In the probability estimates below, we make the very conservative assumption that all bands share a uniform probability of 0.26 (quantified below). The first issue is whether or not X is related to this family. X's DNA fingerprint contains 61 recordable fragments, all of which are present in M and/or the father. If X is unrelated, the probability that each of his bands exists in these parents is 1
−(1−0.26) ² =0.45. Therefore, the probability that M and/or the father have all 61 of X's bands by chance is 0.45 ⁶¹ =7×10 ⁻²² . X is clearly related to this family. The next question is whether an unrelated woman who is not M can be X's mother. X's DNA fingerprint is
It contains 40 "maternal" fragments, of which we assess that about 25 are specifically inherited from the mother. The remaining fragments are shared between mother and father and therefore cannot be used to give evidence as to M's motherhood. All 25 mother-specific fragments in X are present in M. Although M is not related to X, the opportunity to share all 25 fragments is
0.26 ²⁵ = 2×10 ^-15 . Therefore, X and M must be related. The final and most difficult question is whether M's sister, who was not used in the analysis, can be X's mother (the father, of course, must be M's husband).
If a band is shared by any person with an average probability of 0.26, then the corresponding probability that a fragment of one person is also present in a sibling is 0.62. The probability that M is a sister of X's true mother and happens to contain all 25 of X's mother specificity bands is therefore 0.62 ²⁵ =3×10 ⁻⁶ .
We therefore conclude, beyond any reasonable doubt, that M must be the true mother of X. This evidence was provided to the immigration authorities, who discontinued the dispute against X and granted him residence in the UK, allowing him to remain with his family. This difficult case shows that DNA fingerprints can provide clear positive evidence of kinship even when key family members are missing.
This case is simplified by the fact that X has the same father as his compatriot, and that this father transmitted no bands exclusively to X (on average, 1/16 paternity bands are transmitted). was made into Such X-specific fragments clearly weaken the proof of kinship between X and M, but in fact they do not necessarily invalidate the analysis. X does not appear to have more than 5 such paternal fragments in addition to the 25 maternal specific fragments. If X and M are unrelated or if M is an aunt ^of and 9×10 ^-3 . Therefore, this analysis is strong and provides unequivocal evidence for or against the claimed kinship in most such cases. Normally, of course, all family members will be available, for example in paternity disputes or where it is difficult for the family to be reunited due to immigration. DNA
Fingerprints can almost always solve such problems. Quantification of DNA fingerprints M had 61 DNA fragments recorded, compared to 39 fragments specifically inherited from his father. 1/8 of the father's heterozygous DNA fragments are B, S1
or not transmitted to S2, so the correction value for the number of paternal-specific fragments is 39 x 8/7 = 45.
Since the total number of fragments in the DNA fingerprints of M and father are approximately equal, the number of fragments in M shared by father is approximately (61-45)=16. The average probability x of band sharing in M and the father is therefore 16/61=
0.26, consistent with a preliminary estimate obtained from screening a random sample of Northern Europeans (x = 0.2, control 2). Approximately half of the 45 paternal bands were transmitted to B, S1 and S2 (18, 24 and 18, respectively), as expected for heterozygous bands. Of M's 61 bands, more than half are in B, S1, and S2, as expected since some of M's bands are also present in the father and are therefore transmitted to most or all of the children. Yotsute (32, 38 and 38 respectively)
39 pieces) were inherited. If M's DNA fingerprint contains n shared bands transmitted to all children and (61-n) heterozygous bands transmitted to half of the children, then n+0.5(61-n) = 36.3,
For n=12, it matches the estimate of 16 bands common to M and the father (see above). X's DNA fingerprint consists of 21 paternal-specific fragments and 40 M
Consists of bands in common with The proportion of the latter band that is mother-specific and not shared with the father is calculated in two ways. First, the number of mother specificity bands is
It is almost equal to the number of paternal specificities, which is 45/2 = 22.5. Second, n of the 40 maternal bands in X (approximately
12) is a shared maternal/paternal band (see above), leaving X with 28 maternal-specific bands. The number of fragments that X specifically acquired from his mother is therefore approximately
It is 25. Probability of Band Sharing The average probability that a fragment in one individual will be matched by a band of similar electrophoretic mobility and autoradiographic intensity in a second arbitrary person is x
(x=0.2−0.26, see above). Larger minisatellite fragments probably have lower allele frequencies and better electrophoretic separation;
The frequency with which it is shared becomes less, so the fragment sharing probability x is non-uniform. Almost all the fragments are
Since they are inherited independently, the highest probability that all n fragments in an individual are present in any second individual is therefore x ⁿ . Non-uniformity in x reduces this probability. Band sharing between siblings If the shared bands always represent the same allele of the same hypervariable locus, then x = 2q − q ²
is related to the average allele frequency q, which states that the probability that a given band in an individual is also present in a sib is (4+5q- ^6q2 + ^q3 )/4(2-q). Since x=0.26, q is 0.14 and the proportion of bands present in the first sibling that are shared by the record is 0.62. This probability decreases slightly if we assume that the bands shared by any person are never the same allele, ie, many minisatellites have alleles of the same size. Application of DNA fingerprints to the analysis of genetic diseases To determine the possibility of using DNA fingerprints for linkage analysis in humans, in particular for the study of hypervariable DNA fragments that segregate together with disease loci in large kindreds. We investigated the DNA fingerprints of two large families, one segregating for neurofibromatosis and the other for genetic persistence of placental hemoglobin, apparently determined by an autosomal dominant gene not linked to the β-globin gene population. separated). Example 8 Isolation of Hypervariable Minisatellite Fragments in the DNA Fingerprints of a Sibling Group Affected by Neurofibromatosis Blood DNA samples digested with Hin f were isolated to a length of 35 cm.
Electrophoresis was performed on a 0.7% agarose gel, and hybrids were formed using the Southern method with minisatellite probes 33.6 and 33.15. Unaffected father (F), 5 sons (S) and 6 daughters (D)
Figure 7 shows the DNA fingerprint of The affected mothers were not used in the study. Offspring with multiple neurofibromas are indicated by (+); the remaining offsprings are
He showed no signs of neurofibromatosis. Separated paternal (●) and maternal (○) heterozygous DNA fragments are shown, and segregation into offspring was recorded directly from the original autographs taken with short, medium, and long-term irradiation. Only DNA fragments whose positions and relative intensities in each offspring matched those of the parents were recorded. The linked pairs of DNA fragments AB that separate AB or -- into progeny are joined by continuous lines. Alleles separating A- and -B are joined by dotted lines. The single maternal fragment showing evidence of consequential linkage to neurofibromatosis is marked with an asterisk; all six affected offspring inherited this fragment, and four of the five unaffected children Humans do not have this band, resulting in a 10/11 match between inheritance of this band and neurofibromatosis. Materials and Methods DNA Isolation Fresh blood was layered onto an equal volume of 1x SSC (SSC, sodium citrate saline, 0.15M NaC1, 15mM trisodium citrate, pH 7.0), Histopaque−1077 (Sigma). , nucleated cells were collected by centrifugation. Alternatively, thaw frozen blood in double volume of 1x SSC and collect 10,000 g
Nucleated cells and nuclei were pelleted by centrifugation for 15 minutes at . Written by Jeffreys, A.J.
