CN110066791A - The six color fluorescence STR classifying methods and its dedicated kit of a kind of 21 gene locis of synchronous detection - Google Patents
The six color fluorescence STR classifying methods and its dedicated kit of a kind of 21 gene locis of synchronous detection Download PDFInfo
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Abstract
The invention discloses the six color fluorescence STR classifying methods and its dedicated kit of a kind of 21 gene locis of synchronous detection, and this method comprises the following steps: using the genomic DNA of test individual as template, carrying out PCR amplification using kit, obtain pcr amplification product；Pcr amplification product is taken, is denaturalized, then capillary electrophoresis detection, obtains the genotype of 21 gene locis of the genomic DNA of test individual；Then the STR genotyping result of the test individual is obtained.Kit includes primer pair combination, and for the nucleotide sequence of 42 primers of composition primer pair combination successively as shown in sequence 1 to 42 in sequence table, a primer in each primer pair uses fluorescent marker.The kit is suitble to group, the Chinese nation and includes more sites and hereditary information amount, suitable for detecting degradation, outmoded, corrupt, the fracture even sample of fragment loss.The present invention has great application value.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of six color fluorescence STR of 21 gene locis of synchronous detection
Classifying method and its dedicated kit.
Background technique
Short tandem repeat (short tandem repeat, STR) is that one kind is widely present in eukaryotic gene group
In molecular genetic marker, belong to second generation genetic marker.In human genome, just there is a STR bit every about 15-20kb
Point, they account for the 10% of human genome total amount.These STR are usually by 2-6 base composition tandem repeat unit, due to repeating
The difference of unit and number of repetition, they show biggish otherness in not agnate, region crowd, show as heredity
Polymorphism.It is compared with other genetic markers, STR genetic marker not only contains much information, and difference is big between Different Individual, polymorphism
Height, segment are smaller, are easy to expand, and are particularly suitable for some micro or degradation sample.Therefore, the augmentation detection technology based on STR
It is widely used in legal medical expert's individual identification, paternity identification, Population Genetics analysis and building of human DNA database etc..
Earliest mankind's euchromosome STR applied to forensic science, so far have both at home and abroad more molding amplification,
Detection kit and the relevant technologies route, amplification number of loci are continuously increased.Meanwhile euchromosome STR database is built up, and is
Cracking of cases and criminal's Characterizations provide condition.However, for the mixed stain that spot is extracted, euchromosome STR is past
It is past to show deficiency.Y-STR is the important supplement to euchromosome STR, and stable paternal inheritance is presented in they, in mixed stain
The individual identification of male's ingredient, the paternity identification of paternal remote kinsman etc. have special application value.In addition, utilizing Y-
Conserved property of the STR in a family, in case investigation, can be convenient using family where its quick lock in suspect,
So-called " looking for group with Y " provides important clue for investigation.Nowadays, Y-STR mixes male's ingredient in sample in family investigation, men and women
Detection etc. increasingly show the unique value that can not be substituted, and handle a case illustrious military exploits in practice in public security.
Just because the application prospect that STR has, method medical circles and some companies have carried out large-scale exploration to it
Research, the especially U.S. FBI nineties in last century selected 13 euchromosome STR gene locis (including CSF1PO, D3S1358,
D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX and vWA) for building
Vertical DNA data directory system (Combined DNA Index System, CODIS), these STR bit points are commonly known as 13
Core site.But with DNA as identification of means using more and more extensive, the polymorphism in 13 core sites is of the same race
It is had a certain difference between race, has been difficult to meet human DNA requirement for construction data base.For this purpose, U.S. FBI is at original 13 in recent years
Increase 7 STR bit points again on the basis of CODIS: D1S1656, D2S441, D2S1338, D10S1248, D12S391,
D19S433, D22S1045 and 1 Sex Determination site Amelogenin, to upgrade to new CODIS comprising 21 sites
Point, these sites have high polymorphism in group, the Chinese nation, and by the DNA database of multiple countries or poly-talented company
It uses for reference and develops.
What popular commercial kits were representative currently on the market mainly has external ABI'sMiniFi
lerTM, IdentifilerTM、SinofilerTMAnd GlobalFilerTM, the PowerPlex 16 and PowerPlex of Promega
21；The Goldeneye 20A that the country has basic point to recognize reads the MicroReaderTM 21D of micro- gene, middle dolantin connection
AGCUExpressmarker 22, the STRtyper-21G etc. that Ningbo Haier applies.Most of these kits are using multicolored fluorescence
Label, in addition to ABI'sMiniFilerTMOutside, other kit maximum site fragments substantially all in 400bp or
450bp or more.AlthoughMiniFilerTMAmplified fragments are small, and maximum site fragment includes within 300bp
Number of sites is few, and only 9, and proliferation time is in 2h or so.
However, with DNA as identification of means using more and more extensive.User not only counts to the gene position of kit
Mesh, information quantity requirement are higher and higher, more stringent with requirements such as Fan Wei ﹑ proliferation time and detection efficiencies to the Shi of sample.And show
Existing detection site is all made of multicolored fluorescent marker, and maximum site fragment base in 20 or so kits in the market
This proliferation time and takes a long time all in 400bp or 450bp or more, especially encounter have serious degradation, DNA break sample when,
The application of these kits will receive certain restrictions.Therefore, it develops suitable group, the Chinese nation and includes more sites and something lost
Information content is passed, suitable for detecting degradation, outmoded, corrupt, fracture even six color fluorescent marker mini of the sample of fragment loss
STR kit has significant application value.
Summary of the invention
Technical problem to be solved by the invention is to provide suitable group, the Chinese nation and include the detection base in more sites
Because of the kit in site.
