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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
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
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
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
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
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
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
Thomas E, Herrera RJ (1998) Multiplex polymerase chain reaction of Alu polymorphic insertions. Electrophoresis 19(14):2373–2379. doi:10.1002/elps.1150191402
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
Bernard PS, Wittwer CT (2002) Real-time PCR technology for cancer diagnostics. Clin Chem 48(8):1178–1185
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
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
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
Morley AA (2014) Digital PCR: a brief history. Biomol Detect Quantif 1(1):1–2. doi:10.1016/j.bdq.2014.06.001
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
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
Vogelstein B, Kinzler KW (1999) Digital PCR. Proc Natl Acad Sci U S A 96(16):9236–9241
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
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
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
Burkardt HJ (2000) Standardization and quality control of PCR analyses. Clin Chem Lab Med 38(2):87–91. doi:10.1515/CCLM.2000.014
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
Chien A, Edgar DB, Trela JM (1976) Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol 127(3):1550–1557
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
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
McPherson MJ, Moller SG (2006) PCR, 2nd edn. Taylor & Francis e-Library, New York, NY
Myers TW, Gelfand DH (1991) Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry 30(31):7661–7666
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
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
Dieffenbach CW, Lowe TM, Dveksler GS (1993) General concepts for PCR primer design. PCR Methods Appl 3(3):S30–S37
Harisha S (2007) Biotechnology procedures and experiments handbook. Infinity Science Press LLC, Hingham, MA
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
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
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
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-#
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
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
Sarkar G, Sommer S (1990) More light on PCR contamination. Nature 347(6291):340–341. doi:10.1038/347340b0
Sarkar G, Sommer SS (1990) Shedding light on PCR contamination. Nature 343(6253):27. doi:10.1038/343027a0
Sundquist T, Bessetti J (2005) Identifying and preventing DNA contamination in a DNA-typing laboratory. Profiles in DNA 8(2):11–13
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
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
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
Acknowledgments
We thank the Manassas Campus of Northern Virginia Community College for the use of equipment and materials.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6990-6_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6989-0
Online ISBN: 978-1-4939-6990-6
eBook Packages: Springer Protocols