(1979), Call, 18 , 1-10, high molecular weight DNA was produced. Analysis by Southern Method A 5 μg sample of human DNA was analyzed using 20 units of Hin f in the presence of 4 mM spermidine trichloride.
Digested for 2 hours at °C and recovered by phenol extraction and ethanol precipitation. 16 μl of restriction digest
H ₂ O + 4 μl gel loading mixture (Fuicol 400
12.5%, Bromophenol Blue 0.2%, Tris Acetate 0.2M, Sodium Acetate 0.1M, EDTA
1mM, pH 8.3) and 5mg/ml ethidium bromide, dissolved in 2μl of horizontal agarose gel (Tris acetate 40mM, sodium acetate 20mM,
EDTA 0.2mM, ethidium bromide 0.5μg/ml
(0.7% Sigma type agarose in pH 8.3, 0.7 cm thick x 20 cm or 35 cm long). After equilibrating for 10 minutes, the gels were electrophoresed at 2 V/cm for 24-48 hours until all DNA fragments less than 1.5 kb in length were electrophoresed from the gel.
DNA was transferred by blotting onto a nitrocellulose filter (Sart-orius, pore size 0.45 μm). Human minisatellite M13
^32P -labeled single-stranded probe DNA was prepared from recombinant 33.6 and 33.15 and incubated at 65°C in 1xSSC.
Hybridize to Southern blots and autoradiograph as described in the primary application. Data Analysis Separate data and chain analysis were stored on a BBC Model B microcomputer. Table 2 summarizes minisatellite markers in neurofibromatosis families. [Table] The number of different loci recorded (c) is n-a-b
obtained by. The entire DNA fingerprint, including unseparated and therefore unrecorded fragments, is derived from N heterozygous loci (2N fragments). recorded (n-b)
The approximate total number of hypervariable loci detected by a given probe N, assuming that the apparent fragments are a random sample of 2N bands in the DNA fingerprint.
is related to the number of allele pairs by the following formula: N = 1/2 [(n-b) (n-b-1)/2a+1] [Table] Transmission frequency of hypervariable fragments (Figure 7) To study the probes 33.6 and
33.15 the number of fragments detected by the transmissibility 50
% (Table 4) and compared with the predicted number obtained by the binomial distribution ¹¹ Cr·n/2 ¹¹ . Fragments present in all children may be from homozygous loci and were ignored. Maternal fragments that were not passed on to any children had no maternal DNA available, so
I couldn't record it. Average transmission frequency (+S.EM)
You can also get By recording the number of offspring matching for AB (AB or --) using all possible pairwise comparisons of paternal or maternal fragments with initially excluded alleles and linked bands. (i.e., the c locus was analyzed for each parent. See Table 3, c(c-
1)/2 to obtain control comparisons). Pairs of fragments appearing in groups of 0 or 11 children represent alleles or closely linked pairs, respectively. By definition, pairs do not belong to any group. Compare the observed distribution with the predicted value if all c loci were unlinked (U). In this case, the number of pairwise comparisons giving exactly r(AB or --) offspring is given by the binomial distribution ¹¹ Cr/2 ¹¹ ·c(c-1)/2. Furthermore, the c loci are clustered and evenly spaced, and adjacent loci are separated by a recombination frequency θ (10, 20
or 30 cM apart). Therefore, the population is (c-1)ψ
It will spread over the locus unit [in the formula, ψ=-1/21n (1-2θ)]. For the c locus (randomly sampled at one or the other allele), exactly r (AB or --) among 11 sibships.
The number of pairwise comparisons yielding descendants of is given by the following formula: 〓i=c-1 (c-i) ¹¹ Cr i=1[x _i ^11-r (1-x _i ) ^r +x _i ^r (1-x _i ) ^11-r /2] [In the formula, x _i is the recombination fraction between two loci separated by i figure units, and x _i is the mapping function: x _i = 1/2 ( 1−e ^-2i ψ)]. Results DNA Fingerprint Probes The two minisatellite hybrid-forming probes used in this study are detailed in their primary applications.
Probe 33.15 consists of a cloned human minisatellite consisting of 29 repeats of a 16 bp variant of the core sequence. The minisatellite repeat unit in probe 33.6 is a different trimer of the most conserved 11 bp 3' end of the core sequence, repeated 18 times. The sequences of the core and probe repeat units are as follows: A Core GGAGGTGGGCAGGAGG 33.15 AGAGGTGGGCAGGTGG 33.16 AGGGCTGGAGG Probes 33.6 and 3.15 Differences in the sequence and repeat length of the probes result in a long hypervariable minisatellite in the Hin f digest of human DNA. Different patterns of fragments will be detected. This 4bp restriction endonuclease uses small flanking DNA
The separation of different minisatellites is maximized by releasing long tandem-repetitive minisatellites in DNA fragments with . Analysis of DNA fingerprints in a large sibling group To investigate the segregation of individual minisatellite DNA fragments, a large sibling group of 11 British individuals was diagnosed with neurofibromatosis (Recklinghausen).
disease], an autosomal dominant disorder associated with tumors of the peripheral and central nervous system. Genetic marker traits had not yet been linked to the disease. Of 11 children (6 affected, 5 unaffected),
Blood DNA fingerprints detected with probes 33.6 and 33.15 were compared to their unaffected father in Figure 7. Isolation of minisatellite DNA fragments,
Maximized by electrophoresis on a 35 cm long agarose gel. Many of these hypervariable minisatellites, especially very large DNA fragments, have low allelic frequencies and are rarely inherited by unrelated individuals, as mentioned above, and therefore these in families with neurofibromatosis. Many of the fragments exist in a heterozygous state and are transmitted to only some progeny. Even when DNA from the affected mother was not available, minisatellite fragments derived from the mother could be easily identified as fragments present in some offspring but not in the father. Paternal fragments could also be identified in the same way. Separation of 41 paternal and 32 maternal DNA fragments in this sib group using probes 33.6 and 33.15 (Figure 7, Table 3)
was able to record. A number of additional polymorphic fragments were also present, but either electrophoretically removed from the gel or incompletely resolved and therefore could not be reliably recorded. By comparing the segregation patterns of all paternal or maternal DNA fragments in this large sibship group pairwise, allelic pairs of fragments and pairs exhibiting close linkage in competitive conditions can be identified (this The probability of simultaneous separation of a given pair of bands in a sib group is 1/1024). Several examples of allelic pairs of paternal and maternal fragments could be identified with both probes (Figure 7). Also, the probe
33.6 detected linked pairs of fragments in the mother and father.