In order to solve the above technical problems, present invention firstly provides primer pair combinations, it may include for expanding insertion and deletion
The primer pair 1 of site Yindel, is used for amplification of STR gene loci at the primer pair 2 for expanding sex identification site Amel
The primer pair 3 of D3S1358, for amplification of STR gene loci D13S317 primer pair 4, be used for amplification of STR gene loci
The primer pair 5 of D7S820, for amplification of STR gene loci D16S539 primer pair 6, be used for amplification of STR gene loci
The primer pair 7 of D10S1248, for amplification of STR gene loci D5S818 primer pair 8, be used for amplification of STR gene loci
The primer pair 9 of D21S11, for amplification of STR gene loci TPOX primer pair 10, be used for amplification of STR gene loci DXS6795
Primer pair 11, for the primer pair 12 of amplification of STR gene loci D19S433, for amplification of STR gene loci D22S1045's
Primer pair 13, for the primer pair 14 of amplification of STR gene loci D8S1179, for the primer of amplification of STR gene loci D2S441
To 15, for the primer pair 16 of amplification of STR gene loci D12S391, for the primer pair of amplification of STR gene loci D2S1338
17, for the primer pair 18 of amplification of STR gene loci vWA, for the primer pair 19 of amplification of STR gene loci TH01, for expanding
Increase the primer pair 20 of str locus site D18S51 and the primer pair 21 for amplification of STR gene loci CSF1PO；Each primer pair
It can be made of the pair of primers for being located at corresponding gene site two sides.
In above-mentioned primer pair combination, a primer in each pair of primer pair can use fluorescent marker.
In the combination of above-mentioned primer pair, the primer pair 1 can sequence in the primer as shown in sequence 1 in sequence table and sequence table
The composition of primer shown in 2；The primer pair 2 can draw in the primer as shown in sequence 3 in sequence table and sequence table shown in sequence 4
Object composition；The primer pair 3 primer shown in sequence 6 can form in the primer shown in sequence 5 in sequence table and sequence table；Institute
Stating primer pair 4 primer shown in sequence 8 can form in the primer shown in sequence 7 in sequence table and sequence table；The primer pair 5
It primer shown in sequence 10 can form in the primer shown in sequence 9 in sequence table and sequence table；The primer pair 6 can be by sequence
The composition of primer shown in sequence 12 in primer shown in sequence 11 and sequence table in table；The primer pair 7 can be by sequence in sequence table
Primer shown in sequence 14 forms in primer shown in column 13 and sequence table；The primer pair 8 can be by 15 institute of sequence in sequence table
Primer shown in sequence 16 forms in the primer shown and sequence table；The primer pair 9 can draw as shown in sequence 17 in sequence table
Primer shown in sequence 18 forms in object and sequence table；The primer pair 10 can the primer as shown in sequence 19 in sequence table and sequence
Primer shown in sequence 20 forms in list；The primer pair 11 can be in primer and sequence table as shown in sequence 21 in sequence table
The composition of primer shown in sequence 22；The primer pair 12 can sequence 24 in the primer as shown in sequence 23 in sequence table and sequence table
Shown in primer composition；The primer pair 13 can be in the primer as shown in sequence 25 in sequence table and sequence table shown in sequence 26
Primer composition；The primer pair 14 can primer sets shown in sequence 28 in the primer as shown in sequence 27 in sequence table and sequence table
At；The primer pair 15 primer shown in sequence 30 can form in the primer shown in sequence 29 in sequence table and sequence table；Institute
Stating primer pair 16 primer shown in sequence 32 can form in the primer shown in sequence 31 in sequence table and sequence table；The primer
It primer shown in sequence 34 can be formed in the primer shown in sequence 33 in sequence table and sequence table to 17；The primer pair 18 can
Primer shown in sequence 36 forms in the primer shown in sequence 35 in sequence table and sequence table；The primer pair 19 can be by sequence
The composition of primer shown in sequence 38 in primer shown in sequence 37 and sequence table in table；The primer pair 20 can be by sequence in sequence table
Primer shown in sequence 40 forms in primer shown in column 39 and sequence table；The primer pair 21 can be by 41 institute of sequence in sequence table
Primer shown in sequence 42 forms in the primer shown and sequence table.
In any of the above-described primer pair combination, the primer pair 1, the primer pair 2, the primer pair 3, the primer
5 ' ends of a primer in each pair of primer pair in the 4, primer pair 5 and the primer pair 6 can be marked with 6 '-FAM；Institute
State primer pair 7, the primer pair 8, a primer in the primer pair 9 and the primer pair 10 in each pair of primer pair 5 ' ends
End can be marked with HEX；Each pair of primer in the primer pair 11, the primer pair 12, the primer pair 13 and the primer pair 14
5 ' ends of one primer of centering can be marked with TAMRA；The primer pair 15, the primer pair 16,17 and of the primer pair
5 ' ends of a primer in the primer pair 18 in each pair of primer pair can be marked with ROX；The primer pair 19, the primer
5 ' ends of a primer in each pair of primer pair in 20 and the primer pair 21 can be marked with VIG.
Any of the above-described primer pair combination specifically can be by the primer pair 1, the primer pair 2, the primer pair 3, institute
It states primer pair 4, the primer pair 5, the primer pair 6, the primer pair 7, the primer pair 8, the primer pair 9, described draw
Object is to the 10, the primer pair 11, primer pair 12, the primer pair 13, the primer pair 14, the primer pair 15, described
Primer pair 16, the primer pair 17, the primer pair 18, the primer pair 19, the primer pair 20 and 21 groups of the primer pair
At.
The present invention also protects a kind of composite amplification system for detecting 21 gene locis, it may include any of the above-described primer
To combination.21 gene locis can for AMEL, D3S1358, D13S317, D7S820, D16S539, D10S1248,
D5S818、D21S11、TPOX、D19S433、D22S1045、D8S1179、D2S441、D12S391、D2S1338、vWA、TH01、
D18S51, CSF1PO, Yindel and DXS6795.
