JP2003274947A - Personal identification method using polymorphism of human mitochondoria dna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a personal identification method using mtDNA polymorphism, to provide a personal identification method by which a plurality of persons can at once be identified, and to provide a method which contributes to the sensitivity enhancement, cost lowering and simplification of the DNA personal identification. <P>SOLUTION: The types of all polymorphisms can be identified by mixing oligonucleotide primers for detecting six polymorphisms comprising single base polymorphisms at 3010, 4386, 5178, 8794 and 10398 positions among the base sequence of human mitochondoria DNA and a polymorphism related to an insertion or deletion for the base sequence of nine bases among the base sequence ranged from 8272 position to 8289 position, and then subjecting the mixture to a PCR reaction to type all of the polymorphism. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to polymorphisms in human mitochondrial DNA (mtDNA), and its mtD.
The present invention relates to a technology for personal identification using NA polymorphism. This technology is, for example, for personal identification in forensic medicine, paternity examination, epidemiological survey, archaeological survey, etc., and food whose quality is greatly deteriorated when biogenic substances such as hair are mixed as foreign substances into products in the industrial production process. It can be applied to personal identification for manufacturing quality control of medicines and drugs.

[0002]

2. Description of the Related Art In recent years, a technique for identifying an individual from a sequence of nucleic acids contained in a biological substance such as hair or body fluid has been actively applied in the fields of forensic medicine and the like. The most frequently used nucleic acid information for personal identification is
It is a polymorphism of the repeat number of microsatellite or minisatellite (Nakamura, Y. et.al: Science 235, 1616-
1622 (1987): Variable number of tandem repeat (VNT
R) markers for human gene mapping). However, since microsatellite or minisatellite is contained in nuclear DNA, forensic medicine, archeology, or industrial manufacturing process, etc. Often it is difficult to obtain sufficient quantities. This is because, in addition to the small amount of the samples of these biologically-derived substances, the contained DNA is often deteriorated due to a long period of time, heat treatment, drug treatment, or the like.

In such a case, it is conceivable to use mitochondrial DNA (mtDNA) having a copy number several hundred times that of nuclear DNA. Many types of polymorphisms are known in mtDNA because mutations occur frequently. The reason for this phenomenon is thought to be mainly caused by high levels of active oxygen present in mitochondria (Richter, C., Park, JW, Ames, B.
N., Proc.Natl.Acad.Sci.USA 1988, 85,6465-6467.).
Among such mtDNA polymorphisms, the D-LOOP region with large individual differences, particularly the sequence called the hypervariable regions I and II, has been sequenced for the purpose of individual identification (Int J Legal Med 1
993; 106 (2) 85-90).

Mutations in mtDNA observed in both untranslated and translated regions are widely used to characterize human evolutionary processes and to identify individuals (Can,
LR, Stoneking, M., Wilson, AC, Nature 1987, 3
25, 31-36; Quintana-Murci, L., Semio, O., Bandelt,
H.-J., Passrino, G., McElreavey, K., Santachiara-B
enerecetti, AS, Nat.Genet. 1999, 23, 437-441; H
olland, MM, Parsons, TJ Forens. Sci. Rev. 19
99, 11, 21-50.). For the former purpose, studies on base (nt) substitutions in the translated region seem appropriate. This is because mtDNA mutations frequently occur in non-translational control regions, which may make phylogenetic analysis difficult.

Single nucleotide polymorp
Various simple analytical methods are known to detect hisms: SNPs) (Cargill, M. et al: Nature Genetics
22,231-238 (1999): Characterization of single-nuc
leotide polymorphisms incoding regions of human ge
nes). For example, direct sequence, DNA microarray (Patrick, OB et al: Nature Genetics 21,
33-37 (1999): Exploring the new world of the gen
Hybridization method using ome with DNA microarrays, etc., Invader method (Victor, L. et al: Nature
Biotechnology 17, 292-296 (1999): Polymorphism i
dentification and quantitative detection of genomi
c DNA by invasive cleavage of oligonucleotide prob
es), Taqman probe method (Shi, MM: Clin Chem 47, 1
64-172 (2001): Enabling large-scale pharmacogeneti
c studies by high-throughput mutation detection an
d genotyping technologies), Masscode tag method (htt
p: //www.qiagen.com/literature/qiagennews/0201/1016
749_QNews22001p11-12.pdf), allele specific PCR method (Soren, G. et al: Genome Res 10, 258-266 (2000): H.
igh-throughput SNP allele-frequency determination i
n pooled DNA samples by kinetic PCR), restriction fragment length polymorphism (RFLP) method (Jazwinska, EC et al: Am J Hum Ge
net 43,175-181 (1988): Gm typing by immunogloblin h
eavy-chain gene RFLP analysis) etc. are known.

