JP3514779B2 - Method for testing apolipoprotein E genotype and primers and probes suitable for the test - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to apolipoprotein E.
The present invention relates to a method for inspecting a gene type (hereinafter, also referred to as “Apo E”) and a primer and a probe preferably used for the inspection.

[0002]

2. Description of the Related Art In ApoE, three major isotopes E2, E3 and E4 which can be separated by isoelectric focusing and several isotopes such as E5 and E7 which are less frequent are found. This diversity of ApoE is associated with dyslipidemia. That is, E3 is a wild type, and the frequency of the gene ε3 encoding this is about 70%. And the apo E
Of the apoE gene is caused by one or two mutations in the apoE gene, and a simple and accurate method for testing the diversity is desired for definitive diagnosis of abnormal lipid metabolism.
VLD is the conventional method for testing apo E phenotype
Method of protein staining after isoelectric focusing of L or serum (Za
nnis, VI, and Breslow, JL, Biochemistry 20,1033-10
41 1981), or a method in which a sample is treated with sialidase and then subjected to isoelectric focusing, and a mouse antibody against apoE and then an anti-mouse antibody-peroxidase complex are allowed to act on the sample, and then the peroxidase activity is tested ( McDowell, IF
W., Wisdom, GB, and Trimble, ER, Clin.Chem.35,2070-
2073 1989, (hereinafter also referred to as McDowell et al.))
There is. On the other hand, as a method for testing the apoE gene type, a part of the apoE gene was subjected to polymerase chain reaction reported by Saiki, RK et al. (Science 239, 487-491).
Page, 1988, JP-A-61-274697, hereinafter also referred to as PCR method) and then using an allele-specific oligonucleotide probe (Weisgraber KH, Newhouse, Y.
M. and Mahley, RW, Biochem.Biophys.Res.Commun., 15
7.1212-1217 1988 (hereinafter also referred to as the method of Weisgraber et al.), Emi.M., Wu, LL, Robertson, MA, Myers, RL, H
egele, RA, Williams, RR, White, R.and Lalouel, J.-
M., Genomics. 3,373-379 1988 (hereinafter, also referred to as the method of Emi et al.) Or a method in which a DNA fragment amplified by the PCR method is cleaved with a restriction enzyme HhaI and examined by an electrophoresis pattern (Hixson, JE). , and Vernier, DT, J.Lipid Res., 3
1,545-548 1990 (hereinafter also referred to as Hixson et al.). These apoE genotype tests are ε
This is a method for inspecting type 2 and ε4 type, and if these are normal, it is set as ε3 type.

[0003]

In the phenotype test method in which the above sample is subjected to isoelectric focusing and then protein staining, it is likely to be interfered with by a protein that migrates in the same region as apoE. Further, even in the case of the phenotype test method in which the immunoblotting method using an antibody specific to apoE and the isoelectric focusing are combined, it is a homotype unless it is sufficiently treated with sialidase and desialized before the electrophoresis. However, several bands are detected. In the case of heterotype, apo E isotopes do not exist in equal amounts (Utermann, G., Pruin,
N., and Steinmetz, A., Clin. Genet., 15, 63-72 1979)
In some cases, if a sample is stored in a frozen state for a long period of time, it may be denatured, and it is difficult to perform an accurate phenotype test. On the other hand, the conventional genotype inspection method applies the PCR method as described above, but as shown in FIG. 1, the apoE gene is composed of four exons (exons 1 to 4) and three introns sandwiched between them. Is a gene with a very high GC content (Paik.Y.-K., Chang, DJ, Reardon, CA, Dav
ies, GE, Mahley, RW, and Taylor, JM, Proc.Natl.Aca
d.Sci. USA, 82, 3445-3449 1985), it is difficult to amplify a large DNA fragment by the PCR method. Therefore, all of the above-mentioned apoE genotype inspection methods amplify only a short DNA fragment which is a part of exon 4 of the apoE gene to obtain ε2.
This is a method of inspecting only the mold and the ε4 type, and if these are normal, it is set as the ε3 type, but the ε5 type and the ε7 type cannot be inspected. For example, according to the method of Weisgraber et al. And Hixson et al., Ε5 type and ε7 type mutations, which are considered to exist in about 1% of Japanese, are not amplified and therefore cannot be examined. In addition, the method of Emi et al. Cannot be examined because the ε5 type mutation site is not amplified, and a sequence is required when examining the ε7 type, which is troublesome. Furthermore, in the methods of Weisgraber et al. And Emi et al., Which use probes, the hybridization and washing temperatures differ for each of the multiple probes used, so apo E
Testing the genotype is cumbersome because it requires the use of multiple incubators at temperatures suitable for each probe. Therefore, an object of the present invention is to provide a simpler and more accurate apoE isotope test method than the conventional phenotype method and gene type method, and yet another object of the present invention is to provide a suitable method in the above-mentioned test method. It is to provide a probe to be used.

[0004]

[Means for Solving the Problems ]
Therefore, the present inventors have completed the following inventions. (1) A polymerase chain reaction is carried out by using a combination of at least two kinds of primers which are complementary to the apolipoprotein E gene and have a plus-strand primer and a minus-strand primer for a DNA sample to be tested. The ε of the apolipoprotein E gene
DNA fragments containing 2, ε4, ε5, and ε7 mutation sites are amplified, and the amplified DNA fragments contain ε2 type, ε4
Type, the wild-type flop corresponding to each ε5 type, and ε7 type
Lobes and mutant probes, these probes
One is labeled and the other is unlabeled, the number of bases
Allele-specific oligonucleotides with 13 to 15
Ε2 type, ε4 type, ε5 type, and ε7
A method for testing apolipoprotein E gene type, which tests for type. (2) The allele-specific oligonucleotide probe
Is selected from the base sequences of SEQ ID NOs: 5 to 20,
Hive of allele-specific oligonucleotide probes
Rediscovery and wash each at 37 ° C ,
The method described in (1). (3) The method according to (1) or (2), wherein the positive strand primer comprises the base sequence of SEQ ID NO: 1 and the negative strand primer comprises the base sequence of SEQ ID NO: 4. (4) A polymerase chain reaction is carried out by using a combination of at least two kinds of primers which are primers complementary to the apolipoprotein E gene and have a plus-strand primer and a minus-strand primer for a DNA sample to be tested. The ε of the apolipoprotein E gene
A DNA fragment containing 5 mutation sites was amplified, and the amplified DNA fragment was treated with a wild-type probe corresponding to ε5 type and a mutation.
Consists of atypical probes, one of these probes
Labeled and the other unlabeled, with 13-15 bases
Is an allele-specific oligonucleotide probe
A method for testing apolipoprotein E gene type, which is used to test for ε5 type . (5) The allele-specific oligonucleotide probe
Is based on the nucleotide sequences of SEQ ID NOs: 9, 10, 17 and 18.
The allele-specific oligonucleotide
3 lobe hybridizations and 3 washes
The method according to (4), which is performed at 7 ° C. (6) The method according to (4) or (5), wherein the positive strand primer consists of the base sequence of SEQ ID NO: 1 and the negative strand primer consists of the base sequence of SEQ ID NO: 3. (7) A polymerase chain reaction is carried out by using a combination of at least two kinds of primers which are complementary to the apolipoprotein E gene and have a plus-strand primer and a minus-strand primer for a DNA sample to be examined. The ε of the apolipoprotein E gene
A DNA fragment containing 2, ε4 and ε7 mutation sites is amplified, and the amplified DNA fragment contains ε2 type, ε4 type and ε
Wild type probe and mutant type probe corresponding to each of the 7 types
A probe, one of these probes being labeled.
The other is unlabeled and has 13 to 15 bases.
A method for testing apolipoprotein E gene type, which tests for ε2 type, ε4 type and ε7 type using an allele-specific oligonucleotide probe . (8) The allele-specific oligonucleotide probe
Is a salt of SEQ ID NOs: 5-8, 11-16, 19 and 20.
An allele-specific oligonucleotide selected from a base sequence
Hybridization and washing of the rheotide probe
The method according to (7), which is performed at 37 ° C., respectively . (9) The method according to (7) or (8), wherein the positive strand primer comprises the base sequence of SEQ ID NO: 2 and the negative strand primer comprises the base sequence of SEQ ID NO: 4. (10) When the polymerase chain reaction is an asymmetric polymerase chain reaction and the primer used more in the reaction is a primer for a plus strand, an allele-specific probe complementary to the plus strand is used, while When the primer used more frequently in the reaction is a primer complementary to the minus strand, by using an allele-specific probe complementary to the minus strand, the denaturation and neutralization steps in hybridization are omitted. The method according to any one of (1) to (9). (11) A probe set for inspecting apolipoprotein E gene type, which is selected from the nucleotide sequences of SEQ ID NOs: 5 to 20, ε2 type, ε4 type, ε5 type and ε
A probe set comprising a wild-type probe and a mutant-type probe corresponding to each of the 7 types, wherein one of the wild-type probe and the mutant-type probe is labeled and the other is unlabeled. (12) A probe set for testing apolipoprotein E gene type, which comprises SEQ ID NOs: 9, 10, 1
It consists of an allele-specific probe consisting of a wild type probe corresponding to ε5 type and a mutant type probe selected from 7 and 18 nucleotide sequences, and one of the wild type probe and the mutant type probe Is labeled, and either one is unlabeled. (13) A probe set for inspecting apolipoprotein E gene type, which comprises SEQ ID NOs: 5 to 8 and 11
~ 16, 19 and 20 selected from the nucleotide sequences, ε2
A probe comprising a wild-type probe and a mutant-type probe corresponding to each type, ε4 type and ε7-type, wherein either one of the wild-type probe and the mutant-type probe is labeled, and either one is unlabeled, set.