In the composite amplification system, concentration of each primer of the primer pair 1 in the composite amplification system can
It is 0.64 μM.Concentration of each primer of the primer pair 2 in the composite amplification system can be 0.68 μM.The primer pair
Concentration of the 3 each primer in the composite amplification system can be 0.78 μM.Each primer of the primer pair 4 is described multiple
Closing the concentration in amplification system can be 1.12 μM.Concentration of each primer of the primer pair 5 in the composite amplification system
It can be 2.20 μM.Concentration of each primer of the primer pair 6 in the composite amplification system can be 0.73 μM.The primer
Concentration of each primer in the composite amplification system to 7 can be 2.33 μM.Each primer of the primer pair 8 is described
Concentration in composite amplification system can be 0.84 μM.Each primer of the primer pair 9 is dense in the composite amplification system
Degree can be 2.20 μM.Concentration of each primer of the primer pair 10 in the composite amplification system can be 0.89 μM.It is described
Concentration of each primer of primer pair 11 in the composite amplification system can be 1.09 μM.Each primer of the primer pair 12
Concentration in the composite amplification system can be 1.09 μM.Each primer of the primer pair 13 is in the composite amplification system
In concentration can be 2.11 μM.Concentration of each primer of the primer pair 14 in the composite amplification system can be 2.16 μ
M.Concentration of each primer of the primer pair 15 in the composite amplification system can be 0.82 μM.The primer pair 16 it is each
Concentration of the primer in the composite amplification system can be 0.64 μM.Each primer of the primer pair 17 is in the compound expansion
Concentration in increasing system can be 2.51 μM.Concentration of each primer of the primer pair 18 in the composite amplification system can be
1.05μM.Concentration of each primer of the primer pair 19 in the composite amplification system can be 0.73 μM.The primer pair
Concentration of the 20 each primer in the composite amplification system can be 1.00 μM.Each primer of the primer pair 21 is described
Concentration in composite amplification system can be 0.78 μM.
Reagent needed for any of the above-described composite amplification system may also include progress pcr amplification reaction；It is described " to carry out
Reagent needed for pcr amplification reaction " does not include primer needed for pcr amplification reaction.
" carry out pcr amplification reaction needed for reagent " concretely DMSO and/or KCl and/or MgCl2And/or BSA
And/or dNTP and/or Tris-HCl buffer and/or Taq archaeal dna polymerase.The Tris-HCl buffer is 10mM's
Tris-HCl (pH8.3) buffer.The Taq archaeal dna polymerase concretely thermal starting archaeal dna polymerase, antibody closing modification or
Chemical modification.
Any of the above-described composite amplification system can specifically be combined by any of the above-described primer pair and to carry out PCR amplification anti-
Required reagent is answered to form.
The present invention is also protected containing any of the above-described primer pair combination or any of the above-described composite amplification system
Kit；The purposes of the kit can be a1) or a2) or a3): a1) STR parting；A2) individual identification；A3) paternity identification.
The preparation method of any of the above-described composite amplification system or the kit also belongs to protection model of the invention
It encloses；The preparation method may include the step of individually packing each primer in any of the above-described primer pair combination.
X1 protection scope of the present invention) or X2) is also belonged to:
X1) any of the above-described primer pair combination, or, any of the above-described composite amplification system, in reagent preparation box
Application；The purposes of the kit be a1) a2) or a3): a1) STR parting；A2) individual identification；A3) paternity identification；
X2) any of the above-described primer pair combination, or, the application of any of the above-described composite amplification system, is a1) or
A2) or a3): a1) STR parting；A2) individual identification；A3) paternity identification.
The present invention also protects a kind of six color fluorescence STR classifying methods, it may include following steps:
(1) using the genomic DNA of test individual as template, using any of the above-described primer pair combination or any of the above-described institute
It states composite amplification system and carries out PCR amplification, obtain pcr amplification product；
(2) after completing step (1), pcr amplification product is taken, is denaturalized, then capillary electrophoresis detection, obtains test individual
The genotype of 21 gene locis of genomic DNA；
(3) after completing step (2), the STR parting knot of the test individual is obtained according to the genotype of 21 gene locis
Fruit；
21 gene locis can for AMEL, D3S1358, D13S317, D7S820, D16S539, D10S1248,
D5S818、D21S11、TPOX、D19S433、D22S1045、D8S1179、D2S441、D12S391、D2S1338、vWA、TH01、
D18S51, CSF1PO, Yindel and DXS6795.
In the above method, the genomic DNA of the test individual can be the genomic DNA extracted from human tissue or drop
The DNA extracted in solution sample.The tissue can be blood, blood cake, sperm, seminal stain, bone, hair, saliva, salivary stain, sweat
Or the amniotic fluid containing fetal cell.
In the above method, the method for extracting genomic DNA can be conventional method, such as paramagnetic particle method, purifying resin method, phenol chlorine
Imitative method etc..(DNA profiling amount is too low to be may cause certain site primers and does not go out DNA profiling amount, DNA preferably between 0.05-5ng
Template quantity is too high to will lead to nonspecific amplified production generation).
In the above method, the PCR amplification can on various reaction thermal cyclers (such as ABI 9700, ABI Veriti,
Bio-RadmyCycler etc.) it carries out.The Thermal cycling conditions of the PCR amplification can are as follows: 91 DEG C of initial denaturation 1min；95 DEG C of denaturation
5s, 58 DEG C of annealing 45s, 70 DEG C of extension 10s, 28 recycle；60 DEG C are continued to extend 10min；4 DEG C of -12 DEG C of preservations.
In the above method, the data after electrophoresis can be in the Data Analysis Software such as GeneMapperIDx, GeneMarker
Analysis obtains str locus parting map and data.
In the present invention, fluorescent orange label is can be selected in internal standard.Fluorescent marker can be Atto 633.
The present invention uses fluorescent dye primer, is had after the composite amplification system is expanded by above procedure
Each site amplified production mixture of fluorescent marker, the fluorescent marker is capable of emitting under laser excitation to be sequenced instrument or heredity point
The optical signal of analyzer (ABI3130,3100,3500 etc.) identification, therefore the amplified production can pass through sequenator or genetic analysis
Instrument is tested and analyzed.
When being detected using sequenator or genetic analyzer, composite amplification product needs and formamide, molecular weight internal standard
Capillary electrophoresis separation is carried out again after carrying out combined degeneration according to a certain percentage, and middle-molecular-weihydroxyethyl internal standard is a plurality of of fluorescent marker
The DNA fragmentation mixture of known clip size, with its be referring to can calculate composite amplification product clip size and with equipotential base
Because ladder is compared, to analyze and determine the genotype in tested each site of sample.
The Taq archaeal dna polymerase concretely thermal starting archaeal dna polymerase, antibody closing modification or chemical modification it is equal
It can.
