Molecular Methods in Oncology: Targeted Mutational Analysis

  • Jason A. JarzembowskiEmail author
Part of the Molecular Pathology Library book series (MPLB)


Polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) are both molecular techniques that simultaneously exploit and are limited by sequence specificity. Both techniques have high sensitivity and specificity, accomplished through the use of primers and probes custom-designed for a given application. PCR is capable of exponentially amplifying target sequences, either as a direct method of detection or as a preliminary step to create ample material for subsequent assays. FISH allows the identification and localization of specific genetic sequences on chromosomes or other targets. Both techniques work well on formalin-fixed paraffin-embedded tissue, making them ideal for pediatric tumor studies. PCR and FISH are easy to perform and readily set up in most clinical and research laboratories. These assays have found widespread use in pediatric cancer diagnostics including mutational analysis, detecting fusion genes, and measuring gene expression.


Amplification Deletion DNA Fluorescence in situ hybridization Fusion gene Mutation Polymerase chain reaction Primers Probes Translocation 


  1. 1.
    Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana HG. Studies on polynucleotides. XCVI. Repair replications of short synthetic DNAs as catalyzed by DNA polymerases. J Mol Biol. 1971;56:341–61.CrossRefPubMedGoogle Scholar
  2. 2.
    Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–50.CrossRefPubMedGoogle Scholar
  3. 3.
    Saiki RK, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988;239:487–91.CrossRefGoogle Scholar
  4. 4.
    Impraim CC, Saiki RK, Erlich HA, Teplitz RL. Analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues by enzymatic amplification and hybridization with sequence-specific oligonucleotides. Biochem Biophys Res Commun. 1987;142(3):710–6.CrossRefPubMedGoogle Scholar
  5. 5.
    Ren ZP, Sällström J, Sundström C, Nistér M, Olsson Y. Recovering DNA and optimizing PCR conditions from microdissected formalin-fixed and paraffin-embedded materials. Pathobiology. 2000;68(4–5):215–7.CrossRefPubMedGoogle Scholar
  6. 6.
    Greer CE, Lund JK, Manos MM. PCR amplification from paraffin-embedded tissues: recommendations on fixatives for long-term storage and prospective studies. PCR Methods Appl. 1991;1(1):46–50.CrossRefPubMedGoogle Scholar
  7. 7.
    Karlsen F, Kalantari M, Chitemerere M, Johansson B, Hagmar B. Modifications of human and viral deoxyribonucleic acid by formaldehyde fixation. Lab Investig. 1994;71(4):604–11.PubMedGoogle Scholar
  8. 8.
    Wickham CL, Sarsfield P, Joyner MV, Jones DB, Ellard S, Wilkins B. Formic acid decalcification of bone marrow trephines degrades DNA: alternative use of EDTA allows the amplification and sequencing of relatively long PCR products. Mol Pathol. 2000;53(6):336. Erratum in: Mol Pathol 2001 Apr;54(2):120.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Farkas DH, Drevon AM, Kiechle FL, DiCarlo RG, Heath EM, Crisan D. Specimen stability for DNA-based diagnostic testing. Diagn Mol Pathol. 1996;5(4):227–35. Erratum in: Diagn Mol Pathol 1997 Jun;6(3):178.CrossRefPubMedGoogle Scholar
  10. 10.
    Visvikis S, Schlenck A, Maurice M. DNA extraction and stability for epidemiological studies. Clin Chem Lab Med. 1998;36(8):551–5.CrossRefPubMedGoogle Scholar
  11. 11.
    Hung JH, Weng Z. Designing polymerase chain reaction primers using Primer3Plus. Cold Spring Harb Protoc. 2016;2016(9):pdb.prot093096.Google Scholar
  12. 12.
    Hommelsheim CM, Frantzeskakis L, Huang M, Ülker B. PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications. Sci Rep. 2014;4:5052.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Terpe K. Overview of thermostable DNA polymerases for classical PCR applications: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol. 2013;97(24):10243–54.CrossRefPubMedGoogle Scholar
  14. 14.
    Pfeifer JD. Polymerase chain reaction. In: Pfeifer JD, editor. Molecular genetic testing in surgical pathology. Philadelphia: Lippincott Williams & Wilkins; 2006.Google Scholar
  15. 15.