[0005] Here, a combination of at least two kinds of primers which are complementary to the apolipoprotein E gene and have a plus-strand primer and a minus-strand primer means a plus or minus strand of the apolipoprotein E gene. 2 complementary to each specific part
It is a combination of single-stranded DNAs of a species, and the specific part is an upstream part or a downstream part of each end of the DNA fragment to be amplified. That is, the plus-strand primer is complementary to the minus strand of the apoE gene, which is the upstream portion of the DNA fragment to be amplified, while the minus-strand primer is to the downstream portion of the DNA fragment to be amplified. It is complementary to the plus strand of the apoE gene in the portion further downstream than one end of the.

Apolipoprotein E gene of the present invention
In the test method of B type, for example, a DNA fragment containing ε2, ε4, ε5, and ε7 mutation sites may be amplified.
As shown in FIG. 1, ε5 is located most upstream and ε7 is most downstream in these mutation sites. Therefore, in the method for testing apolipoprotein E gene type according to claim 1, the two types In the combination of primers, the plus-strand primer is complementary to the minus strand upstream of the ε5 mutation site of the apoE gene, and the minus-strand primer is complementary to the plus strand downstream of the ε7 mutation site. Target. Then, when amplifying a DNA fragment containing the ε5 mutation site, primer plus strand of apo E gene ε
The minus strand upstream of the 5 mutation site and the minus strand primer are respectively complementary to the plus strand downstream of the ε5 mutation site. In addition, in the method for testing the apolipoprotein E gene type , for example, ε2, ε4 and ε7
A DNA fragment containing a mutation site may be amplified. Figure 1
As shown in, in these mutation sites, ε4 is the most upstream,
Since ε7 is located at the most downstream side, in the inspection method of claim 3, the positive strand primer is ε of the apoE gene.
It is complementary to the minus strand upstream of the 4 mutation sites, and the minus strand primer is complementary to each plus strand downstream of the ε7 mutation site. When the positive strand primer and the negative strand primer have complementarity, a primer dimer is formed and the amplification efficiency of the target apoE-specific DNA fragment is deteriorated. It is desirable that the primer for use has no complementarity.

The DNA sample to be tested is a sample containing the apoE gene prepared from human blood to be tested by a usual method.

According to the above-mentioned inspection method, since the PCR method is used, only a DNA fragment in a portion sandwiched by two kinds of primers is rapidly and simply amplified from a small amount of DNA sample. The D used in this PCR method
When a polymerase that is thermostable at the cleavage temperature of DNA, such as Taq DNA polymerase, is used as NA polymerase, supplementation of the enzyme is hardly required, and thus the PCR method can be made a more rapid and simple method.
Thus, the specific D amplified in large quantities by the PCR method
Since the NA fragment is a part sandwiched between two types of primers, for example, a DNA fragment containing ε2, ε4, ε5 and ε7 mutation sites, a DNA fragment containing ε5 mutation sites, ε2, ε
Various DNAs such as DNA fragments containing 4 and ε7 mutation sites
Each fragment is amplified.

An allele-specific oligonucleotide probe for testing each type (ε2, ε4, ε5, ε7) is a mismatch with the plus or minus strand of the wild-type gene of the apoE gene of each type to be tested. Two types of probes are used: a mutant probe that causes a mutation and a wild-type probe that causes a mismatch with the plus or minus strand of the mutant gene of the apoE gene. Here, a probe having a length of 11 to 19 bases such that the base causing the mismatch is located in the approximate center of the probe is more suitable as the probe. Various ordinary methods can be used for labeling these probes. However, in the method of the present invention, since a large amount of DNA fragments to be reacted with the probe are amplified by the PCR method, it is not necessary to use RI (radioisotope) labeling, and deoxygenation labeled with digoxigenin or biotin is not necessary. Nucleotides such as digoxigenin-11-dU
It may be labeled with TP.

[0010] For example, .epsilon.2, epsilon] 4, when inspecting the ε5 and ε7 type, using each of the wild-type probe and mutant probe. In addition, when inspecting ε5 type, ε5
Type wild type and mutant probes are used. ε
When inspecting type 2, ε4 and ε7 , ε2, ε4 and ε
Each type 7 wild type and mutant probe is used.

For example , in the case of testing ε2, ε4, ε5 and ε7 types, four mutant type probes each causing a mismatch with the plus or minus strand of each wild type gene of ε2, ε4, ε5 and ε7 type , Ε2, ε4, ε5, and ε7 type mutant genes are tested by using at least 8 types of probes, 4 types of wild type probes each of which mismatches with the plus or minus strand of the mutant gene.

Ε amplified with each of these eight types of probes
When hybridized with a DNA fragment containing a 2, ε4, ε5 and ε7 type mutation site, for example, when it reacts only with all wild type probes and does not react with any of the mutant type probes, the tested DNA is derived from the parents. It can be seen that both genes are ε3 type (wild type). Then, when reacted with all wild type probes and one mutant type probe (eg, ε4 type), the tested DN
It can be seen that in A, one of the genes derived from the parents is of ε3 type, and the gene of the other parent is the type of mutant probe reacted (ε4 type). In addition, when it reacts with all wild-type probes and two types of mutant-type probes (eg, reacts with ε2 type and ε4 type), the tested DNA is one of the mutant types in which one of the genes derived from the parents has reacted. It can be seen that it is a species type (ε2 type) and another type (ε4 type) to which the gene derived from the other parent has reacted. In addition, when it reacts with all the wild-type probes other than one wild-type probe and also with a mutant-type probe (eg, ε4 type) for the wild-type probe that has not reacted, the tested DNA is a gene derived from parents. It can be seen that is the mutant probe type (ε4 type) that reacted together.

