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PCR: Identification of Genetic Polymorphisms

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Book cover Molecular Profiling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1606))

Abstract

Polymerase chain reaction (PCR) enables the amplification of a specific sequence of deoxyribonucleic acid (DNA) through the process of three main steps: template DNA denaturation, annealing of the primers to complementary sequences, and primer extension to synthesize DNA strands. By using this method, the target sequence will be copied and amplified at an exponential rate. PCR provides a qualitative method for identifying DNA from fresh or dried cells/body fluids, formalin-fixed archival tissue specimens, and ancient specimens.

Herein we describe basic information for performing successful PCR experiments using the amplification of a human Alu insertion on the PV92 gene locus on chromosome 16 as an example method.

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References

  1. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 51(Pt 1):263–273

    Article  CAS  PubMed  Google Scholar 

  2. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230(4732):1350–1354

    Article  CAS  PubMed  Google Scholar 

  3. Butts EL, Vallone PM (2014) Rapid PCR protocols for forensic DNA typing on six thermal cycling platforms. Electrophoresis 35(21–22):3053–3061. doi:10.1002/elps.201400179

    Article  CAS  PubMed  Google Scholar 

  4. Csakyova V, Szecsenyi-Nagy A, Csosz A, Nagy M, Fusek G, Lango P, Bauer M, Mende BG, Makovicky P, Bauerova M (2016) Maternal genetic composition of a medieval population from a Hungarian-Slavic contact zone in Central Europe. PLoS One 11(3):e0151206. doi:10.1371/journal.pone.0151206

    Article  PubMed  PubMed Central  Google Scholar 

  5. Endo K, Konishi A, Sasaki H, Takada M, Tanaka H, Okumura M, Kawahara M, Sugiura H, Kuwabara Y, Fukai I, Matsumura A, Yano M, Kobayashi Y, Mizuno K, Haneda H, Suzuki E, Iuchi K, Fujii Y (2005) Epidermal growth factor receptor gene mutation in non-small cell lung cancer using highly sensitive and fast TaqMan PCR assay. Lung Cancer 50(3):375–384. doi:10.1016/j.lungcan.2005.08.009

    Article  PubMed  Google Scholar 

  6. Sepp R, Szabo I, Uda H, Sakamoto H (1994) Rapid techniques for DNA extraction from routinely processed archival tissue for use in PCR. J Clin Pathol 47(4):318–323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Thomas E, Herrera RJ (1998) Multiplex polymerase chain reaction of Alu polymorphic insertions. Electrophoresis 19(14):2373–2379. doi:10.1002/elps.1150191402

    Article  CAS  PubMed  Google Scholar 

  8. Wolk DM, Blyn LB, Hall TA, Sampath R, Ranken R, Ivy C, Melton R, Matthews H, White N, Li F, Harpin V, Ecker DJ, Limbago B, McDougal LK, Wysocki VH, Cai M, Carroll KC (2009) Pathogen profiling: rapid molecular characterization of Staphylococcus aureus by PCR/electrospray ionization-mass spectrometry and correlation with phenotype. J Clin Microbiol 47(10):3129–3137. doi:10.1128/JCM.00709-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bernard PS, Wittwer CT (2002) Real-time PCR technology for cancer diagnostics. Clin Chem 48(8):1178–1185

    CAS  PubMed  Google Scholar 

  10. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV (1992) Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 30(3):545–551

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Najioullah F, Viron F, Cesaire R (2014) Evaluation of four commercial real-time RT-PCR kits for the detection of dengue viruses in clinical samples. Virol J 11:164. doi:10.1186/1743-422X-11-164

    Article  PubMed  PubMed Central  Google Scholar 

  12. Punt S, Houwing-Duistermaat JJ, Schulkens IA, Thijssen VL, Osse EM, de Kroon CD, Griffioen AW, Fleuren GJ, Gorter A, Jordanova ES (2015) Correlations between immune response and vascularization qRT-PCR gene expression clusters in squamous cervical cancer. Mol Cancer 14:71. doi:10.1186/s12943-015-0350-0

