Polymerase Chain Reaction and Reverse Transcription&##x2014;Polymerase Chain Reaction

  • Dwight Oliver
Part of the Molecular Pathology Library book series (MPLB, volume 1)


Polymerase chain reaction (PCR) enables one to determine if a specific needle is present in a haystack, and it can be used as a step toward the characterization of the needle. It is a quick, powerful, inexpensive DNA amplification technique that has become a fundamental tool in molecular pathology.


Polymerase Chain Reaction Fluorescence Resonance Energy Transfer Multiplex Polymerase Chain Reaction Moloney Murine Leukemia Virus Polymerase Chain Reaction Technique 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Bell J. The polymerase chain reaction. Immunol Today 1989;10:351–355.CrossRefPubMedGoogle Scholar
  2. 2.
    Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985;230:1350–1354.CrossRefPubMedGoogle Scholar
  3. 3.
    Mullis K, Faloona F, Scharf S, et al. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986;51 (Pt 1):263–273.PubMedGoogle Scholar
  4. 4.
    Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988;239:487–491.CrossRefPubMedGoogle Scholar
  5. 5.
    Saboor SA, Johnson NM, McFadden J. Detection of mycobacterial DNA in sarcoidosis and tuberculosis with polymerase chain reaction. Lancet 1992;339:1012–1015.CrossRefPubMedGoogle Scholar
  6. 6.
    Myerson D, Lingenfelter PA, Gleaves CA, et al. Diagnosis of cytomegalovirus pneumonia by the polymerase chain reaction with archived frozen lung tissue and bronchoalveolar lavage fluid. Am J Clin Pathol 1993;100:407–413.PubMedGoogle Scholar
  7. 7.
    Raad I, Hanna H, Huaringa A, et al. Diagnosis of invasive pulmonary aspergillosis using polymerase chain reaction-based detection of aspergillus in BAL. Chest 2002;121:1171–1176.CrossRefPubMedGoogle Scholar
  8. 8.
    Sundaresan S, Alevy YG, Steward N, et al. Cytokine gene transcripts for tumor necrosis factor-alpha, interleukin-2, and interferon-gamma in human pulmonary allografts. J Heart Lung Transplant 1995;14:512–518.PubMedGoogle Scholar
  9. 9.
    Lordan JL, Bucchieri F, Richter A, et al. Cooperative effects of Th2 cytokines and allergen on normal and asthmatic bronchial epithelial cells. J Immunol 2002;169:407–414.PubMedGoogle Scholar
  10. 10.
    Nogee LM, Dunbar AE 3rd, Wert SE, et al. A mutation in the surfactant protein C gene associated with familial interstitial lung disease. N Engl J Med 2001;344:573–579.CrossRefPubMedGoogle Scholar
  11. 11.
    Pan Q, Pao W, Ladanyi M. Rapid polymerase chain reaction-based detection of epidermal growth factor receptor gene mutations in lung adenocarcinomas. J Mol Diagn 2005;7:396–403.PubMedGoogle Scholar
  12. 12.
    Westra WH, Baas IO, Hruban RH, et al. K-ras oncogene activation in atypical alveolar hyperplasias of the human lung. Cancer Res 1996;56:2224–2228.PubMedGoogle Scholar
  13. 13.
    Pulte D, Li E, Crawford BK, et al. Sentinel lymph node mapping and molecular staging in nonsmall cell lung carcinoma. Cancer 2005;104:1453–1461.CrossRefPubMedGoogle Scholar
  14. 14.
    Bohlmeyer T, Le TN, Shroyer AL, et al. Detection of human papillomavirus in squamous cell carcinomas of the lung by polymerase chain reaction. Am J Respir Cell Mol Biol 1998;18:265–269.PubMedGoogle Scholar
  15. 15.
    Bremnes RM, Sirera R, Camps C. Circulating tumourderived DNA and RNA markers in blood: a tool for early detection, diagnostics, and follow-up? Lung Cancer 2005;49:1–12.CrossRefPubMedGoogle Scholar
  16. 16.
    Eisenstein BI. The polymerase chain reaction. A new method of using molecular genetics for medical diagnosis. N Engl J Med 1990;322:178–183.PubMedCrossRefGoogle Scholar
  17. 17.
    Wenham PR. DNA-based techniques in clinical biochemistry: a beginner’s guide to theory and practice. Ann Clin Biochem 1992;29 (Pt 6):598–624.PubMedGoogle Scholar
  18. 18.