Among them, the RFLP method is one of the most widely used techniques for detecting point mutations in the translation region of mtDNA (Can, LR, Stoneking,
M., Wilson, AC, Nature 1987, 325, 31-36 .: Torro
ni, A., Schurr, TG, Yang, C., Szathmary, EJ
E., Wil-liams, RC, Schanfield, MS, Troup, C.
A., Knowler, WC, Lawrence, DN, Weiss, KM,
Wallace, DC, Genetics 1992, 130, 153-162.). However, when this method is applied to the actual mutation site, even if there is a change in the restriction enzyme cleavage pattern, which base was replaced by which base at the restriction enzyme recognition site (4-6 bp) It can not be identified. In addition, multiple solid-phase fluorescence mini-sequences
scentminisequencing) (Tully, G., Sullivan, KM,
Nixon, P., Stoness, RE, Gill, P., Genomics 1996,
34, 107-113.) And an immunized sequence-specific oligonucleotide probe assay (immobilized sequence).
-specific oligonucleotide probe-based assay) (Sto
neking, M., Hedgecock, D., Higuchi, RG, Vigilan
t, L., Erlich, HA, Am. J. Hum. Genet. 1991, 48,
370-382.) Can also be applied to the analysis of mtDNA polymorphisms.

[0007]

As described above, using nuclear DNA polymorphisms (STR, etc.) for individual identification is often undetectable due to the insufficient amount of DNA obtained from the test material. . Here, if the polymorphism of mtDNA whose copy number is several hundred times that of nuclear DNA can be used for personal identification, many of such problems can be solved. However, among the polymorphisms at the D-LOOP site of mtDNA that have been conventionally used for personal identification, it is known that the frequent occurrence type is about 10%.
It is often difficult to identify an individual only by sequencing the site. In addition, since polymorphism frequently occurs at the D-LOOP site, it is not suitable for application of the commonly known single nucleotide polymorphism analysis method.Therefore, it is usually necessary to use a DNA sequence such as Sanger method for sequence decoding. There is.

On the other hand, in mtDNA, a method of detecting individual polymorphisms is excellent at a site where polymorphisms are present in addition to the hypervariable region. However, the above-mentioned techniques for detecting such polymorphisms are often expensive and time-consuming. Furthermore, it has not been performed so far among the polymorphisms of the entire mtDNA to select ones useful for individual identification and use them for individual identification.
This is because until now, especially in Japanese, the mtDNA sequence has not been sufficiently decoded, and it has not been possible to determine whether the mtDNA polymorphism can be used for individual identification. Also,
There has been no attempt to apply such personal identification by mtDNA to manufacturing quality control.

The present invention has been made in view of the above-mentioned circumstances, and its object is to relate to a polymorphism of mtDNA, a method for identifying an individual by utilizing the polymorphism, and further to By providing a method to analyze multiple
It contributes to high sensitivity, low price, and simplification of DNA individual identification.

[0010]

Means for Solving the Problems, Actions of the Invention, and Effects of the Invention As a result of intensive studies, the present inventors have found a new polymorphism in mtDNA, and in addition to this new polymorphism, known mtDNA Simple amplified product-length polymorphism (AP)
LP method. Watanabe, G., Umetsu, K., Yuasa, I., Suzuki,
T., Hum. Genet. 1997, 99, 34-37 .: Aoshima, T., Um
etsu, K., Yuasa, I., Watanabe, T., Suzuki, T., Ele
We have succeeded in developing a method for typing multiple mtDNA polymorphisms in the same reaction system by applying ctrophoresis 1998, 19, 659-660.

The entire 16,569 base pair base sequence of mtDNA was determined in 1981 and is known as the Cambridge control sequence (Anderson, S., Bankier, AT,
Barrell, BG, de Bruijn, MHL, Coulson, AR,
Drouin, J., Eperon, IC, Nierlich, DP, Ror, B.
A., Sanger, F., Schreier, PH, Smith, AHH, Sta
den, R., & Young, IG: Sequence and organization
of the human mitochondrial genome. Nature 290, 457
-465, 1981).

The present inventors newly discovered single nucleotide polymorphisms at positions 4386 and 8794 by newly determining a plurality of mtDNA sequences. Among them, the polymorphism at position 4386 is one in which thymine (T) is mutated to cytosine (C), and the polymorphism at position 8394 is one in which cytosine (C) is mutated to thymine (T). there were. In addition to the above-mentioned single nucleotide polymorphism, the present inventors have also added an insertion or deletion (non-translated region, cytochrome oxidase II / tRNA ^{Lys) to} the nucleotide sequence of 9 bases between the positions 8272 and 8289. Inner part of) was found.

Furthermore, among mtDNA polymorphisms, positions 3010 and 5178
It was found that single nucleotide polymorphisms at positions 10 and 10398 are useful for personal identification. The frequency of these polymorphisms in Japanese is about 30% to about 60%, as shown in Table 5. By combining these polymorphisms, about 3% to about 38% of Japanese
Can be divided into haplogroups of frequency.

In order to detect single nucleotide polymorphisms, in addition to the multiplex APLP method shown in the examples, known single nucleotide polymorphism analysis methods (for example, direct sequence, hybridization method using DNA microarray, Invader method) , T
aqman probe method, Masscode tag method, allele specific
PCR method, RFLP method, various solid-phase fluorescence mini-sequencing method, immunization sequence-specific oligonucleotide probe assay method, etc. ), And various single nucleotide polymorphism analysis methods developed in the future can be used. 5'as an oligonucleotide for detecting the polymorphism at position 4386
It is preferable that the terminal side has a base sequence complementary to the base sequence of mtDNA, the 3′-terminal side has position 4386, and the base is cytosine (C) or guanine (G). This is because such an oligonucleotide specifically hybridizes to the single nucleotide polymorphism found at position 4386 that we found. Since mtDNA is a double strand and may hybridize to any single strand, the 3'end of the oligonucleotide is C or G.
Any of Here, the oligonucleotide is
It means that several to several tens of nucleotides are linked. Further, one or more bases that are non-complementary to the base sequence of mtDNA can be attached to the 5'end.