A similar linkage is seen in a second kindred (shown below), in which at least one hypervariable region that hybridizes to probe 33.6 contains an internal cleavage site for Hin f and thus can be cleaved. This suggests a long minisatellite/satellite that produces two or more fragments that separate simultaneously as a minisatellite "haplotype" in the family.
The polymorphic DNA fragments recorded using probe 33.15 were not present in the combination of fragments detected by 33.6. Such fragments that hybridized to both probes would be detected as equally sized bands transmitted from the same parent to the same child (ie, "linked"). These two probes therefore hybridize to essentially completely different subsets of human minisatellites. By eliminating alleles and linked fragments, it was concluded that 34 and 25 distinct paternal and maternal loci, respectively, were recorded (Table 3). For approximately 80% of loci, only one of the two alleles is separated, with the second allele probably present in a less-separated complex of relatively short minisatellite fragments. This means that there are large differences in minisatellite allele length, probably due to unequal exchange in these tandem repeat regions. Several allele pairs identified in FIG. 7 do indeed exhibit substantial length differences. From the proportion of matching bands in the alleles, it can be estimated that the total number of heterozygous loci present in the total DNA fingerprint detected by probes 33.6 and 33.15 is approximately 43-66; Approximately half of these are recorded by the father and mother (Table 3). It is not possible to measure the antagonism between the paternal and maternal fragments in this sibship. All of the recorded paternal loci are autosomal and show no specific transmission to daughters (X-linked) or sons (Y-linked). Furthermore, all pairs of paternal DNA fragments AB apparently segregate independently into the offspring;
On average, the same number of (AB, --) or (A-, -B)
give rise to progeny. The exact number follows the expected binomial distribution for unlinked loci. Maternal DNA fragments showed similar behavior. A more detailed analysis shows that the minimum loci-to-locus spacing for these loci is ≦30cM.
It turns out that it must be (in units of 46 figures). Closer spacing results in a significant number of pairs of fragments that tend to segregate simultaneously (linked in a coupled state) or as pseudoalleles (linked in a reciprocal state) (Table 4). Therefore, separable minisatellite loci must be spread over at least half of the 3000 cM long human genome and, therefore, must be scattered across many or all of the human autosomes. It won't happen. One maternal minisatellite fragment (Figure 7) is
Showing weak evidence of coupling linkage with neurofibromatosis, 10/11 children were concordant for this fragment and disease. (θ=10cM, p=0.006). However, since 25 different maternal loci were recorded, the probability that at least one allele at these loci, by chance, exhibits the observed degree of linkage in a coupled or reciprocal state is high (p = 0.24). Example 9 DNA fingerprinting of a wide pedigree: Possible linkage to HPFH DNA fingerprinting analysis was extended to an extensive four-generation pedigree of Gujyarat people segregating for β-thalamic anemia and hereditary hyperfetal hemoglobinaemia (HPFH). Autoradiographs of the label separation patterns shown in Figures 8A and 8B were prepared as follows. A 10 μg sample of blood DNA was digested with Hin f,
Using probe 33.6 (A) or 33.15 (B),
DNA fingerprints were prepared as described in FIG. Electrophoresis was performed on a relatively short (20 cm) agarose gel. The relationships between individuals are shown in Figure 9.
In A, hypervariable fragments a, b, and c are tightly linked and either all present or all absent in each individual in the pedigree. In B, band g and HPFH (H)
are separated at the same time. Individuals 7 and 8 are identical twins and have indistinguishable DNA fingerprints. Materials and methods were the same as in Example 8. Simultaneous separation analysis is shown in Figures 9A and 9B. In Figure 9A, progeny 1-11 of 30 hypervariable fragments from 4 and 27 fragments from 5.
The separation into was screened for possible linkage of the parental fragment pairs AB. Possible instances of linkage showing at least 6/7 (AB, --) progeny were further investigated for additional kinship. Two very clear linkage examples are shown (a-f, presence of fragments a-f in individuals; • absence of fragments). Fragments a-c and e,f each show complete simultaneous separation, and fragment d shows a-c
Although they have a tendency to separate at the same time, sibs 1-4
is uninformative and identical twins 7 and 8 are recombinant with inherited a-c but no d. FIG. 9B shows the inheritance of β-thalassemia trait (〓, 〓), HPFH (H) and minisatellite fragment g. An individual is recorded as having HPFH if the individual exhibits >1% HbF (normal) or >3% HbF (β-thalamic anemia trait). HPFH and β-thalassemia traits are 1
-11 and 5-8 independently separated and unlinked loci. Fragment g separates completely simultaneously with HPFH in the individuals tested. Results As shown in Figures 9A and B, elevated HbF is transmitted independently of the β-thalamic anemia trait and is clearly associated with an autosomal dominant locus not linked to the β-globin gene population. It is measured accordingly. A similar Sardinian ancestry was reported by Gianni et al.
EMBOJ., 2, 921-925 (1983). Figures 8A and 8B show 30 different fragments of grandfather (4) and 27 of grandmother (5).
A fragment was recorded. Their seven descendants (1-1
The study of 1) found that these fragments are derived from at least 22 distinct unlinked paternal autosomal loci and 18 maternal autosomal loci, using criteria recorded for neurofibromatosis families. Remaining
DNA fragments demonstrate allelism or linkage to other fragments, but this demonstration in small sibships is not possible (a given pair of parental DNA fragments can be linked or allelic to a sibship of 7). (has a 1/64 chance of being transmitted by chance as a gene). Additionally, we looked for evidence of linkage in additional members of the family, and two very strong linkage examples are shown in Figures 8 and 9. Fragments a, b and c detected by probe 33.6 are:
4 to its children (1-11) and then to grandchildren (1-8) in a complete chain. The recombinant form is found in 14 informative progeny (p=4×10 ⁻⁹ for bands that separate simultaneously). As mentioned above, this is a minisatellite "haplotype" in which fragments a-c originate from a single hypervariable locus.
means to represent. It also proves that band d detected by probe 33.15 is linked to bands ac. However, one sib group (1-4) does not give information because both parents have fragment d, and the other (5-8)
includes recombinant types (identical twins 7 and 8).
The evidence for linkage between band d and the a-c population is therefore weak (θ=10 cM, p=0.01). motherhood band e
(detected by 33.6) and f (detected by 33.15) show tight linkage in the descendants of 4 and 5 and additional sib groups 15-20 and 17-22 (information of 20 individuals). (no recombinants, θ = 0 cM, p = 10 ^-6 ). Since probes 33.6 and 33.15 detect different combinations of minisatellites and do not form hybrids to fragments e and f, these fragments represent examples of reliable linkage between two different autosomal minisatellite loci. . Finally, two linkage groups (fragments a-c and e-
f) are not alleles at the same locus. One of the individuals is a compound heterozygote with a paternal a-c population and a maternal e-f pair. Both groups are his 4
be transmitted to two of a person's children and do not segregate as alleles, but instead must be derived from two unlinked hypervariable regions. Maternal (5) minisatellite fragments do not show significant linkage to β-thalassemia traits and therefore
It is not tightly linked to the β-globin gene cluster on chromosome 11. In contrast, the 8.6 kb long maternal fragment (g) segregated at the same time as HPFH in an informative sibship of 7 offspring and 3 grandchildren (Figure 9). Showing a tight linkage (θ=0cM, p=2
×10 ^-4 ) No recombinant type was observed in 12 progeny. This linkage is still significant even considering the fact that we examined 17 loci in 5 (probability that an allele of at least one of the 17 recorded loci coincidentally cosegregates with HPFH). is 0.004). Furthermore, we can confirm that fragment g in 5 is only one minisatellite allele and not the two separated DNA fragments listed above.