In the method for testing the ε5 type apolipoprotein E gene type, it was found that it was not ε5 type when it reacted only with the ε5 type wild type probe.
It is found that at least one of the genes derived from the parents is the ε5 type when it reacts with both the wild type and mutant type probes. When only reacting with the ε5 type mutant probe, both genes derived from the parents are found to be ε5 type.

In the test method for the apolipoprotein E gene type of ε2 type, ε4 type and ε7 type, when it reacts with all wild type probes and does not react with any mutant type probe, ε5 type is tested. Although it is not present, the ε5 type is as small as 1%, so it is presumed to be the ε3 type. Then, the other genotypes are tested as described above .

[0015] As the primer, for example, a primer is a nucleotide sequence having a nucleotide sequence or equivalent complementary to these SEQ ID NO: 1, 2, 3 or 4 against the apolipoprotein E gene. Here, a base sequence having equivalent complementarity to the apolipoprotein E gene is
It means a nucleotide sequence in which a mutation occurs due to a deletion, insertion, substitution or the like of a base within a range that does not prevent complementation with the apolipoprotein E gene, and is the same in the following specification.

Nucleotide sequence number of the apo E gene in the portion where the primers of SEQ ID NOs: 1 to 4 have complementarity (the above-mentioned document, Proc. Natl. Acad. Sci, USA, 82, 3445-3449 1985 Fi
(See g.3.) (Hereinafter, this is referred to as the nucleotide number.)
Is the primer of SEQ ID NO: 1 is 5'-2802 to 2823-3 ',
The primer of SEQ ID NO: 2 is 5'-3618 to 3639-3 ', the primer of SEQ ID NO: 3 is 3'-2958 to 2979-5', the primer of SEQ ID NO: 4 is 3'-4220 to 4241-5 ', The primers of SEQ ID NOS: 1 and 2 are for the plus strand, and the primers of SEQ ID NOS: 3 and 4 are for the minus strand.

In the inspection method of the present invention, SEQ ID NO: 1,
A nucleotide sequence of 2, 3 or 4 or a complementarity equivalent to these sequences
It is a nucleotide sequence for the polypoprotein E gene.
It is possible to use some of the primers. example
For example, in the method according to claim 1 or 2, the plus-strand primer is the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having a complementarity equivalent to the nucleotide sequence to the apolipoprotein E gene, and Detection of the apolipoprotein E gene type in which the primer for the minus strand is the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having the same complementarity with the nucleotide sequence for the apolipoprotein E gene
Inspection methods are provided. In addition, according to the method of claim 4,
And the positive strand primer is the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having a complementarity to the apolipoprotein E gene equivalent to the nucleotide sequence, and the negative strand primer is the nucleotide sequence of SEQ ID NO: 4. Apolipoprotein which is a sequence or a nucleotide sequence having complementarity equivalent to the nucleotide sequence to the apolipoprotein E gene
Methods for testing the E genotype are also provided. In addition,
A primer complementary to the lipoprotein E gene
Te, the or the base sequence is a base sequence of SEQ ID NO 1
Etc. have complementarity with the apolipoprotein E gene
Positive strand primer that is a base sequence and the sequence number
3 or the same complementarity as the base sequence
The base sequence for the apolipoprotein E gene
At least two species with a negative strand primer
Using the primer combination for the DNA sample to be tested
By carrying out the polymerase chain reaction,
Amplification of DNA fragment containing ε5 mutation site of thein E gene
And the labeled ε5 mutation was added to the amplified DNA fragment.
Uses site-allele-specific oligonucleotide probes
Apolipoprotein E, characterized by testing
Methods of testing for genotype are also provided.

For example, in the present inspection method, the
When the immers and the primers of SEQ ID NO: 4 are used, as shown in FIG. 1, ε2, ε4, in the DNA fragment sandwiched between the primers of SEQ ID NO: 1 and SEQ ID NO: 4,
Since the ε5 and ε7 mutation sites are present, the DNA fragment containing these mutation sites is amplified by the PCR method. Similarly, in this test method, the primer of SEQ ID NO: 1 and the SEQ ID NO:
When the primer of 3 is used , the ε5 mutation site exists in the DNA fragment sandwiched between the primer of SEQ ID NO: 1 and the primer of SEQ ID NO: 3 as shown in FIG.
A DNA fragment containing the ε5 mutation site is amplified by the PCR method, and the primer of SEQ ID NO: 2 and the primer of SEQ ID NO: 4
1 is used, in the invention of claim 7, as shown in FIG. 1, ε2, ε4 and ε7 are contained in the DNA fragment sandwiched between the primer of SEQ ID NO: 2 and the primer of SEQ ID NO: 4.
Since the mutation sites are present, the DNA fragment containing these mutation sites is amplified by the PCR method.

[0019] The Examples of the allele-specific oligonucleotide probes, for example, a nucleotide sequence or is the base sequence equivalent complementarity of SEQ ID NO: 5-20 are nucleotide sequences having relative apolipoprotein E gene invention
There is a probe.

The probes of SEQ ID NOs: 5 to 12 are probes complementary to the plus strand of the apo E gene, and the probes of SEQ ID NOs: 13 to 20 are probes complementary to the minus strand of the apo E gene. Here, SEQ ID NOS: 5, 6, 13
And the nucleotide numbers of the probes of 14 are 3877-38.
89, and the base of nucleotide number 3883 is a base that causes a mismatch, and the base is G in SEQ ID NO: 5, A in SEQ ID NO: 6, C in SEQ ID NO: 13, and T in SEQ ID NO: 14.
Is. And SEQ ID NOS: 5 and 13 are ε2 wild type probes, and SEQ ID NOS: 6 and 14 are ε2 mutant type probes. Here, the ε2 mutant type probe means the apoE gene ε
It causes a mismatch with the wild type of 2 while ε2
The wild-type probe causes a mismatch with the mutant type of the apoE gene ε2, and so on.

The probes of SEQ ID NOS: 7, 8, 15 and 16 have the nucleotide numbers of 3739 to 3751, and the nucleotide of nucleotide number 3745 is a base that causes a mismatch. 8 is G, SEQ ID NO: 15 is T, and SEQ ID NO: 16 is C. And SEQ ID NOS: 7 and 15 are ε4 wild type probes, and SEQ ID NOS: 8 and 16 are ε4 mutant type probes.

The probes of SEQ ID NOs: 9, 10, 17 and 18 have the nucleotide numbers 2829 to 2843,
The base at nucleotide number 2836 is the base that causes the mismatch, and the base is C in SEQ ID NO: 9, T in SEQ ID NO: 10, G in SEQ ID NO: 17, and A in SEQ ID NO: 18.
And SEQ ID NOS: 9 and 17 are ε5 wild type probes, SEQ ID NOS: 10 and 18 are ε5 mutant type probes, and the probes of SEQ ID NOS: 11, 12, 19 and 20 have the nucleotide numbers of 4136 to 4149, The bases of Nos. 4141 and 4144 are bases that cause a mismatch, and the bases are C in SEQ ID NO: 11, T in SEQ ID NO: 12, G in SEQ ID NO: 19 and A in SEQ ID NO: 20.
And SEQ ID NOS: 11 and 19 are ε7 wild type probes, and SEQ ID NOS: 12 and 20 are ε7 mutant type probes.

Since the probe of the present invention has a length of 13 to 15 bases, hybridization and washing are carried out at 37 ° C.
Can be done at.