    Remick DG, Kunkel SL, Holbrook EA, Hanson CA. Theory and applications of the polymerase chain reaction. Am J Clin Pathol 1990;93:S49–S54.PubMedGoogle Scholar
  19. 19.
    Chakrabarti R, Schutt CE. The enhancement of PCR amplification by low molecular-weight sulfones. Gene 2001;274:293–298.CrossRefPubMedGoogle Scholar
  20. 20.
    Laksanalamai P, Pavlov AR, Slesarev AI, Robb FT. Stabilization of Taq DNA polymerase at high temperature by protein folding pathways from a hyperthermophilic archaeon, Pyrococcus furiosus. Biotechnol Bioeng 2006;93:1–5.CrossRefPubMedGoogle Scholar
  21. 21.
    Breslauer KJ, Frank R, Blocker H, Marky LA. Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci USA 1986;83:3746–3750.CrossRefPubMedGoogle Scholar
  22. 22.
    SantaLucia J Jr, Allawi HT, Seneviratne PA. Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry 1996;35:3555–3562.CrossRefPubMedGoogle Scholar
  23. 23.
    Wallace RB, Shaffer J, Murphy RF, et al. Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch. Nucleic Acids Res 1979;6:3543–3557.CrossRefPubMedGoogle Scholar
  24. 24.
    Chien A, Edgar DB, Trela JM. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol 1976;127:1550–1557.PubMedGoogle Scholar
  25. 25.
    Takagi M, Nishioka M, Kakihara H, et al. Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. Appl Environ Microbiol 1997;63:4504–4510.PubMedGoogle Scholar
  26. 26.
    Davidson JF, Fox R, Harris DD, et al. Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase. Nucleic Acids Res 2003;31:4702–4709.CrossRefPubMedGoogle Scholar
  27. 27.
    Keohavong P, Thilly WG. Fidelity of DNA polymerases in DNA amplification. Proc Natl Acad Sci USA 1989;86:9253–9257.CrossRefPubMedGoogle Scholar
  28. 28.
    Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 1997;63:3741–2751.PubMedGoogle Scholar
  29. 29.
    Khan G, Kangro HO, Coates PJ, Heath RB. Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA. J Clin Pathol 1991;44:360–365.CrossRefPubMedGoogle Scholar
  30. 30.
    Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI. Recent developments in the optimization of thermostable DNA polymerases for efficient applications. Trends Biotechnol 2004;22:253–260.CrossRefPubMedGoogle Scholar
  31. 31.
    Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 1990;93:125–128.CrossRefPubMedGoogle Scholar
  32. 32.
    Eckert KA, Kunkel TA. High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids Res 1990;18:3739–3744.CrossRefPubMedGoogle Scholar
  33. 33.
    McPherson MJ, Møller SG. PCR. Oxford, UK: BIOS Scientific Publications; 2000.Google Scholar
  34. 34.
    Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975;98:503–517.CrossRefPubMedGoogle Scholar
  35. 35.
    Bevan IS, Rapley R, Walker MR. Sequencing of PCRamplified DNA. PCR Methods Appl 1992;1:222–228.PubMedGoogle Scholar
  36. 36.
    Moretti T, Koons B, Budowle B. Enhancement of PCR amplification yield and specificity using AmpliTaq Gold DNA polymerase. Biotechniques 1998;25:716–722.PubMedGoogle Scholar
  37. 37.
    Brandwein M, Zeitlin J, Nuovo GJ, et al. HPV detection using “hot start“ polymerase chain reaction in patients with oral cancer: a clinicopathological study of 64 patients. Mod Pathol 1994;7:720–727.PubMedGoogle Scholar
  38. 38.
    Nuovo GJ, Gallery F, MacConnell P. Detection of amplified HPV 6 and 11 DNA in vulvar lesions by hot start PCR in situ hybridization. Mod Pathol 1992;5:444–448.PubMedGoogle Scholar
  39. 39.
    Chou Q, Russell M, Birch DE, et al. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucleic Acids Res 1992;20:1717–1723.CrossRefPubMedGoogle Scholar
  40. 40.
    Tilston P, Corbitt G. A single tube nested PCR for the detection of hepatitis C virus RNA. J Virol Methods 1995;53:121–129.CrossRefPubMedGoogle Scholar
  41. 41.
