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Kim, Shin, Kim, Park, Yang, and Lee: Rapid and Simple Screening of Mitochondrial DNA in Koreans by the Analysis of Highly Variable Control Region SNPs
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Korean J Leg Med 2013;37(4):183-190.

Published online: 17 January 2013

DOI: https://doi.org/10.7580/kjlm.2013.37.4.183

Rapid and Simple Screening of Mitochondrial DNA in Koreans by the Analysis of Highly Variable Control Region SNPs

Eun Hye Kim1, Kyoung-Jin Shin1, Hye Yeon Kim2, Su Jeong Park2, Woo Ick Yang1, Hwan Young Lee1

1Department of Forensic Medicine and Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea

2DNA Forensic Division, Supreme Prosecutors’ Office, Seoul, Korea

본 연구과제는 2011년도 대검찰청의‘영 상녹화 등 과학수사 연구개발비(1333- 302-260-00)’의 지원을 받아 수행되었 습니다. 책임저자 : 이환영 (120-752) 서울시 서대문구 연세로 50 연세대학교 의과대학 법의학과 전화 : +82-2-2228-2482 FAX : +82-2-362-0860 E-mail : hylee192@yuhs.ac

Received 23 October 2013       Revised 1 November 2013       Accepted 25 November 2013

Copyright © 2013 The Korean Society for Legal Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Human mitochondrial DNA (mtDNA) is generally used to identify highly degraded forensic samples, particularly when the extracted DNA is not sufficient for nuclear DNA analysis. However, direct sequencing, the most widely used mtDNA analysis method, is laborious and time-consuming, and precludes the simultaneous analysis of many samples. Here, we describe a rapid and simple screening method for mtDNA analysis in Koreans using single base extension (SBE) methods. Sixteen highly polymorphic mtDNA SNPs from the control region were selected, and a multiplex SBE system was constructed to analyze them. Because the developed system consists of two duplex PCRs, which produce small amplicons with fewer than 270 bp, it works well with highly degraded samples such as old skeletal remains. Using this multiplex SBE system, 145 different haplotypes were expected to be observed from 593 unrelated Koreans. Seventy-three haplotypes were expected to be observed only once, and the most frequent haplotype was expected to occur 80 times. Since the mean number of pairwise differences was estimated to be 4.55, the developed system could be useful to exclude samples that do not match evidence and reference samples. Therefore, the multiplex SBE system used in this study will be a useful tool to analyze many samples simultaneously and to efficiently screen out non-matching mtDNA sequences in forensic casework.

Keywords: Key Words, Mitochondria DNA, Control region, Single nucleotide polymorphism, Single base extension, Koreans

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Fig. 1.
Multiplex SBE genotyping of various Korean mtDNA samples (a-c). Results for multiplex SBE I were shown on the left, and the results for multiplex SBE II were shown on the right.
kjlm-37-183f1.tif
Fig. 2.
Electropherograms of multiplex SBE using degraded DNA obtained from old skeletal remains (a-d). Results for multiplex SBE I were shown on the left, and the results for multiplex SBE II were shown on the right.
kjlm-37-183f2.tif
Table 1.
Final Concentrations of Primer Sets for Each PCR System∗
System Amplicon Primer Sequences (5’→3’) Amplicon size (bp) Conc. (uM)
Duplex I P11 F15989 CCCAAAGCTAAGATTCTAAT 267 0.8
R16255 CTTTGGAGTTGCAGTTGATG    
P21 F015 CACCCTATTAACCACTCACG 247 0.4
R261 GCTGTGCAGACATTCAATTGTT    
Duplex II P12 F16159-15m (tat)5CATAAAAACCCAATCCACAT 267 0.3
R16410m GAGGATGGTGGTCAAGGGA    
P22 F155m TATTTATCGCACCTACGTTCA 235 1.0
R389 CTGGTTAGGCTGGTGTTAGG    

∗ Monoplex PCR using F15989-R389 produces a 970 bp amplicon; Duplex PCR using F15989-R16410m and F015-R389 produces two amplicons with 422 bp and 375 bp sizes.