Further, as an oligonucleotide for detecting the polymorphism at position 8794, a base sequence complementary to the base sequence of mtDNA is contained at the 5'end side and a 3'end side is provided.
It preferably has position 8794 and the base is thymine (T) or adenine (A). This is because such an oligonucleotide specifically hybridizes to the single nucleotide polymorphism at position 8794 we found. In order to detect insertion or deletion of nucleotide sequences, various electrophoresis methods (agarose gel electrophoresis, acrylamide gel electrophoresis etc.), mass spectrometry (laser TOF-MS method etc.)
Etc. can be used.

In order to detect insertion or deletion of 9 bases between positions 8272 and 8289, a pair of oligonucleotide primer sets facing in opposite directions across the site can be used. By carrying out an amplification reaction using such a primer set, a nucleotide sequence containing positions 8272 to 8289 is synthesized, so if the amplification product is inspected (for example, length, molecular weight, sequence itself, etc.) , Insertion or deletion can be detected. The position and length of such a primer set can be appropriately selected by those skilled in the art. In particular, when the amplification product is separated and confirmed by electrophoresis, the length of the amplification product is generally several tens of bp though it depends on the concentration of gel and the migration time.
It is preferably several hundred bp, more preferably about 40 bp to about 300 bp. 4386 or as above
A novel mtDNA polymorphism is detected by constructing a gene detection kit containing an oligonucleotide for a single nucleotide polymorphism at position 8794 and a polymorphism related to insertion or deletion of 9 nucleotides between positions 8272 and 8289. be able to.

Among these detection methods, particularly when the APLP method is used, the single nucleotide polymorphism and the insertion or deletion of the nucleotide sequence can be detected by a method such as electrophoresis or mass spectrometry. To detect single nucleotide polymorphisms, of the two allele-specific primers, place a sequence that is different from the 5'end of the other primer at the 5'end of the other primer. Amplification products obtained from the two alleles can be amplified as two different products. Then, by distinguishing the two products after the amplification reaction, it is possible to detect which allele is present.

"Two products that are different from each other" are as follows:
There can be two types, different lengths or different 5'end sequences of the primers. Of these, the method of varying the length is a simpler detection method because only the size needs to be confirmed by, for example, electrophoresis after the amplification reaction. Therefore, as a preferred detection method when the lengths are different, in order to detect a single nucleotide polymorphism, two allele-specific primers are used.
Bases complementary to each polymorphic site are placed at the 3'end, and different end sequences are placed at the 5'end so that the lengths of the two products amplified from the alleles are different. I'll do it. As the primer on the other end side, one common to alleles is used. As a method of making the 5'end sequences of the primers different, there is a method of labeling different fluorescent labels on the 5'ends of the two primers.

In addition, in order to detect the insertion or deletion of a base sequence using the APLP method, a pair of primers are provided at positions sandwiching the insertion / deletion site to carry out an amplification reaction. The amplification product having a length different from that of the amplification product of When using the APLP method, the complementary length of the allele-specific primer, the non-complementary length provided at the 5'end, the length of the common primer placed on the opposite side of the amplification product, and the amplification product The length can be set arbitrarily. When the amplification products are separated by the electrophoresis method, the length should be such that they can be separated well depending on the concentration of the electrophoretic gel. Furthermore, when a plurality of polymorphisms are amplified in a single amplification reaction and the amplification products are separated by electrophoresis, (in addition to varying the length of the products amplified from a pair of alleles, ,) The lengths of amplification products amplified from the respective polymorphisms are set to be different from each other.

As described above, when a plurality of polymorphisms are amplified in a single amplification reaction using the present invention, it is possible to design a very large number of types of primers. For example, 8794 5'-ACCTCGGACTTCTG to detect position polymorphisms
CCTC-3 '(SEQ ID NO: 18794C), 5'-ATTGGACCTCGGACT
CTTGCCTT-3 '(SEQ ID NO: 8794T), and 5'-ACAGCGA
A primer set of AAGCCTATAATCAC-3 '(SEQ ID NO: 3: 8794R) can be used. In addition, to detect a polymorphism in the insertion or deletion of 9 bases at positions 8272-8289,
For example, a primer set of 5'-ACAGGAGCATACCACAGTTTC-3 '(SEQ ID NO: 4: 9BPF) and 5'-CTAAGTTAGCTTTACAGTGGG-3' (SEQ ID NO: 5: 9BPR) can be used.

In order to detect the polymorphism at position 4386, for example, 5'-ATCTCCGTGCCACCTAC-3 '(SEQ ID NO: 6: 4386
C), 5'-AGTTGTTCTCCGTGCCAGCTAT-3 '(SEQ ID NO: 7: 4)
386T) and 5'-ATTGCAACATTTTCGGGGTATG-3 '(SEQ ID NO: 8386R) can be used. Also, to detect the polymorphism at position 3010, for example, 5'-
ATTGGATCAGGACTTCCCG-3 '(SEQ ID NO: 9: 3010G), 5'-
GCTACATGGATCAGGACAACCCA-3 '(SEQ ID NO: 10: 3010
A), and 5'-GATCACGTAGGACTTTAATCG-3 '(SEQ ID NO: 1)
The primer set of 1: 3010R) can be used.