Kit provided by the invention is suitble to group, the Chinese nation and includes more sites and hereditary information amount, is suitable for
Detection degradation, outmoded, corrupt, the fracture even sample of fragment loss.The present invention has great application value.
Detailed description of the invention
Fig. 1 is distribution of 21 gene locis on human genome.
Fig. 2 is the parting testing result of DNA standard items 9947A.
Fig. 3 is the parting testing result of DNA standard items 9948.
Fig. 4 is the parting testing result of digestion sample.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Rich day G-1000 thermal cycler is the product of Hangzhou BIOER Technology Co., Ltd.Hot start Taq polymerase is ABI company
Product.
The Tris-HCl (pH8.3) of the 100mM of 10 × Buffer: DMSO containing 100mM, 500mM KCl, 1mg/mL BSA
Buffer.
The preparation of embodiment 1, composite amplification system based on 21 gene locis
One, the screening of 21 gene locis
The present inventor is to meet to need such as the information content, individual identification power and compatibility of str locus site primer
It asks, it is compatible while the number of sites using six color fluorescent markers to increase receivingMiniFilerTM's
All sites and 90% the new site CODIS.
It includes 18 euchromosome STR gene locis, 1 sex identification site that the present inventor, which has finally screened,
AMEL, 1 insertion and deletion site Yindel and 1 individual character chromosome STR gene site totally 21 gene locis inside.18 often contaminate
Colour solid str locus site be D3S1358, D13S317, D7S820, D16S539, D10S1248, D5S818, D21S11, TPOX,
D19S433, D22S1045, D8S1179, D2S441, D12S391, D2S1338, vWA, TH01, D18S51 and CSF1PO.1 individual character
Chromosome STR gene site is DXS6795.
Distribution of 21 gene locis on human genome is as shown in Figure 1.
Two, the preparation of primer pair combination
1, the primary dcreening operation of primer
(1) according to the flanking sequence of 21 gene locis in step 1, using the corresponding primer of Oligo7 software design.
Design of primers principle are as follows: every primer annealing temperature is at 60 DEG C or so；Primer dimer, other interactions or friendship cannot be generated
Fork reaction, amplified production length is in addition to vWA, all within 300bp；Each pair of primer is compared with Blast, guarantees sequence
Specificity.Each gene loci design 2-8 is to primer.
(2) after completing step (1), by Shanghai, invitrogen company synthesizes all primers.
(3) genomic DNA of the blood of Healthy People is extracted using chelex-100 method.
(4) after completing step (2) and (3), respectively with reaction system shown in tabulation 1.
1. reaction system of table
(5) reaction system that step (4) are prepared is placed in rich day G-1000 thermal cycler, carries out PCR amplification, obtains PCR
Amplified production.The Thermal cycling conditions of G-1000 thermal cycler are shown in Table 2.
2. Thermal cycling conditions of table
Note: "-" expression is not present.
(6) pcr amplification product is subjected to 3% agarose gel electrophoresis detection.
The primer that clear single amplified band can be obtained is the primer of primary dcreening operation qualification.
2, the secondary screening of primer
(1) primer of primary dcreening operation qualification is divided into five groups according to amplified fragments size:
First group: Yindel, Amel, D3S1358, D13S317, D7S820 and D16S539；
Second group: D10S1248, D5S818, D21S11 and TPOX；
Third group: DXS6795, D19S433, D22S1045 and D8S1179；
4th group: D2S441, D12S391, D2S1338 and vWA；
5th group: TH01, D18S51 and CSF1PO；
Five groups of primers are marked respectively using five kinds of different fluorescence (6 '-FAM, HEX, TAMRA, ROX and VIG), together
Group primer is marked using same fluorescence.Wherein 6 '-FAM (6 '-Fluoresceincarboxylic acid) are blue-fluorescence element, HEX (chlordene -6-
Methylfluorescein) it is green fluorescein, TAMRA (4- methyl -6- carboxy-rhodamine) is yellow fluorescence element, ROX (carboxyl-X- sieve
It is red bright) it is red fluorescence element, VIG is purple fluorescence element.Internal standard selects fluorescent orange label, fluorescent marker Atto633.
(2) genomic DNA of the blood of Healthy People is extracted using chelex-100 method.
(3) after completing step (1) and (2), respectively with reaction system shown in tabulation 1.
(4) reaction system that step (3) are prepared is placed in rich day G-1000 thermal cycler, carries out PCR amplification, obtains PCR
Amplified production.The Thermal cycling conditions of rich day G-1000 thermal cycler are shown in Table 3.
3. primer secondary screening of table expands thermal cycle conditions
Note: "-" expression is not present.
(5) after completing step (4), 9.5 μ L loading mixtures and 0.5 μ L pcr amplification product are uniformly mixed, are obtained anti-
Answer liquid.Loading mixture is mixed by 0.25 μ L molecular weight internal standard (LIZ-500) and 9.25 μ L deionized formamides.
(6) it after completing step (5), extracts reaction solution, 95 DEG C of denaturation 5min, shifts cooled on ice 4min rapidly, keep DNA complete
It is denaturalized and keeps denatured state (step (6) is optional, and deionized formamide itself can be such that DNA is denaturalized).
(7) after completing step (6), the reaction solution is taken, carries out capillary electrophoresis detection with ABI3130xl genetic analyzer
(voltage 15kV, 60 DEG C of furnace temperature).Capillary Electrophoresis deposition condition are as follows: sample introduction voltage 3kV, sample injection time 10s.
(8) it after completing step (7), usesIDx software analyzes experimental data.Amplification efficiency is high, specificity is high
And the tapering symmetrical primer of peak shape that is formed after Capillary Electrophoresis of amplified production is the primer of secondary screening qualification.
Three, the preparation of primer mixture
1, the preparation of primer mixture
TE buffer is added into the dry powder of the primer of secondary screening qualification, is made into 100 μM of mother liquors；
The upstream and downstream primer (totally 42) for expanding 21 gene locis is mixed into (the primer etc. in same gene site in proportion
Amount), obtain 5 × PrimerSets primer mixture.
2, the genomic DNA of the blood of Healthy People is extracted using chelex-100 method.