In the invention of the inspection method of the present invention, the present invention
Part of the probe can be used . Sanawa
Then, in the test method of the present invention, allele-specific
Gonucleotide probes are selected from the probes of the invention.
Testing for apolipoprotein E genotype
A method is provided. According to this method
High level of all probes in testing for E gene type
Hybridization and washing can be done at 37 ° C
It Therefore, apo E at a time with only one thermostat of 37 ℃
It is convenient because it can check the genotype.

[0025] Here .epsilon.2, epsilon] 4, when inspecting the ε5 and ε7 type, .epsilon.2 probe used is selected from among the probes of the present invention, epsilon] 4, which is wild-type and mutant probes ε5 and ε7 type.

When examining ε5 type, the probes used are wild type and mutant type probes of ε5 type selected from the probes of the present invention , and ε2, ε4 and ε7 types are used.
In the case of examining E. coli, the probes used are wild type and mutant type probes of ε2, ε4 and ε7 types selected from the probes of the present invention.

The probe used may be either one complementary to the positive strand of the apo E gene or one complementary to the negative strand thereof . For example, ε2, ε4, ε5 and
When examining the ε7 type, the probes of SEQ ID NOS: 5 to 12 or the probes of SEQ ID NOS: 13 to 20 may be used, and the probes of SEQ ID NOS: 5, 14, 7, 16, 9, 1 may be used.
8, 11 and 20 may be used, SEQ ID NOS: 5-2
0 probes for each hybridization solution
You may use them one by one.

Further, in the present invention, the above-mentioned object of the present invention is desired.
In some of the inspection methods, when the polymerase chain reaction is an asymmetric polymerase chain reaction and the primer used more in the reaction is a primer for the plus strand, a probe complementary to the plus strand is used, while When the primer used more in the reaction is a minus strand primer, a probe complementary to the minus strand is used , and apolipoprotein E gene is characterized.
A child type inspection method is also provided. According to this method AP
After amplification of the DNA by the CR method, the amplified DNA is
Omitting denaturation and neutralization steps in hybridization
Can be tested for apoE genotype
More convenient.

Here, the asymmetric polymerase chain reaction (hereinafter referred to as APCR method) means that in the polymerase chain reaction, one of the plus-strand primer and the minus-strand primer is 50 to 200 relative to the other primer.
A reaction that uses double amount (Gyllensten, UBand Erlich,
HA, Proc.Natl.Acad.Sci.USA 85,7652-7656,1988
Year). In this APCR method, for example, when a large amount of the plus strand primer is used as compared with the minus strand primer, a large amount of the plus strand is synthesized as compared with the minus strand.

Therefore, by using a probe complementary to the single-stranded DNA composed of the amplified plus strand, the apolipoprotein E gene type can be tested even when the denaturation step and neutralization step of the DNA in the hybridization are omitted. It is possible to Similarly, when a large amount of minus-strand primers are used, the DNA denaturation step and neutralization step in the hybridization can be omitted by using a probe complementary to the minus-strand single-stranded DNA. The type can be checked.

That is, when a large amount of positive strand primers are used in the inspection method of the present invention , for example , SEQ ID NO: 1
When using primers in large quantities, using probes complementary to the plus strand, a place to use a probe of the present invention
In that case, the probes of SEQ ID NOs: 5 to 12 are used.

On the other hand, in the test method of the present invention, when a large amount of the minus strand primer is used, for example , SEQ ID NO: 4
When a large amount of the above-mentioned primer is used, a probe complementary to the minus strand is used, and the probe of the present invention is used.
In some cases, the probes of SEQ ID NOs: 13 to 20 are used.

Further, when the probe is unified into one of the one complementary to the plus strand and the one complementary to the minus strand, it may be necessary to identify a highly stable GT mismatch depending on the probe. Therefore, in such a case, when the probe for which the GT mismatch must be identified is a wild type, a mutant probe of the same genotype (eg, ε2 for ε2) or a GT mismatch is used. In the case where the probe that has to be identified is a mutant type, a wild-type probe of the same genotype may be used together for hybridization without labeling so that it can be clearly identified. For example, when the probe of SEQ ID NO: 8 according to the mutant type of ε4 is labeled and used, unlabeled ε4
Hybridization is performed by adding an appropriate amount of the probe of SEQ ID NO: 7, which is a wild type of the type. Even when the probes are not unified into one kind of strand, the same operation as described above can be performed for the probe for which the GT mismatch must be identified so that the identification can be made more clearly.

[0034]

EXAMPLES Next, preparation of the primer according to claim 4 used in this example will be described prior to a specific example of the present invention.

A. Selection and synthesis known nucleotide sequence of Apo E gene primers (Paik, Y.-K., Chang, D .
J., Reardon, CA, Davies, GE, Mahley, RWand Taylor,
JM, Proc.Natl.Acad.Sci.USA, 82,3445-3449 1985)
More specific oligonucleotide primers were selected by the following method. (1) DNA containing ε2, ε4, ε5 and ε7 mutation sites
Selection of primer combination for amplifying fragment Positive strand primer is upstream from ε5 mutation site, minus strand primer is downstream from ε7 mutation site Repetitive sequence that may inhibit specific amplification, similar The oligonucleotide of SEQ ID NO: 1 was selected as the primer for the positive strand, and the oligonucleotide of SEQ ID NO: 4 was selected as the primer for the negative strand by the method of removing the sequence and palindromic structure by computer. This primer combination is designated as A-1.

(2) Selection of a combination of primers for amplifying a DNA fragment containing the ε5 mutation site: The positive strand primer is located upstream of the ε5 mutation site, and the negative strand primer is located downstream of the ε5 mutation site. The oligonucleotide of SEQ ID NO: 1 was used as the primer for the positive strand and the oligonucleotide of SEQ ID NO: was used as the primer for the negative strand by a computer-based method that removes repetitive sequences, similar sequences, and palindromic structures that may inhibit specific amplification. Three oligonucleotides were selected respectively. This primer combination is designated as A-2.

(3) D containing ε2, ε4 and ε7 mutation sites
Selection of primer combination for amplifying NA fragment Positive strand primer from upstream of ε4 mutation site, minus strand primer from downstream of ε7 mutation site Repetitive sequence that may inhibit specific amplification The oligonucleotide of SEQ ID NO: 2 was selected as the primer for the positive strand and the oligonucleotide of SEQ ID NO: 4 was selected as the primer for the negative strand by the method of removing the similar sequence and the palindromic structure by computer. This primer combination is designated as A-3. Each primer selected by the above method was used as a DNA synthesizer (Gene Assembl manufactured by Pharmacia LKB, Sweden).
ER Plus), and purified by liquid chromatography FPLC (Pharmacia, Sweden) using the anion exchanger MonoQ (Pharmacia, Sweden).

B. Experiments to confirm that the selected primers are specific for the apoE gene. For humans, approximately 3 × 10 9 base pairs of DNA per haploid are used.
Exist, but the nucleotide sequence has been elucidated only in part. Therefore, if the primer theoretically selected by the above method also anneals with the nucleotide sequence of a part that has not yet been elucidated, it will also amplify a part other than the DNA fragment containing the apoE gene,
Such a primer cannot be said to be specific. The following experiment was conducted to confirm that only the specific part of the apoE gene was amplified by each of the selected oligonucleotide primer combinations (A-1, A-2, A-3).

