Open Access, Peer-reviewed
pISSN 2671-5651
eISSN 2671-566X
    Korean J Phys Anthropol. 2018 Sep;31(3):77-82. Korean.
    Published online Sep 28, 2018.
    https://doi.org/10.11637/kjpa.2018.31.3.77
    © 2018 Korean Association of Physical Anthropologists
    Original Article

    Determination of Repeat Numbers of (CA)n in Mitochondrial D-loop using Polymerase Chain Reaction-single Strand Conformational Polymorphism (PCR-SSCP)

    Joo-Young Kim,1 and Dae-Kwang Kim2,3
      • 1Department of Anatomy, College of Medicine, Yeungnam University, Korea.
      • 2Department of Medical Genetics, School of Medicine, Keimyung University, Korea.
      • 3Hanvit institute for Medical Genetics, Korea.
    • Correspondence to: Dae-Kwang Kim (Department of Medical Genetics, School of Medicine, Keimyung University; Hanvit institute for Medical Genetics, Daegu, Republic of Korea), Email: dkkimmd@kmu.ac.kr
    Received July 11, 2018; Revised September 11, 2018; Accepted September 11, 2018.

    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

    Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis is a kind of sensitive mutation detection method that has been usually used in field of medical genetics. A single DNA strand with a mutation or nucleotide polymorphism has a different conformation from its wild-type counterpart, and these conformational differences result in different electrophoretic mobility. In previous study of mitochondrial microsatellite instability in 50 uterine leiomyomas, PCR-SSCP showed 4 types of band mobility at (CA)n of the mitochondrial D-loop. In type 1 and 4, positions of the lower single stand of both were same but those of upper strand were different. In sequencing analysis, repeat number of (CA)n in type 1 was 4, 5 in type 2, 6 in type 3, and 4 in type 4, respectively. Without using expensive sequencing analysis, PCR-SSCP method can be used to detect the repeat number of (CA)n in mitochondrial D-loop.

    Keywords
    PCR-SSCP; Mitochondrial D-loop; (CA)n repeat number

    Figures

    Fig. 1
    Silver stained polyacrylamide gel showing PCR-SSCP patterns at locus (CA)n of mitochondrial D-loop in uterine leiomyomas. 7.5% polyacrylamide gel was used for electrophoresis. At the top of each lane, number 1, 2, 3 and 4 represented the type of SSCP band mobility pattern. (a) The positions of single strands of type 1, 2 and 3 differed respectively. (b) In type 1 and 4, positions of the lower single stand of both were equal but those of upper strand were different.

    Fig. 2
    DNA sequencing analysis. (a) Type 1 of SSCP pattern had CCAG(CA)4CCGC sequencing structure. (b) Type 2 of SSCP pattern had CCAG(CA)5CCGC sequencing structure. (c) Type 3 of SSCP pattern had CCAG(CA)6CCGC sequencing structure. (d) Type 4 of SSCP pattern had CCAA(CA)4CCGC sequencing structure.

    Tables

    Table 1
    Frequencies of mitochondrial D-loop (CA)n repeat polymorphism in Koreans

    References

      1. Attardi G, Schatz G. Biogenesis of mitochondria. Annu Rev Cell Biol 1988;4:289–333.
      1. Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, et al. Sequence and organization of the human mitochondrial genome. Nature 1981;290:457–465.
      1. Wallace DC. Diseases of the mitochondrial DNA. Annu Rev Biochem 1992;61:1175–1212.
      1. Wallace DC. Mitochondrial DNA sequence variation in human evolution and disease. Proc Natl Acad Sci USA 1994;91:8739–8746.
      1. Penta JS, Johnson FM, Wachsman JT, Copeland WC. Mitochondrial DNA in human malignancy. Mutat Res 2001;488:119–133.
      1. Mandavilli BS, Santos JH, Van Houten B. Mitochondrial DNA repair and aging. Mutat Res 2002;509:127–151.
      1. Khrapko K, Coller HA, André PC, Li XC, Hanekamp JS, Thilly WG. Mitochondrial mutational spectra in human cells and tissues. Proc Natl Acad Sci USA 1997;94:13798–13803.
      1. Van Tuyle GC, McPherson ML. A compact form of rat liver mitochondrial DNA stabilized by bound proteins. J Biol Chem 1979;254:6044–6053.
      1. Stoneking M, Hedgecock D, Higuchi RG, Vigilant L, Erlich HA. Population variation of human mtDNA control region sequences detected by enzymatic amplification and sequence-specific oligonucleotide probes. Am J Hum Genet 1991;48:370–382.
      1. Bodenteich A, Mitchell LG, Polymeropoulos MH, Merril CR. Dinucleotide repeat in the human mitochondrial D-loop. Hum Mol Genet 1992;1:140.
      1. Lee JH, Hwang I, Kang YN, Choi IJ, Kim DK. Genetic characteristics of mitochondrial DNA was associated with colorectal carcinogenesis and its prognosis. PLoS One 2015;10:e0118612.
      1. Szibor R, Plate I, Schmitter H, Wittig H, Krause D. Forensic mass screening using mtDNA. Int J Legal Med 2006;120:372–376.
      1. Orita M, Suzuki Y, Sekiya T, Hayashi K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 1989;5:874–879.
      1. Hayashi K, Yandell DW. How sensitive is PCR-SSCP? Hum Mutat 1993;2:338–346.
      1. Hayashi K. Recent enhancements in SSCP. Genet Anal 1999;14:193–196.
      1. Inazuka M, Wenz HM, Sakabe M, Tahira T, Hayashi K. A streamlined mutation detection system: multicolor post-PCR fluorescence labeling and single-strand conformational polymorphism analysis by capillary electrophoresis. Genome Res 1997;7:1094–1103.
      1. Lee JH, Ryu TY, Cho CH, Kim DK. Different characteristics of mitochondrial microsatellite instability between uterine leiomyomas and leiomyosarcomas. Pathol Oncol Res 2011;17:201–205.
      1. Zhang L, Li DY, Liu YP, Wang Y, Zhao XL, Zhu Q. Genetic effect of the prolactin receptor gene on egg production traits in chickens. Genet Mol Res 2012;11:4307–4315.
      1. Raut AA, Kumar A, Kala SN, Chhokar V, Rana N, Beniwal V, et al. Identification of novel single nucleotide polymorphisms in the DGAT1 gene of buffaloes by PCR-SSCP. Genet Mol Biol 2012;35:610–613.
      1. Szibor R, Plate I, Schmitter H, Wittig H, Krause D. Forensic mass screening using mtDNA. Int J Legal Med 2006;120:372–376.
      1. Richard SM, Bailliet G, Páez GL, Bianchi MS, Peltomäki P, Bianchi NO. Nuclear and mitochondrial genome instability in human breast cancer. Cancer Res 2000;60:4231–4237.
      1. Chung U, Lee HY, Yoo JE, Park MJ, Shin KJ. Mitochondrial DNA CA dinucleotide repeats in Koreans: the presence of length heteroplasmy. Int J Legal Med 2005;119:50–53.
      1. Lee JH, Kim DK. Association between Mitochondrial D-loop Polymorphism and Copy Number. Korean J Phys Anthropol 2014;27:131–136.
    MeSH Terms
    DNA
    Genetics, Medical
    Leiomyoma
    Methods
    Microsatellite Instability
    Metrics
    Share
    Figures
    slide
    slide

    1 / 2

    Tables
    slide

    1 / 1

    ORCID IDs
    PERMALINK