In order to detect the polymorphism at position 5178, for example, 5'-GTCGCACCTGAAGCAAGC-3 '(SEQ ID NO: 12: 5178
C), 5'-TGATCAACGCACCTGAAACAAGA-3 '(SEQ ID NO: 1)
3: 5178A), and 5'-ATTGCAAAAAGCAGGTTAGCG-3 '(SEQ ID NO: 14: 5178R) can be used. Further, for detecting the polymorphism at the 10398th position, for example, 5'-CTACAAGAAGGATTAGACTGRG-3 '(SEQ ID NO: 15:
10398G), 5'-ATCACTCTACAAAGAGGATTAGACTGAA-3 '(SEQ ID NO: 16: 10398A), and 5'-CTAGAAGTGAGATGGTAAAT.
A primer set of GC-3 '(SEQ ID NO: 17: 10398R) can be used.

In this example, two kinds of amplification products of 75 bp and 79 bp for the single nucleotide polymorphism at position 3010 and two kinds of amplification products of 85 bp and 90 bp for single nucleotide polymorphism at position 4386 are 5178 and 5178, respectively.
Two types of amplification products of 96 bp and 101 bp for the single nucleotide polymorphism at the position, two types of amplification products of 107 bp and 112 bp for the single nucleotide polymorphism at position 8794, and 122 bp for the insertion or deletion polymorphism of 9 bp And 131 bp, and two kinds of amplification products of 145 bp and 151 bp for the single nucleotide polymorphism at position 10398 are amplified by the amplification reaction, respectively.

Typing based on mtDNA polymorphisms by the multiplex APLP method using the above six primer sets resulted in 2471 individuals from 20 groups consisting of Japanese, Korean, Chinese and German. Mutants of mtDNA could be classified into 18 haplotypes (A1-A3, B1-B8 and C1-C7).

In the case where further subdivision is required for individual identification, single nucleotide polymorphisms that can effectively subdivide the type are selected and analyzed according to the type once categorized.
You can effectively target individuals. If necessary, add the sequence of the D-LOOP region (base sequence determination),
The accuracy of personal identification can be improved. Such sequence methods include the Sanger method, the Maxam Gilbert method,
Pyrosequence method (Ronaghi, M. et al: Science 281, 3
63-365 (1998): A sequencing method based on real-t.
ime pyrophosphate) and the like can be used, but the invention is not limited thereto.

The nucleic acid amplification reaction may be performed in advance before the inspection of the nucleic acid sequence. As such a nucleic acid amplification reaction, a PCR method (Saiki, RK et al: Science 230, 1350-
1354 (1985): Enzymatic amplification of beta-globin
genomic sequences and restriction site analysis f
or diagnosis of sickle cell anemia), LAMP method (Noto
mi, T. et al: Nucleic Acids Res, 28 (12), e63 (2000): L
oop-mediated isothermal amplification of DNA), IC
AN method (http://www.takara.co.jp/news/2000/07-09/00-i
-019.htm), TMA method (Jonas, V. et al: J Clin Microbi
ol 31, 2410-2416 (1993): Detection and identificat
ion of Mycobacterium tuberculosis directly from sp
utum sediments by amplification of rRNA) and the like can be used, but the present invention is not limited thereto.
In addition, during the nucleic acid amplification reaction, an Ampdirect reagent (Nishimura, N. et al:
Ann Clin Biochem 37, 674-680 (2000): Direct PCR fr
om whole blood without DNA isolation) can be used.

According to the present invention, it is possible to identify an individual with respect to a biogenic substance mixed as a foreign substance in an industrial production process. The term “industrial production process” refers to equipment, devices, equipment, fixtures, parts, etc. that are related to the manufacture of products in which biological substances may be mixed as foreign matter in the final product. I mean. Examples of final products include foods, pharmaceuticals, and semiconductor products. In such products, the quality of the product may be significantly deteriorated due to the inclusion of substances of biological origin.Therefore, personal identification is important for investigating the cause or clarifying the responsibility, especially personal identification. It becomes important to do. "Biological substance" means skin, nails, hair (for example, hair, eyelashes, eyebrows, whiskers, pubic hair, axilla, etc.), or body fluid (for example, blood, tears, nasal discharge, saliva, semen, vaginal secretions, etc.) However, the substance is not limited thereto, and any substance containing mtDNA therein may be used.
Personal identification means selecting a smaller number of humans from multiple humans (preferably identifying one human), or that one human is not a human of a certain type. It means both sorting.

Since mtDNA is used in the present invention, personal identification can be performed with a high sensitivity, which is several hundred times higher than that of nuclear DNA. In particular, 3010, 4386, 5178, 8794, or
By using the single nucleotide polymorphism at the 10398th position or the insertion / deletion polymorphism of 9 nucleotides at the 8272-8289th position, Japanese can be effectively separated. Therefore, if polymorphism analysis including these polymorphic sites is used in the first stage of individual identification, it is possible to approach individual identification with less labor. Further, for the above-mentioned 6 polymorphisms, since it is possible to carry out the multiplex APLP method in one PCR reaction system, it is possible to simultaneously detect the amplification product by electrophoresis or mass spectrometry once. Therefore, compared to other methods, it is possible to perform low-cost, high-speed and large-scale personal identification. It should be noted that other personal identification methods can be used in combination if necessary, but the number of persons to be applied can be effectively narrowed down by performing the polymorphism analysis of the present invention in advance, so the labor is significantly reduced. To be done.