    Smit VT, Boot AJ, Smits AM, et al. KRAS codon 12 mutations occur very frequently in pancreatic adenocarcinomas. Nucleic Acids Res 1988;16:7773–7782.CrossRefPubMedGoogle Scholar
  42. 42.
    Herman JG, Graff JR, Myohanen S, et al. Methylationspecific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996;93:9821–9826.CrossRefPubMedGoogle Scholar
  43. 43.
    Curtis CD, Goggins M. DNA methylation analysis in human cancer. Methods Mol Med 2005;103:123–136.PubMedGoogle Scholar
  44. 44.
    Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics 2002;18:1427–1431.CrossRefPubMedGoogle Scholar
  45. 45.
    Bird A. The essentials of DNA methylation. Cell 1992;70:5–8.CrossRefPubMedGoogle Scholar
  46. 46.
    Robertson KD, Jones PA. DNA methylation: past, present and future directions. Carcinogenesis 2000;21:461–467.CrossRefPubMedGoogle Scholar
  47. 47.
    Rhee I, Bachman KE, Park BH, et al. DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature 2002;416:552–556.CrossRefPubMedGoogle Scholar
  48. 48.
    Dammann R, Takahashi T, Pfeifer GP. The CpG island of the novel tumor suppressor gene RASSF1A is intensely methylated in primary small cell lung carcinomas. Oncogene 2001;20:3563–3567.CrossRefPubMedGoogle Scholar
  49. 49.
    Zochbauer-Muller S, Fong KM, Virmani AK, et al. Aberrant promoter methylation of multiple genes in non-small cell lung cancers. Cancer Res 2001;61:249–255.PubMedGoogle Scholar
  50. 50.
    Clark SJ, Harrison J, Paul CL, Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res 1994;22:2990–2997.CrossRefPubMedGoogle Scholar
  51. 51.
    Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE. Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev 2000;13:559–570.CrossRefPubMedGoogle Scholar
  52. 52.
    Richards B, Skoletsky J, Shuber AP, et al. Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs. Hum Mol Genet 1993;2:159–163.CrossRefPubMedGoogle Scholar
  53. 53.
    Scurto P, Hsu Rocha M, Kane JR, et al. A multiplex RT-PCR assay for the detection of chimeric transcripts encoded by the risk-stratifying translocations of pediatric acute lymphoblastic leukemia. Leukemia 1998;12:1994–2005.CrossRefPubMedGoogle Scholar
  54. 54.
    Pallisgaard N, Hokland P, Riishoj DC, et al. Multiplex reverse transcription-polymerase chain reaction for simultaneous screening of 29 translocations and chromosomal aberrations in acute leukemia. Blood 1998;92:574–588.PubMedGoogle Scholar
  55. 55.
    Newton CR, Graham A, Heptinstall LE, et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 1989;17:2503–2516.CrossRefPubMedGoogle Scholar
  56. 56.
    Saiki RK, Bugawan TL, Horn GT, et al. Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature 1986;324:163–166.CrossRefPubMedGoogle Scholar
  57. 57.
    Schaefer BC. Revolutions in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full-length cDNA ends. Anal Biochem 1995;227:255–273.CrossRefPubMedGoogle Scholar
  58. 58.
    Komminoth P, Long AA. In-situ polymerase chain reaction. An overview of methods, applications and limitations of a new molecular technique. Virchows Arch B Cell Pathol Incl Mol Pathol 1993;64:67–73.CrossRefPubMedGoogle Scholar
  59. 59.
    Livak KJ, Flood SJ, Marmaro J, et al. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 1995;4:357–362.PubMedGoogle Scholar
  60. 60.
    Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996;6:986–994.CrossRefPubMedGoogle Scholar
  61. 61.
    Lie YS, Petropoulos CJ. Advances in quantitative PCR technology: 5′ nuclease assays. Curr Opin Biotechnol 1998;9:43–48.CrossRefPubMedGoogle Scholar
  62. 62.
    Holland PM, Abramson RD, Watson R, Gelfand DH. Detection of specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA 1991;88:7276–7280.CrossRefPubMedGoogle Scholar
  63. 63.
    Longley MJ, Bennett SE, Mosbaugh DW. Characterization of the 5′ to 3′ exonuclease associated with Thermus aquaticus DNA polymerase. Nucleic Acids Res 1990;18:7317–7322.CrossRefPubMedGoogle Scholar
  64. 64.
