Advances in Clinical and Experimental Medicine

Title abbreviation: Adv Clin Exp Med
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ISSN 1899–5276 (print)
ISSN 2451-2680 (online)
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Advances in Clinical and Experimental Medicine

2009, vol. 18, nr 1, January-February, p. 85–92

Publication type: review article

Language: English

Application of Selected Methods Based on the Polymerase Chain Reaction in Medical Molecular Diagnostics

Wykorzystanie wybranych metod opartych na reakcji polimerazy do diagnostyki molekularnej chorób genetycznych

Izabela Łaczmańska1,, Karolina Pesz1,, Łukasz Łaczmański2,

1 Department of Genetics, Wroclaw Medical University, Poland

2 Department of Endocrinology and Diabetology, Wroclaw Medical University, Poland


A breakthrough in molecular biology came with the discovery of the polymerase chain reaction. It allowed the amplification of DNA fragments in vitro in an efficient, rapid, and inexpensive way. Furthermore, it is so versatile that the polymerase chain reaction technique has been used for a multitude of purposes and been found to be applicable in a variety of different disciplines. The authors present the most frequently used laboratory methods based on the polymerase reaction and discuss their application in diagnosing genetic disorders.


Rozwój diagnostyki genetycznej jest ściśle związany z odkryciem łańcuchowej reakcji polimerazy (PCR). Pozwala ona na szybką, wydajną i tanią amplifikację in vitro fragmentów DNA. Reakcja prowadzona przez polimerazę stała się ponadto podstawą do stworzenia i rozwoju wielu różnych metod powszechnie stosowanych w laboratoriach. Autorzy przedstawiają najczęściej wykorzystywane metody biologii molekularnej oparte na reakcji polimerazy oraz opisują ich zastosowanie w diagnostyce chorób uwarunkowanych genetycznie.

Key words

polymerase reaction, diagnostic methods

Słowa kluczowe

reakcja polimerazy, metody diagnostyczne

References (38)