As described above, according to the present invention, mtDNA haplotypes are defined and classified to enable high-sensitivity, inexpensive, and quick personal identification. The effects of this feature on each field are as follows. First, in the field of forensic medicine, it becomes easy to distinguish from a very small amount of evidence and evidence that has been exposed to a harsh environment, resulting in a dramatic increase in appraisal ability. This is because the copy number of mitochondrial DNA is several hundred times higher than that of nuclear DNA, and because mitochondrial DNA is circular DNA, it is more stable than nuclear DNA.

In the paternity test, the mitochondrial DNA is maternally inherited, which is useful for the maternal test. Due to its high sensitivity, this method is extremely effective when only a small amount of biological sample of either mother or child is obtained (remaining items, etc.). In epidemiological surveys, by investigating the correlation between each haplotype and disease, there is a possibility that it can be applied to the prediction of disease and lifespan (Tanaka, M., Gong, JS, Zhang, J., Yoneda,
M., Yagi, K., Lancet 1998, 351,185-186 .: Tanaka,
M., Gong, J., Zhang, J., Yamada, Y., Yagi, K., Mec
h. Ageing Dev. 2000, 116, 65-76.). Furthermore, when a certain correlation is found between a specific haplotype and a specific disease in an epidemiological survey, the etiology of the disease can be elucidated, which may lead to diagnosis / treatment of the disease.

In the archaeological survey, by examining the existence probability of haplotypes in each region of the world, useful information on anthropology such as the occurrence and migration of ethnic groups can be obtained (Kazuo U
metsu, Masashi Tanaka, et al: Electrophoresis 22,
3533-3538 (2001): Multiplex amplified product-leng
th polymorphism analysis for rapid detection of hum
an mitochondrial DNA variations, Max Ingman, et a
l: Nature 408, 7 (2000): Mitochondrial genome vari
ation and the origin of modern humans). This is mtDN
This is because A is maternally inherited and the sequence is highly conserved between mother and child.

Further, in product manufacturing process control, it is effective for appraisal of biogenic substances (hair, body fluid, etc.) mixed as foreign substances. If the mtDNA haplotypes of individuals who have the opportunity to contaminate these foreign substances are investigated in advance, when those foreign substances are found in the product, the causative process is identified by matching the haplotypes of the foreign substances. Provides significant evidence. If the responsibility is clarified by this, it is possible to improve the process for reducing defective products including foreign substances and to take appropriate measures (division of liability for compensation, prizes, etc.) by the person in charge of manufacturing. Therefore, the present invention contributes to the improvement of the manufacturing process, and functions beneficially in stably supplying higher quality products.

Further, when a complaint that a foreign substance derived from a living body is mixed in a certain product is generated, if the haplotype of the user of the product is also verified, it is highly possible that the proper complaint processing can be performed. Most of the body-derived foreign substances in the product manufacturing process are body hair such as hair, but the amount of these foreign substances is generally small, and in the manufacturing process of food products, it is possible to undergo severe conditions for DNA such as heat treatment. Therefore, the features of the present invention using mtDNA are particularly effective. Further, the speed of personal identification according to the present invention,
Economic efficiency is a suitable factor for product manufacturing process control.

[0034]

BEST MODE FOR CARRYING OUT THE INVENTION Next, one embodiment of the present invention will be described in detail with reference to the drawings, but the technical scope of the present invention is not limited to the following embodiments, and The present invention can be implemented with various modifications without changing the gist. Moreover, the technical scope of the present invention extends to an equivalent range.

<Selection of polymorphic site> The present inventors
We developed a multiplex APLP method using an allele-specific primer set as a good method to detect several polymorphic sites in Escherichia coli simultaneously. Table 1 shows the six mtDNA polymorphic sites used in this study.

[0036]

[Table 1]

These detection sites appeared to be important mutations in East Asians. Five of the six polymorphic sites (nt3010, nt4386, nt5178, nt8794, and nt10398) are translation regions, and the other one is a non-translation region between cytochrome oxidase II / lysine tRNA (cytochrome oxidase II). / tRNA ^Lys ) (9bp) 9bp repeat mutation (Tanaka, M., Gong, JS, Zha
ng, J., Yoneda, M., Yagi, K., Lancet 1998, 351, 18
5-186 .: Tanaka, M., Gong, J., Zhang, J., Yamada,
Y., Yagi, K., Mech. Ageing Dev. 2000, 116, 65-7
6 .: Ikebe, S., Tanaka, M., Ozawa, T., Mol. Brain R
es. 1995 28,281-295.).

<Preparation of DNA template> As shown in FIG. 1, 247 from healthy individuals among East Asian residents.
One blood sample (including 16 Japanese, 2 Korean, 1 Chinese, and a German (Munich) who previously analyzed alcohol dehydrogenase 2 and aldehyde dehydrogenase 2 (Aoshim
a, T., Umetsu, K., Yuasa, I., Watanabe, T., Suzuki,
T., Electrophoresis 1998, 19, 659-660.). ) Got. Genomic DNA was prepared from each blood sample according to standard protocols.