This was done by examining the DNA fingerprints of all individuals shown in Figure 9, digested with Sau 3A instead of Hinf. All positive fingerprints, as predicted for a single minisatellite fragment,
Fragment g contained a corresponding Sau 3A fragment of similar size (8.2 kb vs. 8.6 kb). Discussion Human pedigree analysis ensures that the DNA fingerprints detected by minisatellite probes allow us to study the segregation of large numbers of heterozygous DNA fragments even in families where one or the other parent is not available for study. Indicates that it can be used. Using two such probes, up to 34 hypervariable loci can be analyzed simultaneously in one individual, which is a much higher genetic potential than can be obtained using traditional methods, including RFLP, in human genetics. The kinetics of labeled trait production can be analyzed. The stable inheritance of the various minisatellite fragments together with the low population frequency of the individual fragments makes them ideally suited for linkage analysis, as shown in the linkage examples found in the two families analyzed. become. These hypervariable minisatellites may be recombination hotspots, but the expected rate of unequal exchange (1
(approximately 0.001 per factor) is not sufficient to significantly disrupt the linkage between minisatellite loci and neighboring genes, such as disease loci. The total number of hypervariable loci detected together by minisatellite probes 33.6 and 33.15 is calculated to be approximately 60. At least one of the two alleles in about half of these loci can be separated in a given DNA fingerprint, so if the DNA fingerprints are different, the spectra of the loci tested will not be identical. Most or all of these loci are not genetically linked and therefore must be scattered over a significant proportion of the human genome. Their exact locations have not yet been determined and must await cloning and regional localization of individual hypervariable minisatellite loci. Curiously, no minisatellites were yet detected on the X or Y chromosomes in either of the families studied. about 43
Different loci were recorded for possible sex in the father of the neurofibromatosis family, along with individual 4 in the HPFH family. Since the X and Y chromosomes together make up about 5% of the male genome, the probability that 43 randomly distributed loci are not present on these chromosomes is (0.95) ⁴³ =0.1. Therefore, the apparent loss of sex-linked minisatellites is not significant. These dispersed hypervariable minisatellite loci are
It is suitable for studying markers linked to disease loci, as demonstrated by the hypothetical example of linkage to neurofibromatosis and HPFH. Unlike traditional single-locus genetic analyses, linkage data cannot be collected among small, unrelated kindreds because different minisatellite alleles are likely associated with disease loci in each kindred. Instead, DNA
Fingerprints alone are especially suitable for the study of linkage of dominant disorders in extended families and, most ideally, in a single large sibling group. To date, probes 33.6 and 33.15 have recorded up to 34 autosomal hypervariable loci in the individual. The probability that at least one of these loci is tightly linked to a given disease locus is 20%, assuming an arbitrary distribution of minisatellites into a 3000 cM long human linkage diagram. For extensive pedigrees such as HPFH families, this probability is approximately 10%, as only one allele at most loci can be recorded, and to detect linkage this allele must be at the disease locus. must be chained in a discounted state. Increasing these probabilities above 50% requires records of more than 104 hypervariable loci in one large sibling group and more than 208 loci in an extended pedigree. These numbers exceed the total number of loci detected by the two minisatellite probes used so far. However, probe 33.6 and
33.15 detected different combinations of hypervariable loci essentially as a whole, suggesting that the total number of human minisatellites containing various core sequences is large. In traditional human pedigree analysis using defined monolocus markers, proof of linkage between the marker and the disease locus usually directly gives the approximate genomic location of the disease gene and analyzes additional pedigrees. This can be further established by The reverse is also true for DNA fingerprints. Further analysis of possible linkages between hypervariable DNA fragments and diseases is made possible by isolating the fragments by preparative gel electrophoresis and cloning. Locus-specific hybridization probe design from isolated minisatellites by using specific sequence segments that directly flank the minisatellites or by using body minisatellites in high stringency hybrid formation can do. Such locus-specific probes can be used to extend linkage data to additional families and to localize minisatellites within the human genome. This approach has now been confirmed by cloning an 8.2 kb Sau 3A minisatellite fragment apparently linked to HPFH in a Gujyarat ancestry. Probes consisting of human minisatellites, each containing a different variant of the core sequence, as described above.
33.5, 33.6 and 33.15 yield different DNA fingerprints,
Thus, various combinations of hypervariable minisatellite regions in human and other vertebrate DNA are detected. In particular, probes 33.6 and 33.15 detect most or all of the different combinations of minisatellites as a result of differences in the length and precise sequence of the repeat cores present in each probe (Third Application and AJ Jeffreys, V. Wilson, S.L.
To test the possibility of detecting additional hypervariable regions using tandem repeats of other core sequences, a series of synthetic A mini-satellite was created. Example 10 Synthesis and cloning of artificial minisatellites
Outlined in Figure. FIG. 10 illustrates a method for producing cloned tandem repeats of the E. coli crossover hot spot initiator sequence (Chi, GCTGGTGG). Polly
The steps for making Chi probes, using standard DNA techniques, are as follows: 1 Synthetic oligonucleotides containing tandem repeats of Chi sequences 8 nucleotides in length are
Mattes et al. (1984) (EMBO J., 3 , 801~
805) and DGBrenner & W.V.Shaw (1985)
Manufactured by the method of EMBO J. 4 , 561-568. A second oligonucleotide consisting of a dimer of complementary sequences of Chi was synthesized. 2 These two oligonucleotides at 10mM
_MgCl2 , 37 in 10mM Tris-HC1 (pH 8.0)
were annealed together at °C to form short double-stranded segments of DNA. The sequence of the second oligonucleotide was chosen to yield an annealed molecule with a 3-nucleotide long 5' overhang suitable for head-to-tail ligation. 3 Phosphorylated the 5' end using T4 polynucleotide kinase and ATP. 4 The annealed DNA fragments were ligated together in a head-to-tail manner using T4 DNA ligase and ATP to produce repeated Chi polymers. 5 The linked polymers were separated by size by electrophoresis on a 1.5% agarose gel, and polymers greater than 150 base pairs in length were isolated by electrophoresis on DE81 paper (Dretzen, G.,
Bellard, M., Sassone-Corri, P. &
Chambon, p., 1981, Anal.Biochem.
112, 295-298). 6. The long polymers recovered were blunt-ended by fill-in repair using the Klenow fragment of E. coli DNA polymerase.