3, with reaction system shown in tabulation 4.2.5 × PCRMasterMix buffer is DMSO containing 25mM, 125mM
KCl、5.0mM MgCl2, (i.e. the concentration of dATP, dTTP, dCTP and dGTP is 0.25mg/mL BSA and 0.5mM dNTP
Tris-HCl (pH8.3) buffer of 25mM 0.5mM).
4. composite amplification reaction system of table
| Component | Volume (μ L) |
| 5 × PrimerSets primer mixture | 5 |
| 2.5 × PCRMasterMix buffer | 10 |
| Hot start Taq polymerase | 0.4(2U) |
| The genomic DNA of the blood of Healthy People | 1 (containing 0.2ng-5ng) |
| Nuclease-free water | It is supplemented to 25 μ L |
4, the reaction system for preparing step 3 is placed in rich day G-1000 thermal cycler, carries out PCR amplification, obtains PCR amplification
Product.The Thermal cycling conditions of rich day G-1000 thermal cycler are shown in Table 3.
5, after completing step 4,9 μ L loading mixtures and 1 μ L pcr amplification product or allelic ladder are mixed
It closes uniformly, obtains reaction solution.Loading mixture is by 0.3 μ L molecular weight internal standard (Salmonplus500) and 9 μ L deionized formamides
It mixes.
6, it after completing step 5, extracts reaction solution, 95 DEG C of denaturation 5min, shifts cooled on ice 4min rapidly, become DNA completely
Property and keep denatured state (step 6 is optional, and deionized formamide itself can be such that DNA is denaturalized).
7, after completing step 6, the reaction solution is taken, carries out capillary electrophoresis detection (voltage with 3500 genetic analyzers
15kV, 60 DEG C of furnace temperature).Capillary Electrophoresis deposition condition are as follows: sample introduction voltage 3kV, sample injection time 10s.
8, it after completing step 7, usesIDx software analyzes experimental data.
There is inclined peak or bifurcated peak for low efficiency in the amplification situation for observing each gene loci, have miscellaneous peak in addition its
Its primer, which interferes with each other, to be amplified the primer at non-specific peak and need to redesign primer.
Each gene loci amplified production is separated according to difference in length in every group, and two neighboring gene loci cannot have weight
It is folded.Composite amplification test is carried out to every group of primer respectively, determines that the group does not have situations such as non-specific amplification phenomenon, no cross reaction
Afterwards, the concentration for adjusting each pair of primer makes each segment peak equalization in group reach 40% or more.By 5 groups of 21 gene locis
Composite amplification adjusts the primer concentration of each gene loci according to product peak height situation, keeps each gene loci peak value whole harmonious
Reach 30% or more, 5 × PrimerSets primer mixture adjusted can be used for above-mentioned 21 gene loci composite amplifications.
By screening, the 42 primers composition of primer pair combination as shown in Table 5,21 screened for detecting step one
Str locus site.Each gene loci upstream and downstream primer sequence corresponding with each gene loci is expanded the 2nd column during see Table 5 for details
With the 3rd column.
Table 5
Four, the preparation of the composite amplification system based on 21 gene locis
Composite amplification system based on 21 gene locis is by DMSO, KCl, Tris-HCl buffer, MgCl2、BSA、
DNTP and primer mixture composition；Primer mixture is mixed by 42 primers in table 5.
In composite amplification system based on 21 gene locis, the concentration of DMSO is 10mM, and the concentration of KCl is 50mM,
The concentration of Tris-HCl buffer is pH8.3,10mM Tris-HCl buffer, MgCl2Concentration be 2.0mM, the concentration of BSA
For 0.1mg/mL, the concentration of dNTP is 0.2mM (i.e. the concentration of dATP, dTTP, dCTP and dGTP are 0.2mM), each gene
The primer concentration in site is shown in Table the 4th column in 5.
Composite amplification system based on 21 gene locis prepared by embodiment 2, embodiment 1 to DNA standard items 9947A and
The parting of DNA standard items 9948 detects
DNA standard items 9947A (concentration is 10ng/ μ L) and DNA standard items 9948 (concentration is 10ng/ μ L) are Xin Haisheng
The product of object Science and Technology Co., Ltd..
1, the acquisition of DNA standard dilutions
It takes 1 μ L DNA standard items (DNA standard items 9947A or DNA standard items 9948), 9 μ L TE buffers is added, obtain
DNA standard dilutions (concentration is 1ng/ μ L).
2, the composite amplification system based on 21 gene locis that in 15 μ L embodiments 1 prepared by step 4 is taken, 0.4 μ L is added
Hot start Taq polymerase (containing 2U), 1 μ L DNA standard dilutions and 8.6 μ L nuclease-free waters, obtain reaction system.
3, the reaction system for obtaining step 2 is placed in rich day G-1000 thermal cycler, carries out PCR amplification, obtains PCR amplification
Product.The Thermal cycling conditions of rich day G-1000 thermal cycler are shown in Table 6.
6. Thermal cycling conditions of table
Note: "-" expression is not present.
4, after completing step 3,9 μ L loading mixtures and 1 μ L pcr amplification product or allelic ladder are mixed
It closes uniformly, obtains reaction solution.Loading mixture is by 0.3 μ L molecular weight internal standard (Salmonplus500) and 9 μ L deionized formamides
It mixes.
5, it after completing step 4, extracts reaction solution, 95 DEG C of denaturation 5min, shifts cooled on ice 4min rapidly, become DNA completely
Property and keep denatured state (step 5 is optional, and deionized formamide itself can be such that DNA is denaturalized).
6, after completing step 5, the reaction solution is taken, carries out capillary electrophoresis detection (voltage with 3500 genetic analyzers
15kV, 60 DEG C of furnace temperature).Capillary Electrophoresis deposition condition are as follows: sample introduction voltage 3kV, sample injection time 10s.
7, it after completing step 6, usesIDx software analyzes experimental data.
Experimental result is shown in Fig. 2, Fig. 3 and table 7.The result shows that the answering based on 21 gene locis prepared using embodiment 1
It closes amplification system and parting, point of genotyping result and disclosed standard items is carried out to DNA standard items 9947A and DNA standard items 9948
Type result is completely the same.Parting detection, inspection are carried out using the composite amplification system based on 21 gene locis prepared by embodiment 1
It is accurate and reliable to survey result.