<Amplification reaction of mtDNA> APLP analysis (Watanabe,
G., Umetsu, K., Yuasa, I., Suzuki, T., Hum. Gene
t.1997, 99,34-37.), the oligonucleotide primer set used was mtDNA (Anderson, S., Barkier, A.
T., Barrell, BG, DeBruijin, MHL, Coulson,
AR, Drouin, J., Eperon, IC, Neirlich, DP,
Roe, BA, Sanger, F., Schreier, PH, Smith,
AJH, Staden, R., Young, IG, Nature 1981, 2
90, 457-465.) And designed at the concentrations shown in Table 2. PCR reaction is 2ng to 20ng Genome D
NA, 1x PCR buffer, 120 μM dNTPs each, appropriate concentration of primers (see Table 2), and 0.5 units HotS
DNA Thermal Cycler 9700 in 20 μL total solution containing tar Taq polymerase (Qiagen, Hilden, Germany)
(Applied Biosystems, Foster City, CA, USA). In Table 2, noncomplementary bases with respect to the base sequence of mtDNA are shown in lowercase letters.

[0040]

[Table 2]

Following the first 15 minutes of denaturation reaction at 95 ° C.,
33 amplification cycles (94 ° C for 10 seconds, 52 ° C for 10 seconds,
One cycle was 5 seconds at 72 ° C. ) And finally 72
An extension reaction was performed at 1 ° C for 1 minute. When the amplification reaction at any of the six polymorphic sites failed, a PCR reaction was performed using the primer set at that site.

<Separation of Amplification Products> Add 3 μL of PCR product solution to 3
6.5 cm polyacrylamide gel (10% T, 5% C) using 75 mM Tris-HCl buffer (pH 8.9) (25 mM Tris, 192 mM glycine; pH 8.3 was used as a running buffer).
Were separated by electrophoresis. The DNA band is SYBR Gold (Molecular Probes, Rockland, ME, US
After staining with A), it was detected by fluorography.

<Direct Sequence of PCR Product>
PCR products from the 9 bp region that differed in mobility were ABI P
rism 310 Sequencer and BigDye Terminator Cycle S
Direct sequencing was performed using the ezuencing FS kit (Applied Biosystems).

<Phylogenetic analysis> Using the haplotype frequencies in each group, the Cavalli-Sforza's chord distance was calculated (Cavalli-Sforza, LL, Edwards, AWF, Am. J. .
Hum. Genet. 1967, 19, 233-257.). The distance matrix is classified into clusters by the neighborhood joining method (Sait
ou, N., Nei, M., Mol. Biol. Evol. 1987, 4, 406-42
Five). These analyzes are based on the computer program PHYLI
P 3.5c (Felsenstein, J., University of Washington,
WA, USA) and TreeView (Page, RDM, Comput. Appl.
Biosci. 1996, 12, 357-358.).

Results and Discussion In order to construct a good system for determining the haplotype of large numbers of DNA samples, we used 6 primer sets to amplify products ranging in size from 75 bp to 151 bp. Were prepared and mixed in one tube. The APLP method is based on 50 m previously analyzed by DNA sequence.
It was evaluated by using the tDNA sample as a test. The results of the APLP method and the sequence analysis were in agreement. AP
The LP method is a very simple method because PCR amplification is performed in a single tube and only SYBR Gold staining and PAGE under non-denaturing are used. Multiplex human mtDNA haplotype
The band pattern after the PLP method and the electrophoretic separation by polyacrylamide gel is shown in FIG. Each of the alleles could be accurately typed by comparison with PCR products with known point mutations.

MtDNA polymorphisms are widely used as maternal genetic markers to explain human evolution and migration. mtDN was analyzed by restriction fragment length polymorphism (RFLP) analysis using two mutation sites, nts5178 and nts10398.
It has been reported that A can be classified into three types (AC), and that in Japanese, the frequency of haplogroup A is significantly higher in centenarians than in younger individuals (Tanaka, M. , Gong, JS, Zhang, J., Yoned
a, M., Yagi, K., Lancet 1998, 351, 185-186 .: Tana
ka, M., Gong, J., Zhang, J., Yamada, Y., Yagi, K.,
Mech. Ageing Dev. 2000, 116, 65-76.). In the current study, these three haplogroups (A, B, and C) added four additional mutation sites,
As shown in Table 3, 18 haplotypes (A1-A3, B1-B
8 and C1-C7).

[0047]

[Table 3]

The numerical values shown in the 9bp ^a) column of Table 3 are: 1 for deletion of 9-bp sequence, 2 for duplication of 9-bp sequence, 3
Shows triplet of 9-bp sequence, 2a shows insertion of 4bp from duplication (heterogeneity), and 2b shows deletion of 3bp from duplication (heterogeneity). The frequencies of these haplotypes in Asian and German residents are shown in Tables 4 and 5. Table 4 shows the number of people who belong to each haplotype.
Shows the proportion of Japanese and foreign nationals belonging to each haplotype, and Table 6 shows the proportion of Japanese and foreign nationals belonging to each haplogroup.

