Skip to main content
SpringerLink
Log in

Molecular Pathology Methods

  • Chapter
Molecular Pathology in Clinical Practice

Abstract

Molecular pathology is based on the principles, techniques, and tools of molecular biology as they are applied to diagnostic medicine in the clinical laboratory. Molecular biology methods were used to elucidate the genetic and molecular basis of many diseases, and these discoveries ultimately led to the field of molecular pathology. As molecular research identifies the most fundamental causes and markers of disease, clinical testing of human and pathogen genetic material has become routine in laboratory medicine. Underlying mutations responsible for genetic diseases, including cancers, are being discovered and used in clinical molecular tests. In this chapter, fundamental and more advanced molecular biology techniques, as practiced in the molecular pathology laboratory, are reviewed.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Fiebelkorn KR, Lee BG, Hill CE, Caliendo AM, Nolte FS. Clinical evaluation of an automated nucleic acid isolation system. Clin Chem. 2002;48(9):1613–5.

    PubMed  CAS  Google Scholar 

  2. Kessler HH, Muhlbauer G, Stelzl E, Daghofer E, Santner BI, Marth E. Fully automated nucleic acid extraction: MagNA Pure LC. Clin Chem. 2001;47(6):1124–6.

    PubMed  CAS  Google Scholar 

  3. Williams SM, Meadows CA, Lyon E. Automated DNA extraction for real-time PCR. Clin Chem. 2002;48(9):1629–30.

    PubMed  CAS  Google Scholar 

  4. Davies J, Reznikoff WS, editors. Milestones in biotechnology: classic papers on genetic engineering. Boston: Butterworth-Heinemann; 1992.

    Google Scholar 

  5. Lm W. A nested reverse-transcriptase polymerase chain reaction assay to detect BCR/ABL. In: Kileen AA, editor. Methods in molecular medicine molecular pathology protocols. Totowa, NJ: Humana Press; 2001. p. 105–14.

    Google Scholar 

  6. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977;74(12):5463–7.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  7. Esch RK. Basic nucleic acid procedures. In: Coleman WB, Tsongalis GJ, editors. Molecular diagnostics for the clinical laboratorian. Totowa, NJ: Humana Press; 1997. p. 55–8.

    Google Scholar 

  8. Schmalzing D, Koutny L, Salas-Solano O, Adourian A, Matsudaira P, Ehrlich D. Recent developments in DNA sequencing by capillary and microdevice electrophoresis. Electrophoresis. 1999;20(15–16):3066–77. doi:10.1002/(SICI)1522-2683(19991001)20:15/16<3066::AID-ELPS3066>3.0.CO;2-X.

    Article  PubMed  CAS  Google Scholar 

  9. Farkas DH. Specimen procurement, processing, tracking, and testing by the Southern blot. In: Farkas DH, editor. Molecular biology and pathology: a guidebook for quality control. San Diego: Academic; 1993. p. 51–75.

    Google Scholar 

  10. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. 1986. Biotechnology. 1992;24:17–27.

    PubMed  CAS  Google Scholar 

  11. Farkas DH. Thermal Cyclers. In: Laboratory instrument evaluation, verification, and maintenance manual. Koenig AS (Editor). Northfield, IL: College of American Pathologists; 1998. p.130–3.

    Google Scholar 

  12. Loeffelholz MJ, Lewinski CA, Silver SR, Purohit AP, Herman SA, Buonagurio DA, Dragon EA. Detection of Chlamydia trachomatis in endocervical specimens by polymerase chain reaction. J Clin Microbiol. 1992;30(11):2847–51.

    PubMed Central  PubMed  CAS  Google Scholar 

  13. DiDomenico N, Link H, Knobel R, Caratsch T, Weschler W, Loewy ZG, Rosenstraus M. COBAS AMPLICOR: fully automated RNA and DNA amplification and detection system for routine diagnostic PCR. Clin Chem. 1996;42(12):1915–23.

    PubMed  CAS  Google Scholar 

  14. Greenfield L, White TJ. Sample preparation methods. In: Persing DH, Smith TF, Tenover FC, White TJ, editors. Diagnostics molecular microbiology principles and applications. Washington, DC: American Society for Microbiology; 1993. p. 126–7.

    Google Scholar 

  15. Liu XY, Nelson D, Grant C, Morthland V, Goodnight SH, Press RD. Molecular detection of a common mutation in coagulation factor V causing thrombosis via hereditary resistance to activated protein C. Diagn Mol Pathol. 1995;4(3):191–7.