Table 2.
SBE Primer Sequences and Final Concentrations of Multiplex SBE System
SBE system Primer Sequences (5’→3’) Conc. (uM)
I 16129F21-21 CAGCCACCATGAATATTGTAC 0.13
16129-03CF20-21† tAGCCACCATGAATATTGCAC 0.10
16129-18TF21-21† CAGTCACCATGAATATTGTAC 0.50
16172F25-30 (t)5CCACCTGTAGTACATAAAAACCCAA 0.50
16182-10YF26-39† (t)13GTACATAAAAACCCAAYCCACATCAA 2.00
16183-11YF28m-46† (t)17aAGTACATAAAAACCCAAYCCACATCAAM 3.20
16189R20-50 (t)29aACTTGCTTGTAAGCATGGGG 0.25
146F23-58 (t)35AGTATCTGTCTTTGATTCCTGCC 0.20
150F21-65 (t)44TGTCTTTGATTCCTGCCTCAT 0.30
150-04CF19-65 (t)46TCTTTGATTCCTGCCCCAT 0.45
152R24-72 (t)48TTGAACGTAGGTGCGATAAATAAT 0.35
II 16217F23-23 ATGCTTACAAGCAAGTACAGCAA 0.40
16223-06YF22-32† (t)10CAAGCAAGTACAGCAAYCAACC 0.60
16261R24-36 (t)12TTTGTTGGTATCCTAGTGGGTGAG 0.20
16261+05AR24-36∗ (t)12TTTGTTGGTATCCTAGTGGATGAG 0.70
16261+13TR24-36∗ (t)12TTTGTTGGTATTCTAGTGGGTGAG 0.60
16261+17AR24-36∗ (t)12TTTGTTGATATCCTAGTGGGTGAG 0.40
16311+08YR26-45† (t)19GCTATGTACGGTAAATGGYTTTATGT 0.80
16311+14GR26-45∗ (t)19GCTATGTACGGTGAATGGCTTTATGT 0.60
16319R25-52 (t)26aGTAATGTGCTATGTACGGTAAATGG 0.80
16319+06GR25-52∗ (t)26aGTAATGTGCTATGTACGGTGAATGG 1.00
16362R17-59 (t)42GGGGGTCATCCATGGGG 0.35
199F27-67 (t)40TTCAATATTACAGGCGAACATACTTAC 0.20
199-04CF27-67∗ (t)40TTCAATATTACAGGCGAACATACCTAC 0.10
199-05TF27-67∗ (t)40TTCAATATTACAGGCGAACATATTTAC 0.50
199-10GF27-67∗ (t)40TTCAATATTACAGGCGAGCATACTTAC 0.12
249R18-73 (t)55AAGCGGCTGTGCAGACAT 0.15

∗ Additional SBE primers for samples with 3% or more polymorphic sites

† Additional SBE primers for 16111 or 16126 mutation with 2% polymorphism frequency

† Degenerate primers were used to amplify samples which have target SNPs in another SNP’ s primer binding site (Degeneracy Y indicated including nucleotide C or T, Degeneracy M indicated including nucleotide A or C)

Table 3.
Statistical Evaluation of 16 Control Region SNPs in the Present Study and 15 Coding Region SNPs of the Previous Report using 593 Unrelated Koreans’ mtDNA Sequences
  Number of haplotypes Discrimination capacity (%) Haplotype diversity Mean number of pairwise differences
16 control region SNPs 145 24.45 0.9691 4.55
15 coding region SNPs∗ 020 03.37 0.8855 2.74
31 SNPs1 174 29.34 0.9836 7.62

∗ 15 coding region SNPs used for East asian haplogroup determination11)

† 31 SNPs consisting of 16 control region SNPs and 15 coding region SNPs

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