M13mp19RFDNA (J.Messing & J.Vieira,
1982. Gene 19 , 269-276) into the Sma site. The ligated DNA was transformed into E. coli JM101 and single-stranded phage DNA was isolated from individual white plaques. 7 The DNA insert of each cloned isolate was isolated using the dideoxynucleotide method (MDBiggin, TJGibon
& H.F.Hong, 1983.Proc.Nat.Acad.Sci.USA
80, 3963-3965) to measure the length, direction, and alignment of the polymer inserts. 5′→3′ orientation in mature single-stranded phage DNA
Recombinant M13 containing 50 tandem repeats of the Chi sequence
A phage was found and is designated M13 Core A (i.e. the insert is poly(Chi) and not poly(complement of Chi)). 8 ³² P-labeled single stranded hybridizing probes were prepared by primer extension method using the same method used to prepare probes from phages 33.6 and 33.15. Examples 11-15 Production of 5 newer artificial minisatellites Using the techniques described above, 5 more core sequences were synthesized and cloned into M13 as a multi-core recombinant, resulting in recombinant M13 core A-F. I got it. Core variants were selected to vary the length and exact sequence of the core. Table 5 shows Clone M13/Core A
-Previously used probes 33/5, 33.6 that described repeat sequences in F as core sequences and early applications
Compare and summarize with 33.15. Underline highly unchanged bases in the core sequence. The orientation of each insert in vector M13mp19 is also shown [→ is
The insertion part is poly(core). ← indicates that the insert is a complementary sequence to poly(core). ] Bases written in lowercase letters indicate departures from the core sequence. Summarize the simple characteristics of each sequence in an “annotation”. [Table] Example 16 M13/core A- to human DNA digested with Hinf
Hybridization of F. In an initial screen, DNA digests from two unrelated individuals were mixed with 33.15 and probe M13.
Each of cores A-F was explored. An 8 μg sample of DNA from two unrelated placentas (1, 2) was
The fragments were digested with Hinf, electrophoresed on a 0.7% agarose gel, plotted using the Southern method, and hybrids were formed for each probe labeled with ³² P by the procedure described in the main application. The resulting autoradiograph is shown in FIG. All probes detected multiple DNA fragments in each individual. Results Probes B, C and D each detected DNA fragment fingerprints that were substantially different between the two individuals. Although the fingerprint varied with the probes, some fragments were picked up by more than one probe and overlapped to some extent with the combination of hypervariable fragments detected by probe 33.15. Probes B-D are all suitable for DNA fingerprinting;
We conclude that together they expand the number of hypervariable minisatellites that can be tested in humans and other vertebrates. Here, the effective fingerprint obtained using core C, which contains only the central most conserved 7 base pair segment of the core sequence, is important. When the tip of the core is further truncated to generate a six base pair repeat in core E, the DNA fingerprint pattern is
It shows a combination of fragments that are completely varied and mostly shared by the two individuals tested (ie, these fragments do not exhibit extreme polymorphic variation). Therefore, Core E does not appear to be as useful a probe for DNA fingerprint analysis as the previously used probes 33.5, 33.6 and 33.15.
This also suggests that a core sequence of at least 7 base pairs is indeed required for effective DNA fingerprinting in general. Also, core (M13/Core G)
Disruption of the most conserved regions at the center of the DNA is likely to reduce the complexity and variability of the DNA fingerprint pattern. M13 Core A (polyChi) generates a novel and strong pattern of hybridizing DNA fragments.
Many of these fragments are extremely large (>
15 kb), are not well separated and may be derived from conventional long satellite sequences containing an optionally present Hin f cleavage site. some DNA
Fragments exhibit individual variability. Example 17 Detailed Examination of Core A In a family affected by neurofibromatosis, which was extensively characterized using probes 33.6 and 33.15, M13.
The pattern produced by Core A was further analyzed (see Example 8 and Figure 7). Figure 12 shows DNA fingerprints from Hin f digests of DNA from a father (F), six daughters (D), and five sons (S). Individuals affected by neurofibromatosis, an autosomal dominant cancer, are marked +. from a sick mother
DNA was not available. Separating fatherhood (●)
and maternal (○) band. Bands joined by solid lines are linked, and bands joined by dotted lines segregate as alleles. Bands marked (x) were previously detected using probe 33.15. Results DNA obtained using probes 33.6 and 33.15
Unlike fingerprints, the level of variability is relatively low;
A large number of fragments are transmitted to all descendants (i.e.
These fragments are common in the population;
(not uncommon, often present in a homozygous state). Separation of 6 heterozygous paternal and 9 heterozygous maternal bands was recorded in a sibship group of 11 children. Allele pairs of one of the paternal bands and the maternal band were previously detected with probe 33.15. After eliminating allelic pairs and linked pairs of bands, 2 new paternal loci and 6 new maternal loci remain unrecorded with probes 33.6 and 33.15, leaving 34 prerecorded paternal loci loci and 25 maternal loci. Therefore, polyChi probes have limited use in human genetic analysis. The results of Examples 16 and 17 underline the top row of Table 5 and confirm the importance of the nine dominant nucleotides discussed in the main uses section. They also go a long way in confirming the prediction made in primary applications that an effective probe core requires a minimum sequence of six nucleotides. Therefore,
Core E represents the minimum availability and includes other sequences of 6 nucleotides, such as the sequence TGGGCA discussed below.
can show marginally improved utility. The increase to 7 nucleotides is quite dramatic within the framework of the study. In an attempt to define the essential elements of a useful core sequence, the variants X and Y used above to represent alternative nucleotides can be usefully expanded into a complete theory as follows: X=A or G P=G not Y=C or T (Q=not A) W=A or T (R=C not) V=C or G (S=not T) (O=any) ( ) = Not used in the following discussion. Using this terminology, embodiments of the invention can be said to include polynucleotides that include the following repetitive core sequence: GPGGCWGGWXG (6). The above sequence, of course, represents the most representative 12 nucleotide sequence. However, the 7-nucleotide core C was also found to have significant utility, and the "accepted"
To include those with variants, one embodiment may also be said to include polynucleotides comprising repeats of the following heptad core sequence: PGGGCWG (7). Of course, P is preferably equal to T as in core C. The other most preferred is A
Will. For clarity, the homology percentages have already been shown in Table 3. For artificial probes of this use, the repeats are precisely repeats, and the homology of the minisatellite as a whole can be demonstrated by the homology of the core sequence. This is not necessarily true for probes 33.6, 33.15 and 33.5 above, whose homology is shown in parentheses. This is due to mutations that occur between repeats. Core F probably shows the lowest homology rate in terms of utility. However, this discrepancy was probably exaggerated by the disruption of the central group present in the core: TGGGCA (8). It is noteworthy that said group is present in the most effective probes B, C and D, and is destroyed in all of the less effective probes A, E and F. Therefore, Eq.