(1) Preparation of DNA sample Human blood having apoE phenotypes of both mother and father-derived genes of type 3 examined by the method of McDowell et al. Was centrifuged at 3000 rpm for 15 minutes to obtain serum. It was removed and blood cells were obtained. About 500 μl of these blood cells
Buffer (10 mM Tris-HCl (pH 7.5), 5 mM Mg)
Cl2, 0.32M sucrose, 1% Triton X-100) was added, and the mixture was centrifuged at 13000 rpm for 20 seconds to remove the supernatant. To the precipitate was added 500 μl of the above buffer solution, 59 μl of 5% SDS and 25 μl of proteinase K (20 mg / ml), and the mixture was heated at 55 ° C. for 2 hours.
After adding 500 μl of phenol / chloroform to the mixture and mixing, the mixture was centrifuged at 8000 rpm for 15 minutes, and 500 μl of phenol / chloroform was added again to the obtained supernatant, and the same operation was performed. Chloroform (500 μl) was added to the supernatant and mixed by inverting to extract phenol, followed by centrifugation at 8000 rpm for 5 minutes. Supernatant 56 μl of 3M NaCl and 1.1
2 ml of cold ethanol was added and the mixture was allowed to stand at -70 ° C for 20 minutes and then centrifuged at 8000 rpm for 10 minutes to obtain a precipitate.
The precipitate was washed with 70% ethanol prepared in advance and stored at -20 ° C, and then dried under reduced pressure. 100 to this
μl buffer (1 mM Tris-HCl (pH 7.5), 0.1
mMEDTA) was added and dissolved to obtain a DNA sample.

(2) Amplification of DNA fragment by PCR method using selected primer combination 50 mM KCl, 1.5 mM MgCl 2, 5% DMSO,
0.001% gelatin, 10 mM Tris-HCl (pH 8.
3), each 200 μM dNTP (dATP, dGTP,
dCTP and dTTP), 0.2 μM each of positive and negative strand primers, DNA sample (0.01-1 μg) and 1.25 units of TaqD.
A reaction solution consisting of NA polymerase (50 μl) was covered with mineral oil and the DNA thermal cycler (DNA ther
mal Cycler) (manufactured by PERKIN ELMER CETUS, USA). Heat denaturation 96 ° C, 3 minutes, annealing 62 ° C,
After 1 minute of extension reaction at 70 ° C. for 4 minutes, heat denaturation 9
An annealing reaction was performed at 6 ° C. for 30 seconds, annealing at 62 ° C. for 1 minute, and extension reaction at 70 ° C. for 3 minutes for 39 cycles. Then 70
The temperature was kept at 7 ° C for 7 minutes to obtain a PCR reaction solution. In this case, as the primers, the combinations (A-1, A-2, A-3) of the three kinds of primers synthesized in the above "A. Selection and synthesis of primers according to claim 4" were used, and 3 Seed P
A CR reaction solution was obtained.

(3) The DNA fragment amplified using the selected combination of primers is the specific DNA of the apoE gene.
Confirmation as a fragment After the amplification reaction, a part of the PCR reaction solution was subjected to 5% polyacrylamide gel electrophoresis together with a size marker and stained with ethidium bromide. The result is shown in FIG. That is, in FIG. 2, lanes 1 and 5 are size markers, and lane 1 contains plasmid pBR322 with restriction enzyme Hinf.
DNA fragments digested with I (1632,517,504,396,3 respectively)
44,298,221,220,154 and 75 base pairs), lane 5 is a fragment obtained by further digesting it with the restriction enzyme EcoRI (998, respectively,
634,517,504,396,344,298,221,220,154 and 75 base pairs)
Is the result of electrophoresis. Meanwhile, Lane 2 to Lane 4
Is the result of electrophoresis of the PCR reaction solution. Lane 2 uses A-1 as a primer combination, lane 3 uses A-2 as a primer combination, and lane 4 uses A-3 as a primer combination. This is the result of the case.

The DNA fragment amplified by the PCR method is a portion sandwiched between two primers. Therefore, ε2, ε4,
When amplifying a DNA fragment containing ε5 and ε7 mutation sites, 5′-2802 to 2823-3 ′ (SEQ ID NO: 1) for the positive strand and 3′-4220 to 4241-5 ′ (SEQ ID NO: 4) for the negative strand ), The size of the amplified DNA fragment is 144
Expected to be 0 bp. In fact, as shown in lane 2 of FIG. 2, DNA was placed at the position corresponding to the expected size.
Fragments were detected. When amplifying a DNA fragment containing the ε5 mutation site, 5'-2802 to 2823-for the positive strand is used.
3 '(SEQ ID NO: 1) and 3'-2958 for the minus strand
Since the primer having the nucleotide number of ˜2979-5 ′ (SEQ ID NO: 3) is used, the size of the DNA fragment to be amplified is expected to be 178 bp. Actually Figure 2
A DNA fragment was detected at a position corresponding to the expected size as shown in lane 3 of the above. When amplifying a DNA fragment containing ε2, ε4 and ε7 mutation sites, 5′-3618 to 3639-3 ′ (SEQ ID NO: 2) for the plus strand and 3′-4220 to 4241-5 ′ for the minus strand ( Since the primer having the nucleotide number of SEQ ID NO: 4) is used, the size of the amplified DNA fragment is expected to be 624 bp. Actually, a DNA fragment was detected at a position corresponding to the expected size as shown in lane 4 of FIG.

Further, in order to confirm that these amplified DNA fragments are specific to apoE, the DNA fragment in lane 2 in FIG. 2 was digested with restriction enzymes EcoRI, HinfI or Sa.
The DNA fragment in lane 3 was digested with HaeII with u3AI, and the DNA fragment in lane 4 was digested with PstI. The result is shown in FIG. That is, in FIG. 3, lane 1 is a size marker,
The plasmid pBR322 is used as a restriction enzyme for HinfI and EcoRI.
2 shows the result of electrophoresis of the fragment digested in step 2. Lane 2 was EcoRI for the DNA fragment of lane 2 in FIG. 2, and lane 3 was Hi.
Lane 4 is the result of digestion with Sau3AI and electrophoresis with nfI, and lane 5 is the result of electrophoresis of the DNA fragment of lane 3 of FIG. 2 digested with HaeII, and lane 6
2 shows the result of electrophoresis of the DNA fragment in lane 4 of FIG. 2 after digestion with PstI.

From the nucleotide sequence of the apoE gene, ε2, ε4,
When the DNA fragments containing the ε5 and ε7 mutation sites were digested with EcoRI, the 1070 bp and 370 bp fragments were Hinf
It is expected that fragments of 1030 bp, 348 bp and 62 bp for I and 909 bp and 531 bp for Sau3AI will be generated. When a DNA fragment containing the ε5 mutation site was digested with HaeII, 103 bp and 75 bp fragments were obtained, and a DNA fragment containing the ε2, ε4 and ε7 mutation sites was digested with Ps.
363 bp, 174 bp and 87 b when digested with tI
It is expected that a fragment of p will result. In fact, as shown in FIG. 3, each DN is located at a position corresponding to the expected size.
The A fragment was confirmed. From the above experimental results, the primer combinations selected in A above (A-1, A-2, A
-3), a part of the apoE gene was specifically amplified, that is, it was confirmed that the primer selected in A above was specific to the apoE gene.

Next, the production of the probe according to claim 8 used in this embodiment will be described. Preparation of C allele-specific oligonucleotide probe Since the nucleotide sequence of the apoE gene and the mutation to be tested in the present invention and its position are known, the wild-type of the apoE gene ε2, ε4, ε5 or ε7 is known based on this knowledge. It is possible to select an oligonucleotide in which the bases causing a mismatch with each of the plus or minus strands of the normal or mutant gene are located in the center of the probe, and further, hybridization and washing temperatures can be carried out at 37 ° C. 13 to 15 Oligonucleotides of SEQ ID NOs: 5 to 20 having a base length were selected as a probe. The probes of SEQ ID NOs: 5 to 20 were used to synthesize the above-mentioned DNA in the same manner as in the synthesis of the above-mentioned primer
It was synthesized using a synthesizer and purified by FPLC using MonoQ.

