Discrimination of haplotype in mitochondrial DNA mixtures using LNA-mediated PCR clamping |
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| Affiliation: | 1. Núcleo de Genética Humana e Molecular (NGHM), Universidade Federal do Espírito Santo, Espírito Santo, Brazil;2. Laboratório de DNA Forense, Departamento de Laboratórios Forenses, Polícia Civil do Espírito Santo, Espírito Santo, Brazil;1. Institute of Forensic Medicine, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu, 610041, PR China;2. Shanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Sciences, Ministry of Justice, Shanghai, 200063, PR China;3. Department of Forensic Science, Medical School of Soochow University, Suzhou, 215123, PR China;4. QIAGEN, Hilden, Germany;5. Department of Forensic Medicine, School of Basic Medical Science, Wenzhou Medical University, Wenzhou, 325035, PR China;6. College of Medicine and Forensics, Xi’an Jiaotong University Health Science Center, Xi’an, 710061, PR China;1. College of Science and Engineering, Flinders University, GPO Box 2100, Adelaide, South Australia, 5001, Australia;2. Forensic Science SA, GPO box 2790, Adelaide, South Australia, 5001, Australia;1. Forensic Science SA, GPO Box 2790, Adelaide, South Australia, 5001, Australia;2. College of Science and Engineering, Flinders University of South Australia, GPO Box 2100, Adelaide, South Australia, 5001, Australia;1. Forensic Science SA, GPO Box 2790, Adelaide, South Australia, 5001, Australia;2. College of Science and Engineering, Flinders University of South Australia, GPO Box 2100, Adelaide, South Australia, 5001, Australia;1. Institute of Forensic Medicine, West China School of Basic Sciences and Forensic Medicine, Sichuan University, Chengdu, China;2. Institute of Forensic Medicine and Laboratory Medicine, Jining Medical University, Jining, China;3. Institute of Criminal Science and Technology, Karamay City Public Security Bureau, Karamay, China;4. Institute of Criminal Investigation, Zhengzhou City Public Security Bureau, Zhengzhou, China;5. Institute of Criminal Science and Technology Research, Lanzhou City Public Security Bureau, Lanzhou, China;6. Department of Criminal Investigation Bureau of Shaanxi Provincial Public Security, Xi’an, China;7. Institute of Forensic Science, Kunming Public Security Bureau, Kunming, China |
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| Abstract: | ![]()
Locked nucleic acid (LNA) has been widely used for various genetic analyses, and has many benefits, in terms of the specificity or sensitivity of amplification, because LNA-containing primers/probes form more stable duplexes with template DNA than probes lacking LNA. Here, we developed a new method for discriminating HV1 haplotypes from mitochondrial DNA (mtDNA) mixtures by applying PCR clamping using LNA. PCR clamping is based on the selective inhibition of amplification using LNA-containing probes, which can discriminate single-nucleotide differences. Before designing probes, we selected 171 sequences with single-nucleotide variations from the HV1 region, and evaluated the specificity of LNA-containing probes for them by predicting Tm values. The differences of Tm between mismatched and exactly matched probe–template duplexes depended markedly on the type of LNA nucleotides for discriminating single-nucleotide differences, and the cytosine LNA nucleotide at the site of variations in the probes was most effective to discriminate these differences. For mixture analysis, each probe targeted one or two variations (16209C, 16217C, 16257A/16261T, 16297C/16298C, 16304C, 16362C, or 16362T) that are particularly common in the Japanese population, and seven designed probes completely inhibited the amplification of exactly matched templates. We prepared mixed samples by mixing DNA from two individuals at a ratio of 1:9, 1:4, 1:1, 4:1, or 9:1, and then performed Sanger sequencing analysis after PCR clamping with each probe. Our method distinguished each haplotype at lower ratios from two-person mixtures, and enabled sensitive detection at 12 pg of total DNA including 600 copies of mtDNA. Moreover, we analyzed three-person mixtures with representative sequences, and detected the minor haplotype of one individual present at a rate of 10% by adding two selected probes. The ability to discriminate haplotypes in mixed samples by using LNA-mediated PCR clamping indicates the potential value of mtDNA analysis in criminal investigations. |
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| Keywords: | Mitochondrial DNA Haplotype Forensic science Mixed sample PCR clamping Locked nucleic acid |
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