It is expected that more effective probes with an overall homology of at least 70% with (6) will be obtained. It is noteworthy that the central 6 polynucleotides of formula (8): TGGGCA (8) are also destroyed in core E. Probably the above 6
The nucleotide core sequence is more effective, but increasing the length from 6 to 7 nucleotides is expected to demonstrate even more dominant properties. Accordingly, embodiments of the invention provide that the core sequence TGGGCA
can be said to include polynucleotides containing repeats of. More generally, the invention can be said to include, as one aspect, polynucleotides according to the above "first" definition in which the sequence TGGGCA is present in all repeat sequences. Viewed from another aspect, the present invention provides that the core has the formula (2)
comprising a modification of a polynucleotide of the "first" definition above representing a sequence of at least six consecutive nucleotides, read in the same 5'→3' sense, selected from the sequences shown in I can do it.
A "core" does not necessarily have the same sequence in each repeating unit, as long as all units contain the sequence TGGGCA. The remaining groups of each unit have at least 70% homology with the sequence of formula (6) (or more preferably (2)) within the constraints of the total unit length. 〓Measurement of twin zygosity at birth The measurement of zygosity in twins is based on epidemiological,
Not only for genetic and obstetric research purposes, but also because of the differences in prediction between identical and fraternal twins.
is important. Identical twins have lower birth rates, lower body weight, more medical complications, and higher mortality than fraternal twins. In the Caucasians, newborns of approx.
30% have different sexes and are therefore dizygotic. When the placental membranes are examined, another 20% of cases are found to be monochorionic, and these are always monozygotic.
The remaining 50% are a relatively constant proportion of the population, are of the same sex, have a dichorionic diamniotic placenta, and can be monozygotic or dizygotic. A variety of methods were used to determine zygosity in these cases, including evaluation of general appearance, fingerprints, skin grafts, taste testing, and measurement of genetic marker traits. The latter is the most reliable, with an accuracy of 95-98%, but because the average heterozygosity of most protein and antigenic variants is relatively low, it is usually necessary to investigate a large number of such marker traits. Follow me. In the example below, DNA from 12 sets of newborn twins
was tested using minisatellite DNA probes as described above. The resulting DNA "fingerprints" vary from person to person, proving that only identical twins exhibit identical patterns. In seven cases where zygosity could be determined from sex observation or placental testing, DNA results were consistent with these findings. Zygosity was determined by DNA analysis in five other twin pairs and two triplets.
could be measured quickly. The DNA probe according to the invention therefore provides one genetic test that can positively measure zygosity in all cases of multiple pregnancies. Example 18 Using single-stranded DNA probes 33.6 and 33.15,
The zygosity of 12 pairs of newborn twins was measured. The details are shown in Table 6. [Table] Umbilical cord blood sample collected at birth or peripheral blood sample (0.5-1.0ml) obtained from each infant the day after birth
was used for DNA extraction. Test the placenta
It was determined whether the placenta was mono- or di-amniotic and mono- or dichorionic. DNA was extracted using standard methods (Old, JM,
Higgs, DR, Genetic Analysis, DJWeatherall,
10-15 μg were digested with Hin f. Place the sample on a 22 cm long 0.6% agarose gel at 45 V at approx.
Electrophoresis was performed for 36 hours until all DNA fragments less than 1.5 kb in length were electrophoretically removed from the gel. DNA was transferred by blotting onto a nitrocellulose filter at 80°C under vacuum.
Baked for an hour. Single-stranded DNA probe, 33.
15 and 33.6 were labeled with ^32P as above. The results are shown in FIG. In FIG. 13, columns 1 and 2 show the DNA banding patterns obtained for each twin of case 1 using the 33.6 single-stranded minisatellite probe. Examples 3 to 19 show "fingerprints" obtained using a single-stranded 33.15 probe; Case 1 shows rows 3 and 4.
Case 2 is in columns 5 and 6; Case 3 is in columns 7 and 8; Case 4 is in columns 9 and 10; Case 5 is in columns 9 and 10;
Case 6 is in columns 11 and 12, case 7 is in columns 13 and 14.
are shown in columns 15 and 16. Columns 17-19 show three triplets: female, male and female. Comparing rows 1 and 2 with rows 3 and 4, the two probes 33.6 and
It can be seen that 33.15 detects various combinations of minisatellite bands. Size labels are given in kilobases. In seven cases (see Table 6), the sex of the twins and their zygosity could be easily determined by testing their placental membranes. All twins with a monochorionic (or monoamniotic) placenta (e.g., case 1) are identical, but only about 50% of identical twins have a monochorionic (or monoamniotic) placenta (2). Nakatsuta. Therefore, the twins in case 1 must be monozygotic and showed identical DNA patterns with the 33.15 and 33.6 probes (Figure 1). In five cases, the twins were of different sexes and showed different band patterns with the 33.15 and 33.6 probes. In cases 3, 5, 7, 9 and 11, the twins have the same sex, have a dichorionic placenta, and can be monozygotic or dizygotic.
Two sets of twins (cases 5 and 9) were identified by DNA analysis.
The cases had different band patterns and were therefore dizygotic, while the other three pairs (cases 3, 7, and 11) had the same band pattern and were found to be monozygotic. Two sets of newborn triplets were similarly studied. In both cases, the mothers were taking fertility drugs to induce pregnancy. Each triplet exhibited a unique bunt pattern (eg, Figure 13, rows 17-19). The results are shown in FIG. In Figure 14, columns 1 and 2 show results using single strand 33.15;
Columns 3 and 4 show the results using duplex 33.15. Example 19 Although single-stranded or double-stranded probes are suitable for each diagnosis, multiple bands could be distinguished using multiple radioactive single-stranded probes (Figure 14). As an alternative to these single-stranded probes, we investigated the possibility of using the corresponding double-stranded probes, which are more familiar to most laboratories. The double-stranded probe used was a double-stranded 600bp Pst I-Aha containing the “core” minisatellite from λ33.15.
fragment and) was a double-stranded 720 bp Hae fragment containing the "core" minisatellite from λ33.6.
These were labeled by nick translation until a specific activity of 0.5-1.0×10 ⁹ cpm/μg DNA was achieved. Prehybridization and hybridization conditions were as described in the standard method above, except that 1×SSC and 10% dextran sulfate were used in the hybridization buffer for the double-stranded probe. Filters were washed in 1×SSC at 65° C. for 1 hour and electrophoresed using an intensifying screen at −70° C. for 1 to 3 days. Discussion of Examples 18 and 19 In the seven cases where zygosity could be determined independently, the DNA results were consistent with the conclusions based on sex observations and placental testing. In the other five pairs, unambiguous measurements of zygosity were possible using minisatellite probes. Similarly, the two sets of triplets studied were found to be trizygotic. Genetic analysis must be used in 50% of cases where twin zygosity cannot be determined due to sex differences (dizygotic) or monochorionic placenta (monozygotic). The amount of information from such a test is proportional to the degree of polymorphism at the loci tested and the number of loci tested. By measuring multiple red blood cell antigens and enzymes, zygosity accuracy on the order of 95-98% can only be achieved if the relevant allele frequencies in the population are known (Nylander, PPS, Twin Birth Phenomenon,
Edited by Barron, SL, and Thomson, AM.