As for the labeling of each probe, digoxigenin-11-dUTP was labeled at its 3'end using a terminal transferase. Specifically, 20 μl of 10X terminal transferase buffer (1.4 mM cacodylate potassium, 10 mM cobalt chloride, 1 mM dithiothreitol, 300 mM Tris-HCl).
(PH 7.2)), 6 μl of 1 mM digoxigenin-11-d
UTP, 100 μl of 2.5 μM oligonucleotide and 68 μl of distilled water were added and stirred, and then the terminal transferase (2
5 U / μl) was added, and the mixture was allowed to react at 37 ° C. for 90 minutes for labeling.

Next, the preparation of the mutant control DNA used in this example will be described. D. Preparation of mutant control DNA
Synthesized by a synthesizer, and F using the anion exchanger MonoQ
Purified by PLC. Here, the oligonucleotides of SEQ ID NOs: 21 and 22 are respectively a plus strand and a minus strand for preparing a control DNA of ε2 mutant type. And the nucleotide number of SEQ ID NO: 21 or 22 is
It is 5'-3858-3887-3 'or 3'-3878-3907-5'. Then, a mutation is formed in T at nucleotide number 3883.

Next, the oligonucleotides of SEQ ID NOs: 23 and 24 are respectively a plus strand and a minus strand for preparing a control DNA of ε4 mutant type. And the nucleotide number of SEQ ID NO: 23 or 24 is 5'-3721-3, respectively.
750-3 'or 3'-3741-3770-5'. Then, a mutation is formed in C at nucleotide number 3745. The oligonucleotides of SEQ ID NOs: 25 and 26 are respectively a positive strand and a negative strand for preparing the control DNA of ε5 mutant type. The nucleotide number of SEQ ID NO: 25 or 26 is 5'-2811-2840-3 'or
It is 3'-2831-2860-5 '. And nucleotide number 28
A mutation is formed in A at 36. The oligonucleotides of SEQ ID NOs: 27 and 28 are respectively a positive strand and a negative strand for preparing the ε7 mutant control DNA. And the nucleotide number of SEQ ID NO: 27 or 28 is 5'-4118-4147-3 'or 3'-4138-416, respectively.
7-5 '. And nucleotide numbers 4141 and 4144.
In A, a mutation is formed.

After annealing the plus and minus strands for preparing various mutant control DNAs,
Taq DNA polymerase acts to form a double-stranded 50 base pair DNA fragment, which is used as a control D for each mutant type.
Used as NA. That is, the composition of the reaction solution of 50 μl was 50 mM KCl, 1.5 mM MgCl 2, 5% DMS.
O, 0.001% gelatin, 10 mM Tris-HCl (pH
8.3), each 200 μM dNTP (dATP, dGT)
P, dCTP and dTTP), 0.2 μM each oligonucleotide, and 1.25 units of Taq DNA polymerase.

As the 0.2 μM oligonucleotide, 0.2 μM each of the oligonucleotides of SEQ ID NOs: 21 and 22 was used to prepare ε2 mutant control DNA, and ε4 mutant control D was used.
When NA is prepared, the oligonucleotides of SEQ ID NOS: 23 and 24 are used at 0.2 μM each, and when the ε5 mutant control DNA is prepared, the nucleotides of SEQ ID NOS: 25 and 26 are used at 0.2 μM, respectively. SEQ ID NO: 27 when preparing ε7 mutant control DNA
And 28 nucleotides were used at 0.2 μM each.

The reaction solution having the above composition is covered with mineral oil, and a DNA thermal cycler is used.
(Manufactured by PERKIN ELMER CETUS, USA). After heat denaturation 96 ° C., 3 minutes, annealing 62 ° C., 1 minute, extension reaction 70 ° C., 4 minutes, heat, denaturation 96 ° C., 30 seconds, annealing 62 ° C., 1 minute, extension reaction 70 ° C.
39 cycles of 3 minutes were performed, and the mixture was kept at 70 ° C. for 7 minutes to prepare various mutant control DNA solutions.

Next, a specific example of the present invention for testing the apoE genotype using the primer of claim 4 prepared in A above and the probe prepared in C above will be described. Example 1 ε2, ε4, ε5 and ε7 type test Each DNA sample was prepared by the method described in B (1) from the blood cells of 9 humans who underwent the apoE phenotype test by the method of McDowell et al. Next, the oligonucleotides of SEQ ID NOs: 1 and 4 selected and synthesized by the method described in A were used as a positive strand primer and a negative strand primer (primer combination A-1), respectively, and B ( Each PCR reaction solution was obtained by performing the PCR method as described in 2). As a result, ε2 and ε of each sample
A DNA fragment containing the 4, ε5 and ε7 mutations was amplified.

As described above, 9 kinds of PCR reaction solutions obtained from DNA samples of 9 humans and each mutant control DNA solution prepared by the method described in D, 0.5 μl each
Each was spotted on a nylon membrane. After air-drying, add 2 to the denaturing solution (0.5M NaOH, 1M NaCl).
Min, followed by a neutralizing solution (1M Tris-HCl (pH 7.5),
1.5 M NaCl) for 2 minutes. 2 at 80 ° C after air drying
Burned for hours. Then prehybridize 3
1 hour at 7 ° C, a kit (DNA labeling & detection kit, manufactured by Boehringer-Mannheim, Germany)
Product No. 1093657), and each of the labeled probes of SEQ ID NOS: 5 to 12 prepared by the method described in C was individually added to each nylon membrane at 1.25 nM, and
Hybridized overnight at ° C.

Since the probes of SEQ ID NOs: 5, 8, 10 or 12 identify GT mismatches, the unlabeled oligonucleotides of SEQ ID NOs: 6, 7, 9 or 11 are respectively labeled with the labeled SEQ ID NOs. Equivalent amounts were added to 5, 8, 10 or 12 probes and hybridization was performed. Subsequently, at 37 ° C., a cleaning solution (0.1-6 × S
SC, 0.1% SDS) and washed twice for 10 minutes. Here, 1 × SSC is a 0.015 M sodium citrate solution (pH 7.0) containing 0.15 M NaCl. Hybridization and wash temperatures were 37 ° C. for all types of tests. Then, a non-RI test was performed using the above Boehringer-Mannheim kit. The result is shown in FIG.

In FIG. 4, 2W, 2M, 4W, 4M, 5W,
5M, 7W and 7M are symbols indicating the type of probe, the numbers indicate the type of mutation, W indicates the wild type, and M indicates the mutation type. That is, 2W, 2M, 4W, 4M,
5W, 5M, 7W or 7M is SEQ ID NO: 5, 6, 7,
It means 8, 9, 10, 11 or 12 probes. The parentheses after human DNA (NO. 1 to 9) indicate 9 human apoE phenotypes, for example, 3
/ 3 means that both parents' genes are type 3,
3/4 means that the gene derived from one parent is type 3 and the gene derived from the other parent is type 4. The black circles in FIG. 4 indicate that they reacted with the probe.