    Article  PubMed  PubMed Central  Google Scholar 

  13. Morley AA (2014) Digital PCR: a brief history. Biomol Detect Quantif 1(1):1–2. doi:10.1016/j.bdq.2014.06.001

    Article  PubMed  PubMed Central  Google Scholar 

  14. Huggett JF, Cowen S, Foy CA (2015) Considerations for digital PCR as an accurate molecular diagnostic tool. Clin Chem 61(1):79–88. doi:10.1373/clinchem.2014.221366

    Article  CAS  PubMed  Google Scholar 

  15. Sedlak RH, Cook L, Huang ML, Magaret A, Zerr DM, Boeckh M, Jerome KR (2014) Identification of chromosomally integrated human herpesvirus 6 by droplet digital PCR. Clin Chem 60(5):765–772. doi:10.1373/clinchem.2013.217240

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Vogelstein B, Kinzler KW (1999) Digital PCR. Proc Natl Acad Sci U S A 96(16):9236–9241

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Farrar JS, Wittwer CT (2015) Extreme PCR: efficient and specific DNA amplification in 15-60 seconds. Clin Chem 61(1):145–153. doi:10.1373/clinchem.2014.228304

    Article  PubMed  Google Scholar 

  18. Sheel Kumar V, Webster M (2015) Extreme PCR: a breakthrough innovation for outbreaks? Clin Chem 61(4):674–676. doi:10.1373/clinchem.2014.236950

    Article  PubMed  Google Scholar 

  19. Birch L, English CA, Burns M, Keer JT (2004) Generic scheme for independent performance assessment in the molecular biology laboratory. Clin Chem 50(9):1553–1559. doi:10.1373/clinchem.2003.029454

    Article  CAS  PubMed  Google Scholar 

  20. Burkardt HJ (2000) Standardization and quality control of PCR analyses. Clin Chem Lab Med 38(2):87–91. doi:10.1515/CCLM.2000.014

    Article  CAS  PubMed  Google Scholar 

  21. Raggi CC, Pinzani P, Paradiso A, Pazzagli M, Orlando C (2003) External quality assurance program for PCR amplification of genomic DNA: an Italian experience. Clin Chem 49(5):782–791

    Article  CAS  PubMed  Google Scholar 

  22. Chien A, Edgar DB, Trela JM (1976) Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol 127(3):1550–1557

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Hu CY, Allen M, Gyllensten U (1992) Effect of freezing of the PCR buffer on the amplification specificity: allelic exclusion and preferential amplification of contaminating molecules. PCR Methods Appl 2(2):182–183

    Article  CAS  PubMed  Google Scholar 

  24. Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonak J, Lind K, Sindelka R, Sjoback R, Sjogreen B, Strombom L, Stahlberg A, Zoric N (2006) The real-time polymerase chain reaction. Mol Asp Med 27(2–3):95–125. doi:10.1016/j.mam.2005.12.007

    Article  CAS  Google Scholar 

  25. McPherson MJ, Moller SG (2006) PCR, 2nd edn. Taylor & Francis e-Library, New York, NY

    Google Scholar 

  26. Myers TW, Gelfand DH (1991) Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry 30(31):7661–7666

    Article  CAS  PubMed  Google Scholar 

  27. Nair AJ (2008) Introduction to biotechnology and genetic engineering. Infinity Science Press LLC, Hingham, MA. http://www.globalspec.com/reference/65995/203279/introduction-to-biotechnology-and-genetic-engineering

  28. Apte A, Daniel S (2003) PCR primer design. In: Dieffenbach CW, Dveksler GS (eds) PCR primer: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 61–74