The genotyping result of table 7. 9947A and 9948
| Gene loci title | 9947A genotype | 9948 genotype |
| Yindel | - | 2 |
| AMEL | X, X | X, Y |
| D3S1358 | 14,15 | 15,17 |
| D13S317 | 11,11 | 11,11 |
| D7S820 | 10,11 | 11,11 |
| D16S539 | 11,12 | 11,11 |
| D10S1248 | 13,15 | 12,15 |
| D5S818 | 11,11 | 11,13 |
| D21S11 | 30,30 | 28,30 |
| TPOX | 8,8 | 8,9 |
| DXS6795 | 12,13 | 11 |
| D19S433 | 14,15 | 13,14 |
| D22S1045 | 11,14 | 16,18 |
| D8S1179 | 13,13 | 12,13 |
| D2S441 | 10,14 | 11,12 |
| D12S391 | 18,20 | 18,24 |
| D2S1338 | 19,23 | 23,23 |
| vWA | 17,18 | 17,17 |
| TH01 | 8,9.3 | 6,9.3 |
| D18S51 | 15,19 | 15,18 |
| CSF1PO | 10,12 | 10,11 |
Note: "-" indicates no genotyping result
Composite amplification system based on 21 gene locis prepared by embodiment 3, embodiment 1 is to triplet family
Parting detection
The blood sample (sample is by blood card blood stain) of triplet family is provided by the identification of Guangdong Hua Da forensic.
1, the acquisition of the genomic DNA of family blood sample
Extract the genomic DNA of the blood sample of triplet family respectively using chelex-100 method.
2, the composite amplification system based on 21 gene locis that in 15 μ L embodiments 1 prepared by step 4 is taken, 0.4 μ L is added
Hot start Taq polymerase (containing 2U), the genomic DNA of 1 μ L family blood sample and 8.6 μ L nuclease-free waters, obtain reaction system.
3, the reaction system for obtaining step 2 is placed in rich day G-1000 thermal cycler, carries out PCR amplification, obtains PCR amplification
Product.The Thermal cycling conditions of rich day G-1000 thermal cycler are shown in Table 6.
4, after completing step 3,9 μ L loading mixtures and 1 μ L pcr amplification product or allelic ladder are mixed
It closes uniformly, obtains reaction solution.Loading mixture is by 0.3 μ L molecular weight internal standard (Salmonplus500) and 9 μ L deionized formamides
It mixes.
5, it after completing step 4, extracts reaction solution, 95 DEG C of denaturation 5min, shifts cooled on ice 4min rapidly, become DNA completely
Property and keep denatured state (step 5 is optional, and deionized formamide itself can be such that DNA is denaturalized).
6, after completing step 5, the reaction solution is taken, carries out capillary electrophoresis detection (voltage with 3500 genetic analyzers
15kV, 60 DEG C of furnace temperature).Capillary Electrophoresis deposition condition are as follows: sample introduction voltage 3kV, sample injection time 10s.
7, it after completing step 6, usesIDx software analyzes experimental data.
8 are shown in Table to the genotyping result of above-mentioned triplet family.The result shows that tested female, son, father are in 18 sites of detection
In meet genetic development, calculating accumulative paternity index (CPI) is 5.8460151 × 109, calculate relative parentage possibility (RCP)
It is 99.9999999828943%, it can be assumed that parent child relationship.
The genotyping result of 8. triplet family of table
Note: "-" indicates no genotyping result
Composite amplification system based on 21 gene locis prepared by embodiment 4, embodiment 1 detects digestion sample
Parting
1, the acquisition of digestion sample
The male's DNA sample (concentration is 7.5ng/ μ L) for taking 15 μ L to purify, uses endonuclease enzymatic treatment for small fragment, obtains
To digestion sample.
2, the composite amplification system based on 21 gene locis that in 15 μ L embodiments 1 prepared by step 4 is taken, 0.4 μ L is added
Hot start Taq polymerase (containing 2U), 1 μ L digestion sample and 8.6 μ L nuclease-free waters, obtain reaction system.
3, the reaction system for obtaining step 2 is placed in rich day G-1000 thermal cycler, carries out PCR amplification, obtains PCR amplification
Product.The Thermal cycling conditions of rich day G-1000 thermal cycler are shown in Table 6.
4, after completing step 3,9 μ L loading mixtures and 1 μ L pcr amplification product or allelic ladder are mixed
It closes uniformly, obtains reaction solution.Loading mixture is by 0.3 μ L molecular weight internal standard (Salmonplus500) and 9 μ L deionized formamides
It mixes.
5, it after completing step 4, extracts reaction solution, 95 DEG C of denaturation 5min, shifts cooled on ice 4min rapidly, become DNA completely
Property and keep denatured state (step 5 is optional, and deionized formamide itself can be such that DNA is denaturalized).
6, after completing step 5, the reaction solution is taken, carries out capillary electrophoresis detection (voltage with 3500 genetic analyzers
15kV, 60 DEG C of furnace temperature).Capillary Electrophoresis deposition condition are as follows: sample introduction voltage 3kV, sample injection time 10s.
7, it after completing step 6, usesIDx software analyzes experimental data.
Genotyping result is shown in Table 9 and Fig. 4.The result shows that 21 gene locis in digestion sample can accurately be divided
Type can further prove composite amplification system provided by the invention to the parting ability of degradation sample, wherein larger segment
Peak value is integrally lower than smaller fragment, and peak height harmony is slightly poorer than standard items 9947A and standard items 9948, but meets and tie to experiment
The expection of fruit.
The genotyping result of 9. digestion sample of table
| Detection site | Genotyping result |
| Yindel | 1 |
| AMEL | X, Y |
| D3S1358 | 16,16 |
| D13S317 | 8,11 |
| D7S820 | 10,12 |
| D16S539 | 10,13 |
| D10S1248 | 12,14 |
| D5S818 | 9,11 |
| D21S11 | 31.2 33.2 |
| TPOX | 8,8 |
| DXS6795 | 13 |
| D19S433 | 13,14 |
| D22S1045 | 16,17 |
| D8S1179 | 10,12 |
| D2S441 | 10,11 |
| D12S391 | 18,19 |
| D2S1338 | 17,23 |
| vWA | 14,18 |
| TH01 | 8,9 |
| D18S51 | 13,13 |
| CSF1PO | 12,12 |
<110>Shenzhen Hua Da legal medical expert Science and Technology Ltd.