[0049]

[Table 4]

[0050]

[Table 5]

[0051]

[Table 6]

99% or more of mtDNA has 10 mtDNA haplotypes (A1, A2, B1, B2, B3, B5, C1, C2, C3, and C5).
Was composed of. A phylogenetic network showing the evolutionary relationship of mtDNA haplotypes is shown in FIG. Haplotypes B1 (presumed ancestral haplotypes) and C1 were distributed in all populations. However, the haplotype
A1, A2, B2, B3 and C2 were mostly restricted to Asians, and B5 and C5 were almost exclusively identified to Germans only. Haplogroup A was all confirmed by Asians but not by Germans. Haplotypes A1 and A2 were widely distributed in Asian groups.

The frequency of haplotype A2 in Japanese is
Spreading from 26% to 44%, it was the least common (17%) among Chinese. Conversely, Chinese people have A1 frequency (1
2%) showed the highest value. Haplotype B2 is
It was frequently observed in all Japanese, especially Okinawa (21.7%), but was low in two Koreans (3.1%), Chinese and German (0%). It was speculated that haplotype B2 first occurred in prehistoric times in southern Japan, increased in frequency due to genetic drift, and then spread to northern Japan and South Korea.

The deletion frequency of 9 bp (CCCCCTCTA) in Japanese varied from 19% in Tajimi to 7% in Tsushima. This value corresponds to the previously reported value (Harihara, S., Hirai,
M., Suutou, Y., Shimizu, K., Omoto, K., Hum. Biol.
1992, 64, 161-166; Horai, S., Murayama, K., Hayasa
ka, K., Matsubayashi, S., Hattori, Y., Fucharoen,
G., Harihara, S., Park, KS, Omoto, K., Pan, I.
H., Am. J. Hum. Genet. 1996, 59, 579-590.). 9bp deletion is a haplotype
The characteristics of individuals belonging to B3 and C3 are shown. Furthermore, for five individuals with irregular bands, the cause of the irregularity was clarified by a sequence. Three of them had triplicated 9 bp regions.

In the other two, self-production of heteroduplexes was observed. For one person, a heterologous (heteropla) containing an insertion of approximately 4 bp.
smic) (CCCCCCCCCCCTACCCCCTCTA: SEQ ID NO: 1)
8), the other one was heterogeneous (CCCCCCCCCCCTCTCTA: SEQ ID NO: 19) containing a deletion of about 3 bp. Similar changes have been reported previously (Wrischnik,
LA, Higuchi, RG, Stoneking, M., Erlich, H.
A., Arnheim, N., Wilson, AC, Nucleic AcidsRes.
1987, 15, 529-542; Watkins, WS, Bamshad, M., Di
xon, ME, Bhaskara, RB, Naidu, JM, Reddy,
PG, Prasad, BVR, Das, PK, Perry, PC,
Gai, PB, Bhanu, A., Kusuma, YS, Lum, JK,
Fischer, P., Jorde, LB, Am. J. Phys. Anthropol.
1999, 109, 1467-158; Finnila, S., Majamaa, K., J.
Hum. Genet. 2001, 46, 64-69.). We have COII-tRNA
^It was confirmed that the chain length mutation of the 9 bp repeating sequence in the intermediate region of ^Lys occurred several times.

Code distance of Cavalli Sforza (Ca
Fig. 4 shows a phylogenetic tree constructed by using the valli-Sforza's chord distance) and the neighborhood joining method. This phylogenetic tree reveals that Japanese residents have strong genetic relatives with the East Asian population analyzed this time. Clusters in this rootless phylogenetic network were observed in Germany, northern China, South Korea, and Japan. The relationship between Germany and Okinawa was farthest.
Interestingly enough, the phylogenetic tree obtained from this simple experiment showed that conventional genetic markers (Omoto, K., Saito
u, N., Am. J. Phys. Anthropol. 1997, 102, 437-44
6.) and untranslated region of mtDNA (Horai, S., Murayama, K.,
Hayasaka, K., Matsubayashi, S., Hattori, Y., Fuch
aroen, G., Harihara, S., Park, KS, Omoto, K., P
an, IH, Am. J. Hum. Genet. 1996, 59, 579-590.)
It resembled a phylogenetic tree obtained from huge data from.

<Conclusion> Before using the APLP method, it is necessary to determine the specificity, optimal concentration, and annealing temperature of the oligonucleotide primer due to PCR conditions. Once these conditions are optimized, reproducible results are obtained. The PCR-APLP method is a rapid, easy, and sensitive method for examining known point mutation sites in mtDNA. Moreover, this method only requires PCR and gel electrophoresis, and does not require expensive fluorescent or biotin-labeled primers, restriction enzymes, and expensive equipment. Thus, since the PCR-APLP method can be carried out in one PCR reaction system,
Compared with other methods, the amplified product can be analyzed simultaneously by electrophoresis or mass spectrometry.
It was confirmed that it is a low-cost, high-speed, and mass-identification method.