Obstetrical Epidemiology. London, Academic
Press, 1983: 143-165). Similarly, several different
Analysis of large numbers of restriction enzyme site polymorphisms using DNA probes yields a significant percentage of monozygotic “false positive” diagnoses (Derom, et al.
C., Bakker, E., Vliet-ineck, R.Derom, R.,
Van der Berghe, H., Thiery, M., Pearson,
P., Determination of zygosity in newborn twins using DNA variants, J.Med.Genet., 1985, 22:279
~282). The minisatellite probe according to the invention overcomes this problem. That is,
This is because the hypervariable DNA segments to be detected are large and can vary significantly. As mentioned above, hybridizing minisatellite fragments are rarely shared among randomly selected individuals. We have already shown that the probability that two unrelated individuals exhibit identical DNA fingerprints with probes 33.6 and 33.15, which detect different combinations of hypervariable loci, is therefore astronomical (p<< 10 ^-18 , see Example 4). For siblings who share about half of the fragments, the probability of such a “false positive” diagnosis of genetic identity is <
10 ^-8 . Therefore, when single-stranded hybridizing probes 33.6 and 33.15 are used in combination,
Higher accuracy than traditional genetic testing. Even using the more common double-stranded minisatellite probes (Figure 14), which do not hybridize to some of the relatively weak bands detected by single-stranded probes, it is possible to detect "mistakes" from DNA fingerprints (Figure 14). Sufficient information can be obtained to reduce the ivy monozygotic rate to <10 ^-4 . Another advantage of this zygosity measurement method is that
Only a very small sample is required for analysis. Usually half a ml of peripheral blood or umbilical cord blood,
Sufficient DNA was obtained. The availability of this unambiguous means of measuring zygosity in newborns and older twins and triplets allows for more accurate epidemiological studies of the determinants and effects of different types of multiple pregnancies. is possible. In the example below, the molecular weight label is not obtained in the autoradiograph, but as in the other results,
A valid general range was 1.5-20kb. Application of DNA fingerprints to forensic medicine Due to the individual specificity of DNA fingerprints detected by minisatellite probes 33.6 and 33.15,
DNA fingerprints become ideally suited for personal identification in forensic medicine. The only uncertainty is that DNA
for example, whether they survive in a sufficiently undegraded state to perform DNA fingerprint analysis in dried blood or semen scars. Home Office Central Research to determine the analyzability of forensic specimens.
Preliminary studies were carried out on DNA samples supplied by Dr Peter Gill of the Establishment, Aldermaston. Example 20 Dried blood and semen scars on cloth were subjected to various tests before DNA extraction (dissolved in SDS in the presence of 1 MDTT, then extracted with phenol and precipitated with ethanol) performed by Dr. Gill. It was left at room temperature for an hour. Similarly, DNA was extracted from fresh hair roots and vaginal swabs taken before and after intercourse. DNA
Samples were digested with Hin f, electrophoresed on a 0.8% agarose gel, and blotted onto nitrocellulose filters. Filter the ³² P-labeled single-stranded probe 33.15 using standard techniques of the invention as described above.
It was made into a hybrid. The samples electrophoresed were as follows: 1. 40 μl of semen scar on cloth, 4 weeks later. 2 40 μl of fresh semen. 3. 60μl of fresh blood. 4 60 μl of blood scar on cloth, after 4 weeks. 5 60 μl of blood scar on cloth, 2 years later. 6. 15 hair roots. note. Samples 1-6 were collected from the same person. 7 Blood scar on cloth 60 μl, male, fresh. 8 Vaginal swab taken from 7 and 11 1 hour after intercourse. 9 Same as 8 except that the sample was collected 7 hours after intercourse. 10 Vaginal swabs taken from 11. 11 Blood scar on cloth 60 μl, female, fresh. The obtained DNA fingerprint is shown in Figure 15. Sufficient DNA (approximately 0.5-3 μg) was extracted from each sample for analysis. DNA remains largely undegraded in dried blood scars up to two years old, and in dried semen up to two months old, producing DNA fingerprints identical to those obtained from fresh blood and semen from the same individual. occured. They were also able to obtain DNA fingerprints from something as small as 15 hair roots. Vaginal swabs taken after sex are not degraded either.
A DNA fingerprint was generated. However, the swab pattern primarily matched that of female rather than male blood. This indicates that most of the DNA collected from the swabs was from vaginal epithelial cells that were detached during swabbing, and not from semen. Nevertheless, three additional non-female bands were detected in the postcoital sample. These corresponded to the major bands in male blood and had to be derived from semen.
Such bands could be detected in samples taken 7 hours after intercourse. Discussion The results of these preliminary experiments indicate that DNA remains intact in various forensic specimens and therefore
It is shown that DNA fingerprints can be applied to at least some forensic samples for identification purposes. For example, it becomes possible to positively identify a rapist based on semen scars on the victim's clothing. Using vaginal swabs from rape victims is less reliable, as vaginal DNA can be contaminated. However, removal of this female DNA by SDS lysis prior to the 2-DTT or mercaptoethanol-mediated reduction of the semen required for semen DNA isolation results in a more pronounced DNA fingerprint. In this case, identification of the rapist is made possible by DNA fingerprint analysis of semen scars and/or vaginal swabs. Then, in a recent study conducted in collaboration with Dr. Gill, using the pre-separation technique generally described above,
Clear “fingerprint” of semen DNA from post-coital vaginal swab
It was confirmed that this was obtained. Figure 15A shows DNA fingerprints (column 3) from two vaginal swabs taken 6.5 hours after intercourse. Column 1 shows the fingerprint from the male partner's blood sample, and column 2 shows the DNA fingerprint from the blood obtained from the female. Female cell nuclei from the swab were collected using SDS/
Preferential lysis was achieved by preincubation in a proteinase K mixture. Sperm nuclei are unaffected by this treatment and can therefore be separated from the female component by centrifugation. Then, the semen nucleus
Lysis was achieved by treatment with an SDS/Proteinase K/DTT mixture. It is clear that this separation operation was completely effective. Semen from semen-contaminated vaginal swabs
The DNA fingerprint was a perfect match to the fingerprint obtained from the other man's blood. Examples of DNA fingerprints of economically important animals obtained using human minisatellite probes 21-25 DNA samples were prepared from blood collected from dogs, cats, sheep, pigs, horses, and cows. 8 μg of the sample was treated with an appropriate restriction endonuclease (unless otherwise specified, Hin
f), and the restriction digest was recovered by ethanol precipitation after phenol extraction. Restriction digests were redissolved in water, electrophoresed on 0.7% agarose gels, denatured, transferred to Schleicher-Schuell nitrocellulose filters, and ^filtered as described above.