<120>the six color fluorescence STR classifying methods and its dedicated kit of a kind of 21 gene locis of synchronous detection
<160> 42
<170> PatentIn version 3.5
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<400>29
attggagcta agtggctgtg gtg 23
<210>30
<211>25
<212>DNA
<213>artificial sequence
<220>
<223>
<400>30
acaaaaggct gtaacaaggg ctaca 25
<210>31
<211>25
<212>DNA
<213>artificial sequence
<220>
<223>
<400>31
ctccagagag aaagaatcaa cagga 25
<210>32
<211>24
<212>DNA
<213>artificial sequence
<220>
<223>
<400>32
agatttttca gcctccatat cact 24
<210>33
<211>23
<212>DNA
<213>artificial sequence
<220>
<223>
<400>33
gccagtccca gaggcccttg tca 23
<210>34
<211>25
<212>DNA
<213>artificial sequence
<220>
<223>
<400>34
gtgggaggga gccagtggat ttgga 25
<210>35
<211>26
<212>DNA
<213>artificial sequence
<220>
<223>
<400>35
gatggatgga tagatggata gataga 26
<210>36
<211>21
<212>DNA
<213>artificial sequence
<220>
<223>
<400>36
gagggatcat ttacttcaag c 21
<210>37
<211>26
<212>DNA
<213>artificial sequence
<220>
<223>
<400>37
gtgcaggtca cagggaacac agactc 26
<210>38
<211>23
<212>DNA
<213>artificial sequence
<220>
<223>
<400>38
gctctggggt gattcccatt ggc 23
<210>39
<211>23
<212>DNA
<213>artificial sequence
<220>
<223>
<400>39
ctggtgtgtg gagatgtctt aca 23
<210>40
<211>24
<212>DNA
<213>artificial sequence
<220>
<223>
<400>40
tcactctgag tgacaaattg agac 24
<210>41
<211>22
<212>DNA
<213>artificial sequence
<220>
<223>
<400>41
gtgccagact gagccttctc ag 22
<210>42
<211>25
<212>DNA
<213>artificial sequence
<220>
<223>
<400>42
gcaaggcagc agctcatcag gttgc 25
Claims (10)
1. primer pair combine, including for expanding insertion and deletion site Yindel primer pair 1, for expanding sex identification site
The primer pair 2 of Amel, for amplification of STR gene loci D3S1358 primer pair 3, be used for amplification of STR gene loci D13S317
Primer pair 4, for the primer pair 5 of amplification of STR gene loci D7S820, for the primer of amplification of STR gene loci D16S539
To the 6, primer pair 7 for amplification of STR gene loci D10S1248, the primer pair 8 for amplification of STR gene loci D5S818,
For the primer pair 9 of amplification of STR gene loci D21S11, for the primer pair 10 of amplification of STR gene loci TPOX, for expanding
The primer pair 11 of str locus site DXS6795, for amplification of STR gene loci D19S433 primer pair 12, be used for amplification of STR
The primer pair 13 of gene loci D22S1045, for amplification of STR gene loci D8S1179 primer pair 14, be used for amplification of STR base
Because the primer pair 15 of site D2S441, for amplification of STR gene loci D12S391 primer pair 16, be used for amplification of STR gene position
The primer pair 17 of point D2S1338, for amplification of STR gene loci vWA primer pair 18, be used for amplification of STR gene loci TH01
Primer pair 19, the primer pair 20 for amplification of STR gene loci D18S51 and drawing for amplification of STR gene loci CSF1PO
Object is to 21；
Each primer pair is made of the pair of primers for being located at corresponding gene site two sides.
2. primer pair combination as described in claim 1, it is characterised in that: a primer fluorescence mark in each pair of primer pair
Note.
3. primer pair combination as claimed in claim 1 or 2, it is characterised in that:
Primer shown in sequence 2 forms in the primer shown in sequence 1 in sequence table of primer pair 1 and sequence table；
Primer shown in sequence 4 forms in the primer shown in sequence 3 in sequence table of primer pair 2 and sequence table；
Primer shown in sequence 6 forms in the primer shown in sequence 5 in sequence table of primer pair 3 and sequence table；
Primer shown in sequence 8 forms in the primer shown in sequence 7 in sequence table of primer pair 4 and sequence table；
Primer shown in sequence 10 forms in the primer shown in sequence 9 in sequence table of primer pair 5 and sequence table；
Primer shown in sequence 12 forms in the primer shown in sequence 11 in sequence table of primer pair 6 and sequence table；
Primer shown in sequence 14 forms in the primer shown in sequence 13 in sequence table of primer pair 7 and sequence table；
Primer shown in sequence 16 forms in the primer shown in sequence 15 in sequence table of primer pair 8 and sequence table；
Primer shown in sequence 18 forms in the primer shown in sequence 17 in sequence table of primer pair 9 and sequence table；
Primer shown in sequence 20 forms in the primer shown in sequence 19 in sequence table of primer pair 10 and sequence table；
Primer shown in sequence 22 forms in the primer shown in sequence 21 in sequence table of primer pair 11 and sequence table；
Primer shown in sequence 24 forms in the primer shown in sequence 23 in sequence table of primer pair 12 and sequence table；
Primer shown in sequence 26 forms in the primer shown in sequence 25 in sequence table of primer pair 13 and sequence table；
Primer shown in sequence 28 forms in the primer shown in sequence 27 in sequence table of primer pair 14 and sequence table；
Primer shown in sequence 30 forms in the primer shown in sequence 29 in sequence table of primer pair 15 and sequence table；
Primer shown in sequence 32 forms in the primer shown in sequence 31 in sequence table of primer pair 16 and sequence table；
Primer shown in sequence 34 forms in the primer shown in sequence 33 in sequence table of primer pair 17 and sequence table；
Primer shown in sequence 36 forms in the primer shown in sequence 35 in sequence table of primer pair 18 and sequence table；
Primer shown in sequence 38 forms in the primer shown in sequence 37 in sequence table of primer pair 19 and sequence table；
Primer shown in sequence 40 forms in the primer shown in sequence 39 in sequence table of primer pair 20 and sequence table；
Primer shown in sequence 42 forms in the primer shown in sequence 41 in sequence table of primer pair 21 and sequence table.