    Google Scholar 

  29. Dieffenbach CW, Lowe TM, Dveksler GS (1993) General concepts for PCR primer design. PCR Methods Appl 3(3):S30–S37

    Article  CAS  PubMed  Google Scholar 

  30. Harisha S (2007) Biotechnology procedures and experiments handbook. Infinity Science Press LLC, Hingham, MA

    Google Scholar 

  31. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13:134. doi:10.1186/1471-2105-13-134

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Batzer MA, Stoneking M, Alegria-Hartman M, Bazan H, Kass DH, Shaikh TH, Novick GE, Ioannou PA, Scheer WD, Herrera RJ et al (1994) African origin of human-specific polymorphic Alu insertions. Proc Natl Acad Sci U S A 91(25):12288–12292

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Comas D, Plaza S, Calafell F, Sajantila A, Bertranpetit J (2001) Recent insertion of an Alu element within a polymorphic human-specific Alu insertion. Mol Biol Evol 18(1):85–88

    Article  CAS  PubMed  Google Scholar 

  34. Stellwagen E, Stellwagen NC (2002) The free solution mobility of DNA in Tris-acetate-EDTA buffers of different concentrations, with and without added NaCl. Electrophoresis 23(12):1935–1941. doi:10.1002/1522-2683(200206)23:12<1935::AID-ELPS1935>3.0.CO;2-#

    Article  CAS  PubMed  Google Scholar 

  35. Lum A, Le Marchand L (1998) A simple mouthwash method for obtaining genomic DNA in molecular epidemiological studies. Cancer Epidemiol Biomark Prev 7(8):719–724

    CAS  Google Scholar 

  36. Mulot C, Stucker I, Clavel J, Beaune P, Loriot MA (2005) Collection of human genomic DNA from buccal cells for genetics studies: comparison between cytobrush, mouthwash, and treated card. J Biomed Biotechnol 2005(3):291–296. doi:10.1155/JBB.2005.291

    Article  PubMed  PubMed Central  Google Scholar 

  37. Sarkar G, Sommer S (1990) More light on PCR contamination. Nature 347(6291):340–341. doi:10.1038/347340b0

    Article  CAS  PubMed  Google Scholar 

  38. Sarkar G, Sommer SS (1990) Shedding light on PCR contamination. Nature 343(6253):27. doi:10.1038/343027a0

    Article  CAS  PubMed  Google Scholar 

  39. Sundquist T, Bessetti J (2005) Identifying and preventing DNA contamination in a DNA-typing laboratory. Profiles in DNA 8(2):11–13

    Google Scholar 

  40. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J (1990) Rapid and simple method for purification of nucleic acids. J Clin Microbiol 28(3):495–503

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Carbonero F, Nava GM, Benefiel AC, Greenberg E, Gaskins HR (2011) Microbial DNA extraction from intestinal biopsies is improved by avoiding mechanical cell disruption. J Microbiol Methods 87(1):125–127. doi:10.1016/j.mimet.2011.07.014

    Article  CAS  PubMed  Google Scholar 

  42. Vingataramin L, Frost EH (2015) A single protocol for extraction of gDNA from bacteria and yeast. BioTechniques 58(3):120–125. doi:10.2144/000114263

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank the Manassas Campus of Northern Virginia Community College for the use of equipment and materials.

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Authors and Affiliations

  1. Northern Virginia Community College, Manassas Campus, 6901 Sudley Road, Manassas, VA, 20109, USA

    Amanda M. Harbison & Jenny Ngoc Tran Nguyen

Authors
  1. Amanda M. Harbison
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  2. Jenny Ngoc Tran Nguyen
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Corresponding author

Correspondence to Amanda M. Harbison .

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Editors and Affiliations

  1. Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia, USA

    Virginia Espina

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Harbison, A.M., Nguyen, J.N.T. (2017). PCR: Identification of Genetic Polymorphisms. In: Espina, V. (eds) Molecular Profiling. Methods in Molecular Biology, vol 1606. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6990-6_13

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  • DOI: https://doi.org/10.1007/978-1-4939-6990-6_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6989-0

  • Online ISBN: 978-1-4939-6990-6

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