    Förster T. Zwischemolekulare energiewanderung und fluoreszenz. Ann Physik 1948;2:55–67.CrossRefGoogle Scholar
  65. 65.
    Stryer L, Haugland RP. Energy transfer: a spectroscopic ruler. Proc Natl Acad Sci USA 1967;58:719–726.CrossRefPubMedGoogle Scholar
  66. 66.
    Grinvald A, Haas E, Steinberg IZ. Evaluation of the distribution of distances between energy donors and acceptors by fluorescence decay. Proc Natl Acad Sci USA 1972;69:2273–2277.CrossRefPubMedGoogle Scholar
  67. 67.
    Oliver DH, Thompson RE, Griffin CA, Eshleman JR. Use of single nucleotide polymorphisms (SNP) and real-time polymerase chain reaction for bone marrow engraftment analysis. J Mol Diagn 2000;2:202–208.PubMedGoogle Scholar
  68. 68.
    Lay MJ, Wittwer CT. Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. Clin Chem 1997;43:2262–2267.PubMedGoogle Scholar
  69. 69.
    Temin HM, Mizutani S. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 1970;226:1211–1213.CrossRefPubMedGoogle Scholar
  70. 70.
    Baltimore D. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature 1970;226:1209–1211.CrossRefPubMedGoogle Scholar
  71. 71.
    Spiegelman S, Burny A, Das MR, et al. Characterization of the products of DNA-directed DNA polymerases in oncogenic RNA viruses. Nature 1970;227:563–567.CrossRefPubMedGoogle Scholar
  72. 72.
    Shinnick TM, Lerner RA, Sutcliffe JG. Nucleotide sequence of Moloney murine leukaemia virus. Nature 1981;293:543–548.CrossRefPubMedGoogle Scholar
  73. 73.
    Reddy EP, Smith MJ, Aaronson SA. Complete nucleotide sequence and organization of the Moloney murine sarcoma virus genome. Science 1981;214:445–450.CrossRefPubMedGoogle Scholar
  74. 74.
    Kotewicz ML, D’Alessio JM, Driftmier KM, et al. Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli. Gene 1985;35:249–258.CrossRefPubMedGoogle Scholar
  75. 75.
    Verma IM, Baltimore D. Purification of the RNA-directed DNA polymerase from avian myeloblastosis virus and its assay with polynucleotide templates. Methods Enzymol 1974;29:125–130.CrossRefPubMedGoogle Scholar
  76. 76.
    Houts GE, Miyagi M, Ellis C, et al. Reverse transcriptase from avian myeloblastosis virus. J Virol 1979;29:517–522.PubMedGoogle Scholar
  77. 77.
    Verma IM. Studies on reverse transcriptase of RNA tumor viruses III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNase H. J Virol 1975;15:843–854.PubMedGoogle Scholar
  78. 78.
    Marcus SL, Modak MJ. Observations on templatespecific conditions for DNA synthesis by avian myeloblastosis virus DNA polymerase. Nucleic Acids Res 1976;3:1473–1486.PubMedGoogle Scholar
  79. 79.
    Sambrook J, Russell DW. Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001:A4.24.Google Scholar
  80. 80.
    Broackes-Carter FC, Mouchel N, Gill D, et al. Temporal regulation of CFTR expression during ovine lung development: implications for CF gene therapy. Hum Mol Genet 2002;11:125–131.CrossRefPubMedGoogle Scholar
  81. 81.
    Dagnon K, Pacary E, Commo F, et al. Expression of erythropoietin and erythropoietin receptor in non-small cell lung carcinomas. Clin Cancer Res 2005;11:993–999.PubMedGoogle Scholar
  82. 82.
    Singhal S, Wiewrodt R, Malden LD, et al. Gene expression profiling of malignant mesothelioma. Clin Cancer Res 2003;9:3080–3097.PubMedGoogle Scholar
  83. 83.
    Lam KM, Oldenburg N, Khan MA, et al. Significance of reverse transcription polymerase chain reaction in the detection of human cytomegalovirus gene transcripts in thoracic organ transplant recipients. J Heart Lung Transplant 1998;17:555–565.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2008

Authors and Affiliations

  • Dwight Oliver
    • 1
  1. 1.Department of Pathology and Laboratory MedicineUniversity of Texas-Houston Medical SchoolHoustonUSA

Personalised recommendations