As shown in FIG. 4, the DNA amplified by the PCR method using the primers according to the present invention from the human DNA samples of 9 persons reacted only with the probe expected from the result of the phenotype test. For example, human DNA
In (NO. 1), the phenotype is 3/4, that is, since one of the genes derived from parents is ε3 type (wild type), this gene is 2W, 4W, 5W and 7W.
And the gene from the other parent is ε4 type,
It is expected that this gene will react with 2W, 4M, 5W and 7W. And as shown in FIG. 4, human DNA
As for (NO. 1), 2W, 4W, 4M, 5 as expected
Reacting with 5 probes of W and 7W. In human DNA (NO. 2 and 4), the phenotype is 3
/ 3 and, as expected, 4 of 2M, 4M, 5M and 7M
Not reacting with one probe.

In order to test the ε2, ε4, ε5 and ε7 types, for example, a DNA fragment containing the ε5 mutation site was amplified using a combination of the primers of SEQ ID NOS: 1 and 3 and a DNA sample, while A combination of the primers of Nos. 2 and 4 was used for the DNA sample to amplify the DNA fragments containing the ε2, ε4 and ε7 mutation sites, which were mixed and
It is also possible to test the apoE genotype using an allele-specific oligonucleotide probe for a DNA fragment containing the 2, ε4, ε5 and ε7 mutation sites.

Example 2 ε4 type test requiring no denaturation step a. Amplification of apoE-specific DNA fragment by PCR method (APCR method) using positive strand primer in 50 times amount of negative strand primer. McDowell et al. According to the method, each DNA sample was prepared from the blood cells of humans (3 persons) who underwent the apoE phenotype test by the method described in B (1), and amplified using the APCR method. 50 μl reaction solution composition is 50 mM KCl, 1.5 mM
MgCl2, 5% DMSO, 0.001% gelatin,
10 mM Tris-HCl (pH 8.3), 200 μM dN each
TP (dATP, dGTP, dCTP and dTTP),
SEQ ID NO: 2 plus strand primer (250 nM)
And a primer for the minus strand, which is SEQ ID NO: 4 (5n
M), human DNA sample (0.01-1 μg) and 1.2.
5 units of Taq DNA polymerase. The reaction solution of this composition is covered with mineral oil and the DNA thermal Cycler (DNA thermal cycler) (Perkin Electronics, USA) is used.
CETUS). After heat denaturation 96 ° C., 3 minutes, annealing 62 ° C., 1 minute, extension reaction at 70 ° C., 4 minutes, heat denaturation 96 ° C., 30 seconds, annealing 62
C., 1 minute, extension reaction: 70.degree. C., 3 minutes: 39 cycles. Then, the temperature was kept at 70 ° C. for 7 minutes to obtain an APCR reaction solution.

B. Ε4 type test without denaturation and neutralization step The APCR reaction solution obtained in a.
Reaction solution and denaturing solution (0.5M NaOH, 1M NaCl)
0.5 .mu.l of each of two types in which equal amounts of and were mixed (the former has no denaturing step and the latter has a denaturing step) was spotted on a nylon membrane. Ordinary PCR using equal amounts of two types of primers for plus and minus strands
The same procedure is performed for the PCR reaction solution obtained by performing the method, and 0.5 μm of each of two types, one with and without the denaturation step, is used.
l spotted. Also, an ε4 type control DNA solution (0.5 μl) prepared by the method described in D was mixed with the above denaturing solution in an equal amount, and 0.5 μl of the denaturing step was spotted. After air drying, the neutralization solution (1M Tris-HCl,
1.5M NaCl) for 2 minutes, air-dried, and baked at 80 ° C. for 2 hours. Then pre-hybridize 37
1 hour at ℃, a kit manufactured by Boehringer-Mannheim, Germany (DNA labeling & detection kit,
According to the product number 1093657), 1.25 nM of the labeled oligonucleotide probe of SEQ ID NO: 7 or 8 prepared by the method described in C was added and hybridized at 37 ° C. overnight. Since the probe of SEQ ID NO: 8 discriminates GT mismatch, an unlabeled oligonucleotide of SEQ ID NO: 7 was added to the labeled probe of SEQ ID NO: 8 in an equal amount to carry out hybridization. Subsequently, at 37 ° C., a washing solution (5 × SSC, 0.1% SD
It was washed twice with S) for 10 minutes. Then, a non-RI test was performed using the above Boehringer-Mannheim kit. The result is shown in FIG.

In FIG. 5, parentheses and black circles after 4W, 4M and human DNA have the same meanings as explained in FIG.

As shown in FIG. 5, it was possible to test the apoE genotype of the DNA amplified by the APCR method even if the denaturation step was omitted. However, for DNA amplified by the usual PCR method, the apoE genotype could be tested only when the denaturation step was performed.

According to the test method of the present invention, the ε2 type, ε4 type, ε5 type and ε7 type of the apolipoprotein E gene can be easily and accurately tested. Inspection of the present invention
According to the method, the ε5 type of the apolipoprotein E gene can be easily and accurately tested. Inspection method of the present invention
According to the method, the apolipoprotein E gene ε2 type, ε
Type 4 and ε7 type can be tested easily and accurately. According to the probe of the present invention , apolipoprotein E
Suitably employed et al is the examination of gene, using this probe
And 3 for hybridization and washing of all probes in the apolipoprotein E genotype test.
It can be carried out at 7 ° C. Therefore, the apoE genotype can be tested at one time in only one 37 ° C. thermostat. In addition, when APCR is applied, DNA
After amplification, the apoE genotype can be examined even when the denaturation and neutralization steps in the hybridization of the amplified DNA are omitted, which is more convenient.

[Sequence listing] SEQ ID NO: 1 Sequence length: 22 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array GAACTTGTTC CACACAGGAT GC 22 SEQ ID NO: 2 Sequence length: 22 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array GGAGTTGAAG GCCTACAAAT CG 22 SEQ ID NO: 3 Sequence length: 22 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CTCCTCCTGC ACCTGCTCAG AC 22 SEQ ID NO: 4 Sequence length: 22 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array GCCCACTGGC GCTGCATGTC TT 22 SEQ ID NO: 5 Sequence length: 13 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CCAGGCGCTT CTG 13 SEQ ID NO: 6 Sequence length: 13 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CCAGGCACTT CTG 13 SEQ ID NO: 7 Sequence length: 13 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array GGCCGCACAC GTC 13 SEQ ID NO: 8 Sequence length: 13 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array GGCCGCGCAC GTC 13 SEQ ID NO: 9 Sequence length: 15 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array GCTTGCTCCA CCTTG 15 SEQ ID NO: 10 Sequence length: 15 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array GCTTGCTTCA CCTTG 15 SEQ ID NO: 11 Sequence length: 14 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CTGCTCCTCC AGCT 14 SEQ ID NO: 12 Sequence length: 14 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CTGCTTCTTC AGCT 14 SEQ ID NO: 13 Sequence length: 13 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CAGAAGCGCC TGG 13 SEQ ID NO: 14 Sequence length: 13 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CAGAAGTGCC TGG 13 SEQ ID NO: 15 Sequence length: 13 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array GACGTGTGCG GCC 13 SEQ ID NO: 16 Sequence length: 13 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array GACGTGCGCG GCC 13 SEQ ID NO: 17 Sequence length: 15 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CAAGGTGGAG CAAGC 15 SEQ ID NO: 18 Sequence length: 15 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CAAGGTGAAG CAAGC 15 SEQ ID NO: 19 Sequence length: 14 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array AGCTGGAGGA GCAG 14 SEQ ID NO: 20 Sequence length: 14 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array AGCTGAAGAA GCAG 14 SEQ ID NO: 21 Sequence length: 30 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CCGCGATGCC GATGACCTGC AGAAGTGCCT 30 SEQ ID NO: 22 Sequence length: 30 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CCCCGGCCTG GTACACTGCCA GGCACTTCT 30 SEQ ID NO: 23 Sequence length: 30 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CTGGGCGCGG ACATGGAGGA CGTGCGCGGC 30 SEQ ID NO: 24 Sequence length: 30 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CCGCGGTACT GCACCAGGCG GCCGCGCACG 30 SEQ ID NO: 25 Sequence length: 30 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CCACACAGGA TGCCAGGCCA AGGTGAAGCA 30 SEQ ID NO: 26 Sequence length: 30 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CCGGCTCTGT CTCCACCGCT TGCTTCACCT 30 SEQ ID NO: 27 Sequence length: 30 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array TGGCGGAGGT GCGCGCCAAG CTGAAGAAGC 30 SEQ ID NO: 28 Sequence length: 30 Sequence type: Nucleic acid Number of chains: Single chain Sequence type: Other nucleic acids Synthetic DNA Array CAGGCGTATCT GCTGGGCCTG CTTCTTCAG 30

[Brief description of drawings]

FIG. 1 ApoE gene and its mutated portions, SEQ ID NOS: 1 to
It is a schematic diagram showing the position of the primer of No. 4.