P-label human minisatellite probe 33.6 and
33.15 was used to form hybrids. Example 21 Dog Obtained from a dog family using probe 33.6
The DMA fingerprint is shown in Figure 16. The samples electrophoresed were as follows: Row 1 Beagle father Row 2 Greyhound mother examples 3, 4 and 5 "Greagle" pups of these parents (female, male and male respectively) ). As complex as that obtained from human DNA,
Detailed DNA fingerprints were obtained from each dog. The DNA fingerprints showed significant variability, as seen by comparing the patterns obtained from the father and mother (columns 1, 2). All three puppies each have different DNA fingerprints consisting of bands that can all be traced to their father and/or mother. These DNA fingerprints are therefore useful for paternity testing and pedigree analysis in dogs, as well as in humans. The DNA of samples 1 to 3 was slightly degraded,
Thereby, the intensity of the largest (slowest migrating) band was preferentially reduced. Despite this degradation, transmission of the band from parents to offspring could be easily measured. Example 22 Cat Figure 17 shows DMA fingerprints from a family of short-haired domestic cats using (left) probe 33.6 and (right) probe 33.15. Row 1 Mother Row 2 Father Row 3 Kitten Probes 33.6 and 33.15 produce informative DNA fingerprints. Most bands in kittens can be recorded as maternal or paternal, so these DNA fingerprints are suitable for pedigree testing in cats as well as individual identification. Example 23 Sheep DNA fingerprints obtained from various sheep using probes 33.6 (left) and 33.15 (right) are shown in Figure 18.
The samples were: Row 1 Crossbred female row 2 Crossbred female row 3 Dorset female row 4 Hampshire x Dorset female row 5 Dorset male Individual specificity from each sheep using both probes
DNA fingerprints were obtained. DNA fingerprints are human
Although weaker and less complex than a DNA fingerprint,
Still useful for identification and genetic purposes. Probe 33.6 detects one or two very strong polymorphic bands in each sheep in addition to a range of weak bands. These bands are probably derived from a single minisatellite locus showing extremely high probe 33.6 homology. Example 24 Pigs and horses Figure 19 shows three different pigs (columns 1-3) and three different horses (columns 4-6) [sex and breeding:
DNA fingerprint from [unidentified]. Probes 33.6 and 33.15 contain species- and individual-specific DNA.
Produces fingerprints. Although the DNA fingerprint with probe 33.15 in particular is weak and contains far fewer bands compared to the corresponding human DNA fingerprint, the combination of both probes is nevertheless useful for individual identification. Example 25 Cows Figure 20 shows the DNA fingerprint of Hin fDNA digests obtained using probe 33.6 for the cow family and additional cows. The samples are as follows: [column 1 human] column 2 dam column 3 sire column 4 calves 1 and 2 column 5 Angus bull column 6 Flesian bull Same as for sheep, pigs and horses. , a fairly simple but still individual-specific DNA fingerprint was obtained from each animal. In the calf, all bands could be traced back to the dam and/or sire, confirming the animal's pedigree. Figure 21 shows probes for the same bovine DNA.
The results using 33.15 are shown. The individual columns indicate: 1 Mother cow 2 Father cow 3 Calfs 1 and 2 4 Angus bull 5 Fresian bull [6. Human] Probe 33.15 is a cow digested with Hin f.
Hybridization in DNA produces a strong, inseparable, complex pattern of DNA bands. In Bgl digests, this strong signal is mainly confined to extremely large DNA fragments;
This suggests that this signal is derived from a tufted sequence or region, probably a conventional satellite DNA. Brief autoradiography of the Hin f digest reveals periodicity between the 45-50 base pair "rungs" (data not shown) and a "ladder" toward the bottom of the gel. Shows a band. Therefore, a strong signal is probably of length 45−
It is derived from a satellite with a 50 base pair repeating unit. A strong signal is Pvu ,
Significantly disrupted in bovine DNA digested with Dde or Alu , satellite DNA repeat units contain one or more sites for each of these restriction endonucleases, but not Hin f or Alu.
This suggests that it does not contain a site for Bgl. These are precisely the 1.720 bovine satellites, consisting of 100,000 tandem repeats of 46 base pair repeat units (E. Poschl and REStreek, “Prototype sequence of bovine 1.720 satellite DNA”, J. Mol. Biol. 143 , 147–
153:1980). A comparison of the sequence of the 1.720 repeat unit with the core probe 33.6 repeat unit and 33.15 repeat unit is shown below. (matched parts are marked *). This region in the 1.720 repeat shows an excellent match with the repeat unit of 33.15, but a less perfect match with 33.6. This is the 1.720 satellite DNA probe
The reason why it is detected by 33.15 but not by 33.6 (Fig. 20) will be explained. [1.720
Satellite repeat units (similar to those of myoglobin λ33 minisatellites) contain too many microtides on each side of the core (H+J>15 in equation (1)) to act as multilocate probes according to the present invention. do not〕. To detect bovine minisatellites that hybridize to probe 33.15, DNA was Alu
Alternatively, digestion with Dde reduced the 1.720 satellite to a 46 base pair monomer that was removed by electrophoresis from the bottom of the gel. Figure 21 shows the clear distribution of each of these restriction enzymes from bovine DNA.
This shows that a DNA fingerprint was actually obtained (No. 3).
However, almost all of these bands lost the Alu and Dde sites in each repeat (possibly due to mutations in one or the other of the underlined bases above), resulting in overlapping Alu and Dde sites.
by destroying the normal
1.720 satellite embedded within 1.720DNA
It is derived from a mutant block of DNA. This would explain the following facts: a Indeed identical bovine DNA fingerprints were obtained using Alu and Dde . b Very large fragments consistently hybridize more strongly than smaller ones, as they contain correspondingly more 1.720 satellite repeat units. Very large bovine DNA fragments
(contains 440 of these units). In humans,
Band intensity does not increase continuously with size (Figure 21). c Almost all of the bovine DNA fingerprint fragments are 1.720
It is removed by Hha , which cuts once within the repeat unit (see above, data not shown). d The sire (No. 2) has almost no hybridization fragments in the Alu digest. This means that 1.720 containing these mutant repeat blocks
This suggests that a region of satellite DNA has been deleted in this animal. e The DNA fingerprints of the mother cow (No. 1) and calf (No. 5) are almost identical. The mother cow was likely heterozygous for the deletion and had coincidentally transmitted the non-deleted chromosome (and thus the entire band) to the calf, equivalent to that seen in the father cow. These bands are therefore all linked and not transmitted independently to offspring as occurs in humans. Nevertheless, the five cows tested were
showing different “satellite” DNA fingerprint patterns,
Although these unusual types of fingerprints are useful for individual identification, they cannot be used to obtain multilocus signature information. In equation (1), some probes can function effectively even if n is not necessarily equal to 3. In such probes, at least one pair of (J.
core. The repeat of K) can be separated from at least two further repeats. Therefore, “non-core”
In pairs separated by DNA sequences (J. Core.
K) Sufficiently long probes can be constructed in which sequences are aligned.