    Kwok S, Higuchi R. Avoiding false positives with PCR. Nature. 1989;339(6221):237–8. Erratum in: Nature 1989 Jun 8;339(6224):490.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Langerak AW, Groenen PJ, Brüggemann M, Beldjord K, Bellan C, Bonello L, et al. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations. Leukemia. 2012;26(10):2159–71.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Rezuke WN, Abernathy EC, Tsongalis GJ. Molecular diagnosis of B- and T-cell lymphomas: fundamental principles and clinical applications. Clin Chem. 1997;43(10):1814–23.PubMedGoogle Scholar
  18. 18.
    Matsuda K, Sugano M, Honda T. PCR for monitoring of minimal residual disease in hematologic malignancy. Clin Chim Acta. 2012;413(1–2):74–80.CrossRefPubMedGoogle Scholar
  19. 19.
    Van Deerlin VM, Leonard DG. Bone marrow engraftment analysis after allogeneic bone marrow transplantation. Clin Lab Med. 2000;20(1):197–225.CrossRefPubMedGoogle Scholar
  20. 20.
    Diaz-Cano SJ, Blanes A, Wolfe HJ. PCR techniques for clonality assays. Diagn Mol Pathol. 2001;10(1):24–33.CrossRefPubMedGoogle Scholar
  21. 21.
    Kristensen LS, Hansen LL. PCR-based methods for detecting single-locus DNA methylation biomarkers in cancer diagnostics, prognostics, and response to treatment. Clin Chem. 2009;55(8):1471–83.CrossRefPubMedGoogle Scholar
  22. 22.
    Berg KD, Glaser CL, Thompson RE, Hamilton SR, Griffin CA, Eshleman JR. Detection of microsatellite instability by fluorescence multiplex polymerase chain reaction. J Mol Diagn. 2000;2(1):20–8.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Jensen EC. Real-time reverse transcription polymerase chain reaction to measure mRNA: use, limitations, and presentation of results. Anat Rec (Hoboken). 2012;295(1):1–3.CrossRefGoogle Scholar
  24. 24.
    Mayer G, Müller J, Lünse CE. RNA diagnostics: real-time RT-PCR strategies and promising novel target RNAs. Wiley Interdiscip Rev RNA. 2011;2(1):32–41.CrossRefPubMedGoogle Scholar
  25. 25.
    Bridge JA. Reverse transcription-polymerase chain reaction molecular testing of cytology specimens: pre-analytic and analytic factors. Cancer. 2017;125(1):11–9.Google Scholar
  26. 26.
    Merckx J, Wali R, Schiller I, Caya C, Gore GC, Chartrand C, et al. Diagnostic accuracy of novel and traditional rapid tests for influenza infection compared with reverse transcriptase polymerase chain reaction: a systematic review and meta-analysis. Ann Intern Med. 2017;167(6):394–409.CrossRefPubMedGoogle Scholar
  27. 27.
    Wilkin F, Gagné N, Paquette J, Oligny LL, Deal C. Pediatric adrenocortical tumors: molecular events leading to insulin-like growth factor II gene overexpression. J Clin Endocrinol Metab. 2000;85(5):2048–56.PubMedGoogle Scholar
  28. 28.
    Beiske K, Ambros PF, Burchill SA, Cheung IY, Swerts K. Detecting minimal residual disease in neuroblastoma patients-the present state of the art. Cancer Lett. 2005;228(1–2):229–40.CrossRefPubMedGoogle Scholar
  29. 29.
    Lianidou ES, Markou A. Molecular assays for the detection and characterization of CTCs. Recent Results Cancer Res. 2012;195:111–23.CrossRefPubMedGoogle Scholar
  30. 30.
    Schrappe M. Detection and management of minimal residual disease in acute lymphoblastic leukemia. Hematology Am Soc Hematol Educ Program. 2014;2014(1):244–9.PubMedGoogle Scholar
  31. 31.
    Vo KT, Edwards JV, Epling CL, Sinclair E, Hawkins DS, Grier HE, et al. Impact of two measures of micrometastatic disease on clinical outcomes in patients with newly diagnosed ewing sarcoma: a report from the children's oncology group. Clin Cancer Res. 2016;22(14):3643–50.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Arya M, Shergill IS, Williamson M, Gommersall L, Arya N, Patel HR. Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn. 2005;5(2):209–19.CrossRefPubMedGoogle Scholar
  33. 33.