FIG. 2 shows an amplification reaction solution of a DNA fragment of ApoE gene and a polyacrylamide gel electrophoresis ethidium bromide stained gel of a size marker.

FIG. 3 shows a polyacrylamide gel electrophoresis ethidium bromide-stained gel of a fragment obtained by digesting a DNA fragment of the apoE gene with various restriction enzymes.

FIG. 4 shows the test results of the apo E genotype in Example 1.

FIG. 5 shows the results of ε4 type inspection in Example 2.

[Explanation of symbols]

1 SEQ ID NO: 1 primer 2 Primer of SEQ ID NO: 2 3 Primer of SEQ ID NO: 3 4 SEQ ID NO: 4 primer

─────────────────────────────────────────────────── ─── Continuation of the front page (56) References Genomics, Vol. 8, No. 4 (1990) p. 684-692 J. Lipid Res. , Vol. 32, No. 1 (1991. Jan.) p. 183-187 Biochem. Biophys. Res. Commun. , Vol. 157, No. 3 (1988) p. 1212-1217 Clin. Chim. Acta, Vol. 189, No. 2 (1990) p. 153-157 Genomics, Vol. 3, No. 4 (1988) p. 373-379 J.I. Lipid Res. , Vol. 29, No. 9 (1988) p. 1231-1237 Hum. Genet. , Vol. 83, No. 4 (1989) p. 364-368 Clin. Chem. , Vol. 36, No. 12 (1990) p. 2087-2092 J. Biochem. , Vol. 105, No. 4 (1989) p. 491-493 J. Biochem. , Vol. 105, No. 1 (1989) p. 51-54 (58) Fields investigated (Int.Cl. ⁷ , DB name) GenBank / EMBL / DDBJ / GeneSeq PubMed

Claims (13)

(57) [Claims] 1. A polymerase chain reaction is carried out by using a combination of at least two kinds of primers which are complementary to the apolipoprotein E gene and have a plus-strand primer and a minus-strand primer for a DNA sample to be examined. DNA containing the ε2, ε4, ε5, and ε7 mutation sites of the apolipoprotein E gene
A fragment is amplified, and the amplified DNA fragment is added with ε2 type,
Wild corresponding to each of ε4 type, ε5 type, and ε7 type
Type probe and mutant type probe.
One is labeled and the other is unlabeled
Allele-specific oligonuclides with cardinality of 13-15
Ε2 type, ε4 type, ε5 type, and
A method for testing apolipoprotein E gene type for testing ε7 type . 2. The allele-specific oligonucleotide
The probe is selected from the nucleotide sequences of SEQ ID NOs: 5 to 20.
And said allele-specific oligonucleotide probe
Hybridization and washing at 37 ° C
Performing method of claim 1. 3. The method according to claim 1 or 2, wherein the positive strand primer comprises the nucleotide sequence of SEQ ID NO: 1 and the negative strand primer comprises the nucleotide sequence of SEQ ID NO: 4. 4. A polymerase chain reaction is carried out by using a combination of at least two kinds of primers which are complementary to the apolipoprotein E gene and have a plus-strand primer and a minus-strand primer for a DNA sample to be examined. By doing so, a DNA fragment containing the ε5 mutation site of the apolipoprotein E gene is amplified, and the amplified DNA fragment is treated with a wild-type probe corresponding to ε5 type.
And mutated probes, among these probes
One is labeled and the other is unlabeled, with a base number of 13
~ 15 allele-specific oligonucleotide pro
Probe for ε5 type , apolipoprotein E
Genotype test method. 5. The allele-specific oligonucleotide
The probes are nucleotide sequences of SEQ ID NOs: 9, 10, 17 and 18.
Selected from the column, said allele-specific oligonucleosides
It does the hybridization and washing of the tide probe
The method according to claim 4, wherein each method is performed at 37 ° C. 6. The method according to claim 4, wherein the positive strand primer comprises the base sequence of SEQ ID NO: 1 and the negative strand primer comprises the base sequence of SEQ ID NO: 3. 7. A primer complementary to the apolipoprotein E gene, which is a combination of at least two kinds of primers having a plus-strand primer and a minus-strand primer, is used for a DNA sample to be tested for polymerase chain reaction. By doing so, a DNA fragment containing the ε2, ε4, and ε7 mutation sites of the apolipoprotein E gene is amplified, and the amplified DNA fragment contains wild-type probes and mutations corresponding to ε2 type, ε4 type, and ε7 type, respectively.
Type probe, one of which is the standard
And the other is unlabeled and has 13-15 bases
Use an allele-specific oligonucleotide probe
A method for testing apolipoprotein E gene type, which comprises testing ε2 type, ε4 type, and ε7 type . 8. The allele-specific oligonucleotide
The probes are SEQ ID NOs: 5-8, 11-16, 19 and 2
Selected from the base sequence 0, and the allele-specific orientation
Hybridization of gonucleotide probes and
The method according to claim 7, wherein each washing is performed at 37 ° C. 9. The method according to claim 7, wherein the positive strand primer comprises the nucleotide sequence of SEQ ID NO: 2 and the negative strand primer comprises the nucleotide sequence of SEQ ID NO: 4. 10. When the polymerase chain reaction is an asymmetric polymerase chain reaction and the primer used more in the reaction is a primer for a plus strand, an allele-specific probe complementary to the plus strand is used, , If the primer used most in the reaction is a primer complementary to the minus strand, by using an allele-specific probe complementary to the minus strand, the denaturation and neutralization steps in hybridization can be omitted. The method according to any one of claims 1 to 9, characterized by: 11. A probe set for examining apolipoprotein E genotype, which comprises SEQ ID NOs: 5 and 2.
0 type, ε2 type, ε4 type, ε5 type
And a ε7-type wild type probe and a mutated probe , respectively, wherein either one of the wild type probe and the mutated probe is labeled, and the other one is unlabeled. 12. A probe set for examining apolipoprotein E genotype, comprising SEQ ID NOs: 9 and 1.
Selected from 0, 17 and 18 nucleotide sequences , paired with ε5 type
A probe set comprising an allele-specific probe comprising a corresponding wild-type probe and a mutant-type probe, wherein either one of the wild-type probe and the mutant-type probe is labeled, and either one is unlabeled. 13. A probe set for testing apolipoprotein E genotype, which comprises SEQ ID NOs: 5 to 5.
Selected from the nucleotide sequences of 8, 11 to 16, 19 and 20 , consisting of wild type probes and mutant type probes corresponding to ε2 type, ε4 type and ε7 type, respectively. A probe set in which one of the mutant probes is labeled and the other is unlabeled.