4. the primer pair as described in claims 1 to 3 is any combines, it is characterised in that:
The primer pair 1, the primer pair 2, the primer pair 3, the primer pair 4, the primer pair 5 and the primer pair 6
In 5 ' ends of a primer in each pair of primer pair marked with 6 '-FAM；
A primer in the primer pair 7, the primer pair 8, the primer pair 9 and the primer pair 10 in each pair of primer pair
5 ' ends marked with HEX；
One in the primer pair 11, the primer pair 12, the primer pair 13 and the primer pair 14 in each pair of primer pair
5 ' ends of primer are marked with TAMRA；
One in the primer pair 15, the primer pair 16, the primer pair 17 and the primer pair 18 in each pair of primer pair
5 ' ends of primer are marked with ROX；
It uses 5 ' ends of a primer in the primer pair 19, the primer pair 20 and the primer pair 21 in each pair of primer pair
VIG label.
5. a kind of composite amplification system for detecting 21 gene locis, including any primer pair combination of Claims 1-4；
21 gene locis be AMEL, D3S1358, D13S317, D7S820, D16S539, D10S1248, D5S818, D21S11,
TPOX、D19S433、D22S1045、D8S1179、D2S441、D12S391、D2S1338、vWA、TH01、D18S51、CSF1PO、
Yindel and DXS6795.
6. composite amplification system as claimed in claim 5, it is characterised in that:
Concentration of each primer of the primer pair 1 in the composite amplification system is 0.64 μM；
Concentration of each primer of the primer pair 2 in the composite amplification system is 0.68 μM；
Concentration of each primer of the primer pair 3 in the composite amplification system is 0.78 μM；
Concentration of each primer of the primer pair 4 in the composite amplification system is 1.12 μM；
Concentration of each primer of the primer pair 5 in the composite amplification system is 2.20 μM；
Concentration of each primer of the primer pair 6 in the composite amplification system is 0.73 μM；
Concentration of each primer of the primer pair 7 in the composite amplification system is 2.33 μM；
Concentration of each primer of the primer pair 8 in the composite amplification system is 0.84 μM；
Concentration of each primer of the primer pair 9 in the composite amplification system is 2.20 μM；
Concentration of each primer of the primer pair 10 in the composite amplification system is 0.89 μM；
Concentration of each primer of the primer pair 11 in the composite amplification system is 1.09 μM；
Concentration of each primer of the primer pair 12 in the composite amplification system is 1.09 μM；
Concentration of each primer of the primer pair 13 in the composite amplification system is 2.11 μM；
Concentration of each primer of the primer pair 14 in the composite amplification system is 2.16 μM；
Concentration of each primer of the primer pair 15 in the composite amplification system is 0.82 μM；
Concentration of each primer of the primer pair 16 in the composite amplification system is 0.64 μM；
Concentration of each primer of the primer pair 17 in the composite amplification system is 2.51 μM；
Concentration of each primer of the primer pair 18 in the composite amplification system is 1.05 μM；
Concentration of each primer of the primer pair 19 in the composite amplification system is 0.73 μM；
Concentration of each primer of the primer pair 20 in the composite amplification system is 1.00 μM；
Concentration of each primer of the primer pair 21 in the composite amplification system is 0.78 μM.
7. containing any primer pair combination of Claims 1-4 or the examination of the composite amplification system of claim 5 or 6
Agent box；The purposes of the kit be a1) a2) or a3): a1) STR parting；A2) individual identification；A3) paternity identification.
8.X1) or X2):
X1) any primer pair combination of Claims 1-4, or, the composite amplification system of claim 5 or 6, is preparing
Application in kit；The purposes of the kit be a1) a2) or a3): a1) STR parting；A2) individual identification；A3) parental right
Identification；
X2) any primer pair combination of Claims 1-4, or, any composite amplification system of claim 5 or 6 is answered
With, be a1) a2) or a3): a1) STR parting；A2) individual identification；A3) paternity identification.
9. a kind of six color fluorescence STR classifying methods, include the following steps:
(1) it using the genomic DNA of test individual as template, is wanted using any primer pair combination of Claims 1-4 or right
It asks 5 or 6 composite amplification systems to carry out PCR amplification, obtains pcr amplification product；
(2) after completing step (1), pcr amplification product is taken, is denaturalized, then capillary electrophoresis detection, obtains the gene of test individual
The genotype of 21 gene locis of group DNA；
(3) after completing step (2), the STR genotyping result of the test individual is obtained according to the genotype of 21 gene locis；
21 gene locis be AMEL, D3S1358, D13S317, D7S820, D16S539, D10S1248, D5S818,
D21S11、TPOX、D19S433、D22S1045、D8S1179、D2S441、D12S391、D2S1338、vWA、TH01、D18S51、
CSF1PO, Yindel and DXS6795.
10. method as claimed in claim 9, it is characterised in that: the genomic DNA of the test individual is from human tissue
The DNA extracted in the genomic DNA or degradation sample of extraction.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810068950.6A CN110066791A (en) | 2018-01-24 | 2018-01-24 | The six color fluorescence STR classifying methods and its dedicated kit of a kind of 21 gene locis of synchronous detection |
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| CN201810068950.6A CN110066791A (en) | 2018-01-24 | 2018-01-24 | The six color fluorescence STR classifying methods and its dedicated kit of a kind of 21 gene locis of synchronous detection |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112195228A (en) * | 2020-09-28 | 2021-01-08 | 苏州阅微基因技术有限公司 | X-STR fluorescent amplification system, kit and application |
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| CN112195228A (en) * | 2020-09-28 | 2021-01-08 | 苏州阅微基因技术有限公司 | X-STR fluorescent amplification system, kit and application |
| CN112195228B (en) * | 2020-09-28 | 2022-02-22 | 苏州阅微基因技术有限公司 | X-STR fluorescent amplification system, kit and application |
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