  1. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N: Enzymatic amplification of betaglobin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985, 20, 230, 1350–1354.
  2. Sommer SS, Groszbach AR, Bottema CD: PCR amplification of specific alleles (PASA) is a general method for rapidly detecting known single−base changes. Biotechniques 1992, 12, 82–87.
  3. Okayama H, Curiel DT, Brantly ML, Holmes MD, Crystal RG: Rapid, nonradioactive detection of mutations in the human genome by allele−specific amplification. J Lab Clin Med 1989, 114, 105–113.
  4. Lubiński J, Górski B, Huzarski T, Byrski T, Gronwald J, Serrano−Fernández P, Domagała W, Chosia M, Uciński M, Grzybowska E, Lange D, Maka B, Mackiewicz A, Karczewska A, Bręborowicz J, Lamperska K, Stawicka M, Gozdecka−Grodecka S, Bębenek M, Sorokin D, Wojnar A, Haus O, Sir J, Mierzwa T, Niepsuj S, Gugała K, Góźdź S, Sygut J, Kozak−Klonowska B, Musiatowicz B, Posmyk M, Kordek R, Morawiec M, Zambrano O, Waśko B, Fudali L, Skret J, Surdyka D, Urbański K, Mituś J, Ryś J, Szwiec M, Rozmiarek A, Dziub
  5. Grzybowska E, Siemińska M, Zientek H, Kalinowska E, Michalska J, Utracka−Hutka B, Rogozińska−Szczepka J, Kaźmierczak−Maciejewska M: Germline mutations in the BRCA1 gene predisposing to breast and ovarian cancers in Upper Silesia population. Acta Biochim Pol 2002, 49, 351–356.
  6. Janiszewska H, Haus O, Lauda−Swieciak A, Pasińska M, Laskowski R, Szymański W, Górski B, Lubiński J: Frequency of three BRCA1 gene founder mutations in breast/ovarian cancer families from the Pomerania−Kujawy region of Poland. Clin Genet 2003, 64, 502–508.
  7. Chan PC, Wong BY, Ozcelik H, Cole DE: Simple and rapid detection of BRCA1 and BRCA2 mutations by multiplex mutagenically separated PCR. Clin Chem 1999, 45, 1285–1287.
  8. Jacobson DR, Moskovits T: Rapid, nonradioactive screening for activating ras oncogene mutations using PCRprimer introduced restriction analysis (PCR−PIRA). PCR Methods Appl 1991, 1, 146–148.
  9. Balmer D, Arredondo J, Samaco RC, LaSalle JM: MECP2 mutations in Rett syndrome adversely affect lymphocyte growth, but do not affect imprinted gene expression in blood or brain. Hum Genet 2002, 110, 545–552.
  10. Matijevic T, Knezevic J, Slavica M, Pavelic J: Rett syndrome: from the gene to the disease. Eur Neurol 2009, 61, 3–10.
  11. Górski B, Cybulski C, Huzarski T et al.: Breast cancer predisposing alleles in Poland. Breast Cancer Res Treat 2005, 92, 19–24.
  12. Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB: Methylation−specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 1996, 93, 9821–9826.
  13. Gutierrez MI, Siraj AK, Bhargava M, Ozbek U, Banavali S, Chaudhary MA, El Solh H, Bhatia K: Concurrent methylation of multiple genes in childhood ALL: Correlation with phenotype and molecular subgroup. Leukemia 2003, 17, 1845–1850.
  14. Hayslip J, Montero A: Tumor suppressor gene methylation in follicular lymphoma: a comprehensive review. Mol Cancer 2006, 6, 44–45.
  15. Nicholls RD, Knoll JH, Butler MG, Karam S, Lalande M: Genetic imprinting suggested by maternal heterodisomy in nondeletion Prader–Willi syndrome. Nature 1989, 16, 342, 281–285.
  16. Sutcliffe JS, Nakao M, Christian S, Orstavik KH, Tommerup N, Ledbetter DH, Beaudet AL: Deletions of a differentially methylated CpG island at the SNRPN gene define a putative imprinting control region. Nat Genet 1994, 8, 52–58.
  17. Gille JJ, Hogervorst FB, Pals G, Wijnen JT, van Schooten RJ, Dommering CJ, Meijer GA, Craanen ME, Nederlof PM, de Jong D, McElgunn CJ, Schouten JP, Menko FH: Genomic deletions of MSH2 and MLH1 in colorectal cancer families detected by a novel mutation detection approach. Br J Cancer 2002, 87, 892–897.
  18. Hogervorst FB, Nederlof PM, Gille JJ, McElgunn CJ, Grippeling M, Pruntel R, Regnerus R, van Welsem T, van Spaendonk R, Menko FH, Kluijt I, Dommering C, Verhoef S, Schouten JP, van’t Veer LJ, Pals G: Large genomic deletions and duplications in the BRCA1 gene identified by a novel quantitative method. Cancer Res 2003, 63, 1449–1453.
  19. Belogianni I, Apessos A, Mihalatos M, Razi E, Labropoulos S, Petounis A, Gaki V, Keramopoulos A, Pandis N, Kyriacou K, Hadjisavvas A, Kosmidis P, Yannoukakos D, Nasioulas G: Characterization of a novel large deletion and single point mutations in the BRCA1 gene in a Greek cohort of families with suspected hereditary breast cancer. BMC Cancer 2004, 4, 61.
  20. Nakagawa H, Hampel H, de la Chapelle A: Identification and characterization of genomic rearrangements of MSH2 and MLH1 in Lynch syndrome (HNPCC) by novel techniques. Hum Mutat 2003, 22, 258.
  21. Nygren AO, Ameziane N, Duarte HM, Vijzelaar RN, Waisfisz Q, Hess CJ, Schouten JP, Errami A: Methylation−specific MLPA (MS−MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nucleic Acids Res 2005, 33, 128.
  22. Eldering E, Spek CA, Aberson HL, Grummels A, Derks IA, de Vos AF, McElgunn CJ, Schouten JP: Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways. Nucleic Acids Res 2003, 31, 153.
  23. Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G: Relative quantification of 40 nucleic acid sequences by multiplex ligation−dependent probe amplification. Nucleic Acids Res 2002, 30, 57.
  24. Higuchi R, Dollinger G, Walsh PS, Griffith R: Simultaneous amplification and detection of specific DNA sequences. Biotechnology (N Y) 1992, 10, 413–417.
  25. Lee LG, Connell CR, Bloch W: Allelic discrimination by nick−translation PCR with fluorogenic probes. Nucleic Acids Res 1993, 21, 3761–3766.
  26. Livak KJ, Flood SJ, Marmaro J, Giusti W, Deetz K: 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.
  27. Valasek MA, Repa JJ: The power of real−time PCR. Adv Physiol Educ 2005, 29, 151–159.
  28. Morrison TB, Weis JJ, Wittwer CT: Quantification of low−copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 1998, 24, 954–958.
  29. Vandesompele J, De Paepe A, Speleman F: Elimination of primer−dimer artifacts and genomic coamplification using a two−step SYBR green I real−time RT−PCR. Anal Biochem 2002, 303, 95–98.
  30. Hiyoshi M, Hosoi S: Assay of DNA denaturation by polymerase chain reaction−driven fluorescent label incorporation and fluorescence resonance energy transfer. Anal Biochem 1994, 221, 306–311.
  31. Chen Q, Lentz BR: Fluorescence resonance energy transfer study of shape changes in membrane−bound bovine prothrombin and meizothrombin. Biochemistry 1997, 36, 4701–4711.
  32. Lee WI, Kantarjian H, Glassman A, Talpaz M, Lee MS: Quantitative measurement of BCR/abl transcripts using real−time polymerase chain reaction. Ann Oncol 2002, 13, 781–788.
  33. Venter JC, Adams MD, Myers EW et al.: The sequence of the human genome. Science 2001, 291, 1304–1351.
  34. Ahmadian A, Ehn M, Hober S: Pyrosequencing: history, biochemistry and future. Clin Chim Acta 2006, 363, 83–94.
  35. Shendure J, Mitra RD, Varma C, Church GM: Advanced sequencing technologies: methods and goals. Nat Rev Genet 2004, 5, 335–344.
  36. Pastinen T, Partanen J, Syvänen AC: Multiplex, fluorescent, solid−phase minisequencing for efficient screening of DNA sequence variation. Clin Chem 1996, 42, 1391–1397.
  37. Fiorentino F, Magli MC, Podini D, Ferraretti AP, Nuccitelli A, Vitale N, Baldi M, Gianaroli L: The minisequencing method: an alternative strategy for preimplantation genetic diagnosis of single gene disorders. Mol Hum Reprod 2003, 9, 399–410.
  38. Ronaghi M, Uhlén M, Nyrén P: A sequencing method based on real−time pyrophosphate. Science 1998, 281, 363–365.