[0058]

[Sequence list]                                SEQUENCE LISTING        <110> Masashi, TANAKA <120> Multiplex amplified product-length polymorphism       analysis for rapid detection of human mitochondrial DNA       variations <130> APLP analysis of human mtDNA <140> P02013TAN <141> 2002-03-22 <160> 19 <170> PatentIn Ver. 2.1 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 8794C <400> 1 acctcggact tctgcctc 18    <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 8794T <400> 2 attggacctc ggactcttgc ctt 23    <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 8794R <400> 3 acagcgaaag cctataatca c 21    <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 9BPF <400> 4 acaggagcat accacagttt c 21    <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 9BPR <400> 5 ctaagttagc tttacagtgg g 21    <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 4386C <400> 6 atctccgtgc cacctac 17    <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 4386T <400> 7 agttgttctc cgtgccagct at 22    <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 4386R <400> 8 attgcaacat tttcggggta tg 22    <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 3010G <400> 9 attggatcag gacttcccg 19    <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 3010A <400> 10 gctacatgga tcaggacaac cca 23    <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 3010R <400> 11 gatcacgtag gactttaatc g 21    <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 5178C <400> 12 gtcgcacctg aagcaagc 18    <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 5178A <400> 13 tgatcaacgc acctgaaaca aga 23    <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 5178R <400> 14 attgcaaaaa gcaggttagc g 21    <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 10398G <400> 15 ctacaagaag gattagactg rg 22    <210> 16 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 10398A <400> 16 atcactctac aaagaggatt agactgaa 28    <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 10398R <400> 17 ctagaagtga gatggtaaat gc 22    <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 4bp-ins <400> 18 cccccccccc ctaccccctc ta 22    <210> 19 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 3bp-del <400> 19 cccccccccc ctcta 15

[Brief description of drawings]

FIG. 1 is a map showing the geographical distribution of East Asian residents when mtDNA mutations are detected. Note that small islands are indicated by white circles.

FIG. 2 is a photograph after electrophoresis showing the APLP band pattern when the mtDNA haplotype was confirmed. Lane 1 is A1, Lane 2 is A2, Lane 3 is B1, Lane 4
Is B2, lane 5 is B3, lane 6 is C1, lane 7 is
C2, lane 8 are C3, and lane M is a 10-bp DNA ladder marker.

FIG. 3 is a diagram showing a phylogenetic tree network of mtDNA. The most probable root is the haplotype
B1, but Cambridge control sequence (Anderson, S., Ba
rkier, AT, Barrell, BG, DeBruijin, MHL,
Coulson, AR, Drouin, J., Eperon, IC, Neirlic
h, DP, Roe, BA, Sanger, F., Schreier, PH,
Smith, AJH, Staden, R., Young, IG, Nature
1981290, 457-465.) Corresponds to haplotype C1. The size of each circle is approximately proportional to the frequency of occurrence in Japanese. In addition, the dotted line in the figure is a connection of haplotypes of unrelated ones.

FIG. 4 is a diagram phylogenetically showing the neighboring zygosity trees of 20 inhabitants based on the Cavalli-Sforza'schord genetic distance of Cavalli-Sforza. The distance is proportional to the length of each branch.

Continued front page    (72) Inventor Masatsugu Tanaka             1-18 Koyo 1-chome, Chikusa-ku, Nagoya, Aichi (72) Inventor Kazuo Umezu             2-16 Iida, Yamagata City, Yamagata Prefecture F-term (reference) 4B024 AA11 CA01 HA12                 4B063 QA12 QA18 QA19 QA20 QQ42                       QR08 QR32 QR42 QR55 QR62                       QS25 QS34 QX01

Claims (11)

[Claims] 1. A method for detecting a gene, which comprises detecting at least one single nucleotide polymorphism at position 4386 or position 8794 in the nucleotide sequence of human mitochondrial DNA. 2. A method for detecting a gene, which comprises detecting a polymorphism due to insertion or deletion in a nucleotide sequence of 9 bases from 8272 to 8289 in the human mitochondrial DNA. 3. A method for detecting a gene, which comprises detecting at least one single nucleotide polymorphism at position 3010, position 5178, or position 10398 in a human mitochondrial DNA base sequence. 4. A method for identifying an individual using a polymorphism in human mitochondrial DNA, characterized by identifying an individual based on the method for detecting a gene according to any one of claims 1 to 3. 5. Among the nucleotide sequences of human mitochondrial DNA, 3010th position, 4386th position, 5178th position and 8794th position.
Position, or the single nucleotide polymorphism at the 10398th position, or the polymorphism due to insertion or deletion with respect to the nucleotide sequence of 9 bases from the 8272nd position to the 8289th position, nucleic acid amplification is performed in the same reaction system, A method for detecting a gene, which comprises detecting a polymorphism in a plurality of human mitochondrial DNAs by detecting. 6. The human according to claim 4 or 5, wherein an individual who is a source of the foreign substance is identified with respect to a biological substance mixed as a foreign substance in an industrial production process. An individual identification method using a polymorphism in mitochondrial DNA. 7. The human mitochondrial DNA polymorphism according to claim 4 or 5, wherein the individual is identified in personal identification, paternity examination, epidemiological survey, or archaeological survey in the field of forensic medicine. Personal identification method using. 8. A human mitochondrial D containing a nucleotide sequence complementary to the nucleotide sequence of human mitochondrial DNA.
An oligonucleotide having the 4386-position of NA at the 3'end, wherein the base corresponding to the 4386-position is cytosine or guanine. 9. A human mitochondrial D containing a base sequence complementary to the base sequence of human mitochondrial DNA.
An oligonucleotide having the 8794th position of NA at the 3'end, wherein the base corresponding to the 8794th position is thymine or adenine. 10. A pair of oligonucleotide primer sets directed in opposite directions with a base sequence between at least positions 8272 and 8289 in the base sequence of human mitochondrial DNA interposed therebetween. 11. A gene detection kit comprising the oligonucleotide according to any one of claims 8 to 10.