    Faner R, Casamitjana N, Colobran R, Ribera A, Pujol-Borrell R, Palou E, et al. HLA-B27 genotyping by fluorescent resonance emission transfer (FRET) probes in real-time PCR. Hum Immunol. 2004;65(8):826–38.CrossRefPubMedGoogle Scholar
  34. 34.
    Edwards MC, Gibbs RA. Multiplex PCR: advantages, development, and applications. PCR Methods Appl. 1994;3(4):S65–75.CrossRefPubMedGoogle Scholar
  35. 35.
    Bottema CD, Sommer SS. PCR amplification of specific alleles: rapid detection of known mutations and polymorphisms. Mutat Res. 1993;288(1):93–102.CrossRefPubMedGoogle Scholar
  36. 36.
    Matsuda K. PCR-based detection methods for single-nucleotide polymorphism or mutation: real-time PCR and its substantial contribution toward technological refinement. Adv Clin Chem. 2017;80:45–72.CrossRefPubMedGoogle Scholar
  37. 37.
    Ugozzoli L, Wallace RB. Allele-specific polymerase chain reaction. Methods. 1991;2(1):42–8.CrossRefGoogle Scholar
  38. 38.
    Quiros RM, Ding HG, Gattuso P, Prinz RA, Xu X. Evidence that one subset of anaplastic thyroid carcinomas are derived from papillary carcinomas due to BRAF and p53 mutations. Cancer. 2005;103(11):2261–8.CrossRefPubMedGoogle Scholar
  39. 39.
    Wilson RC, Wei JQ, Cheng KC, Mercado AB, New MI. Rapid deoxyribonucleic acid analysis by allele-specific polymerase chain reaction for detection of mutations in the steroid 21-hydroxylase gene. J Clin Endocrinol Metab. 1995;80(5):1635–40.PubMedGoogle Scholar
  40. 40.
    Le Van Kim C, Colin Y, Brossard Y, Cartron JP. Rh haemolytic disease of the newborn and Rh genotyping by RFLP--and allele-specific--PCR. Transfus Clin Biol. 1995;2(4):317–24.CrossRefGoogle Scholar
  41. 41.
    Vogelstein B, Kinzler KW. Digital PCR. Proc Natl Acad Sci U S A. 1999;96(16):9236–41.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Huang A, Zhang X, Zhou SL, Cao Y, Huang XW, Fan J, Yang XR, Zhou J. Detecting circulating tumor DNA in hepatocellular carcinoma patients using droplet digital PCR is feasible and reflects intratumoral heterogeneity. J Cancer. 2016;7(13):1907–14.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Olmedillas-López S, García-Arranz M, García-Olmo D. Current and emerging applications of droplet digital PCR in oncology. Mol Diagn Ther. 2017;21(5):493–510.CrossRefPubMedGoogle Scholar
  44. 44.
    Sofronova JK, Ilinsky YY, Orishchenko KE, Chupakhin EG, Lunev EA, Mazunin IO. Detection of mutations in mitochondrial DNA by droplet digital PCR. Biochemistry (Mosc). 2016;81(10):1031–7.CrossRefGoogle Scholar
  45. 45.
    Baylin SB, Makos M, Wu JJ, Yen RW, de Bustros A, Vertino P, et al. Abnormal patterns of DNA methylation in human neoplasia: potential consequences for tumor progression. Cancer Cells. 1991;3(10):383–90.PubMedGoogle Scholar
  46. 46.
    Goyama S, Kitamura T. Epigenetics in normal and malignant hematopoiesis: an overview and update 2017. Cancer Sci. 2017;108(4):553–62.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    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(18):9821–6.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Langer-Safer PR, Levine M, Ward DC. Immunological method for mapping genes on Drosophila polytene chromosomes. Proc Natl Acad Sci U S A. 1982;79(14):4381–5.CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Bauman JG, Wiegant J, Borst P, van Duijn P. A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome labelled RNA. Exp Cell Res. 1980;128(2):485–90.CrossRefPubMedGoogle Scholar
  50. 50.
    Cheung SW, Tishler PV, Atkins L, Sengupta SK, Modest EJ, Forget BG. Gene mapping by fluorescent in situ hybridization. Cell Biol Int Rep. 1977;1(3):255–62.CrossRefPubMedGoogle Scholar
  51. 51.
    Manning JE, Hershey ND, Broker TR, Pellegrini M, Mitchell HK, Davidson N. A new method of in situ hybridization. Chromosoma. 1975;53(2):107–17.CrossRefPubMedGoogle Scholar
  52. 52.
    Rudkin GT, Stollar BD. High resolution detection of DNA-RNA hybrids in situ by indirect immunofluorescence. Nature. 1977;265(5593):472–3.CrossRefPubMedGoogle Scholar
  53. 53.
    Wu M, Davidson N. Transmission electron microscopic method for gene mapping on polytene chromosomes by in situ hybridization. Proc Natl Acad Sci U S A. 1981;78(11):7059–63.CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Bayani J, Squire JA. Fluorescence in situ hybridization (FISH). Curr Protoc Cell Biol. 2004;Chapter 22:Unit 22.4.PubMedGoogle Scholar
  55. 55.
    Tucker JD. Reflections on the development and application of FISH whole chromosome painting. Mutat Res Rev Mutat Res. 2015;763:2–14.CrossRefPubMedGoogle Scholar
  56. 56.
    Swennenhuis JF, Terstappen L. Sample preparation methods following cellsearch approach compatible of single-cell whole-genome amplification: an overview. Methods Mol Biol. 2015;1347:57–67.CrossRefPubMedGoogle Scholar
  57. 57.
    van Rijk A, Svenstroup-Poulsen T, Jones M, Cabeçadas J, Cigudosa JC, Leoncini L, et al. Double-staining chromogenic in situ hybridization as a useful alternative to split-signal fluorescence in situ hybridization in lymphoma diagnostics. Haematologica. 2010;95(2):247–52.CrossRefPubMedGoogle Scholar
  58. 58.
    van Gijlswijk RP, van de Corput MP, Bezrookove V, Wiegant J, Tanke HJ, Raap AK. Synthesis and purification of horseradish peroxidase-labeled oligonucleotides for tyramide-based fluorescence in situ hybridization. Histochem Cell Biol. 2000;113(3):175–80.CrossRefPubMedGoogle Scholar
  59. 59.
    Knight SJ, Flint J. Perfect endings: a review of subtelomeric probes and their use in clinical diagnosis. J Med Genet. 2000;37(6):401–9.CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Ducray F, Idbaih A, Wang X-W, Cheneau C, Labussiere M, Sanson M. Predictive and prognostic factors for gliomas. Expert Rev Anticancer Ther. 2011;11(5):781–9.CrossRefPubMedGoogle Scholar
  61. 61.
    Bridge RS, Rajaram V, Dehner LP, Pfeifer JD, Perry A. Molecular diagnosis of Ewing sarcoma/primitive neuroectodermal tumor in routinely processed tissue: a comparison of two FISH strategies and RT-PCR in malignant round cell tumors. Mod Pathol. 2006;19(1):1–8.CrossRefPubMedGoogle Scholar
  62. 62.
    Tubbs RR, Pettay J, Roche P, et al. Concomitant oncoprotein detection with fluorescence in situ hybridization (CODFISH): a fluorescence-based assay enabling simultaneously visualization of gene amplification and encoded protein expression. J Mol Diagn. 2000;2:78–83.CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Weber-Matthiesen K, Deerberg J, Poetsch M, Grote W, Schlegelberger B. Numerical chromosome aberrations are present within the CD30+ Hodgkin and Reed-Sternberg cells in 100% of analyzed cases of Hodgkin's disease. Blood. 1995;86(4):1464–8.PubMedGoogle Scholar
  64. 64.
    Nolte M, Werner M, Ewig M, et al. Fluorescence in situ hybridization (FISH) is a reliable diagnostic tool for detection of the 9;22 translocation. Leuk Lymphoma. 1996;22(3–4):287–94.Google Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Division of Pediatric PathologyMedical College of WisconsinMilwaukeeUSA

Personalised recommendations