    Article  PubMed  CAS  Google Scholar 

  16. Lindeman R, Hu SP, Volpato F, Trent RJ. Polymerase chain reaction (PCR) mutagenesis enabling rapid non-radioactive detection of common beta-thalassaemia mutations in Mediterraneans. Br J Haematol. 1991;78(1):100–4.

    Article  PubMed  CAS  Google Scholar 

  17. Sorscher EJ, Huang Z. Diagnosis of genetic disease by primer-specified restriction map modification, with application to cystic fibrosis and retinitis pigmentosa. Lancet. 1991;337(8750):1115–8. doi:0140-6736(91)92785-Z [pii].

    Article  PubMed  CAS  Google Scholar 

  18. Khanna M, Park P, Zirvi M, Cao W, Picon A, Day J, Paty P, Barany F. Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors. Oncogene. 1999;18(1):27–38. doi:10.1038/sj.onc.1202291.

    Article  PubMed  CAS  Google Scholar 

  19. McMillin DE, Muldrow LL, Laggette SJ. Simultaneous detection of toxin A and toxin B genetic determinants of Clostridium difficile using the multiplex polymerase chain reaction. Can J Microbiol. 1992;38(1):81–3.

    Article  PubMed  CAS  Google Scholar 

  20. Jama M, Nelson L, Pont-Kingdon G, Mao R, Lyon E. Simultaneous amplification, detection, and analysis of common mutations in the galactose-1-phosphate uridyl transferase gene. J Mol Diagn. 2007;9(5):618–23. doi:10.2353/jmoldx.2007.070027.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  21. Farkas DH, Miltgen NE, Stoerker J, van den Boom D, Highsmith WE, Cagasan L, McCullough R, Mueller R, Tang L, Tynan J, Tate C, Bombard A. The suitability of matrix assisted laser desorption/ionization time of flight mass spectrometry in a laboratory developed test using cystic fibrosis carrier screening as a model. J Mol Diagn. 2010;12(5):611–9. doi:10.2353/jmoldx.2010.090233.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  22. Sugita T, Nakajima M, Ikeda R, Niki Y, Matsushima T, Shinoda T. A nested PCR assay to detect DNA in sera for the diagnosis of deep-seated trichosporonosis. Microbiol Immunol. 2001;45(2):143–8.

    Article  PubMed  CAS  Google Scholar 

  23. Ferrie RM, Schwarz MJ, Robertson NH, Vaudin S, Super M, Malone G, Little S. Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene. Am J Hum Genet. 1992;51(2):251–62.

    PubMed Central  PubMed  CAS  Google Scholar 

  24. Bugawan TL, Begovich AB, Erlich HA. Rapid HLA-DPB typing using enzymatically amplified DNA and nonradioactive sequence-specific oligonucleotide probes. Immunogenetics. 1991;34(6):413.

    Article  PubMed  CAS  Google Scholar 

  25. Jarvius J, Nilsson M, Landegren U. Oligonucleotide ligation assay. Methods Mol Biol. 2003;212:215–28.

    PubMed  CAS  Google Scholar 

  26. Nickerson DA, Kaiser R, Lappin S, Stewart J, Hood L, Landegren U. Automated DNA diagnostics using an ELISA-based oligonucleotide ligation assay. Proc Natl Acad Sci U S A. 1990;87(22):8923–7.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  27. Tsiatis AC, Norris-Kirby A, Rich RG, Hafez MJ, Gocke CD, Eshleman JR, Murphy KM. Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications. J Mol Diagn. 2010;12(4):425–32. doi:10.2353/jmoldx.2010.090188.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  28. Baltimore D. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature. 1970;226(5252):1209–11.

    Article  PubMed  CAS  Google Scholar 

  29. Bernard PS, Wittwer CT. Real-time PCR technology for cancer diagnostics. Clin Chem. 2002;48(8):1178–85.

    PubMed  CAS  Google Scholar 

  30. Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol. 2000;25(2):169–93. doi:JME00927 [pii].

    Article  PubMed  CAS  Google Scholar 

  31. Lay MJ, Wittwer CT. Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. Clin Chem. 1997;43(12):2262–7.

    PubMed  CAS  Google Scholar 

  32. Olek A, Oswald J, Walter J. A modified and improved method for bisulphite based cytosine methylation analysis. Nucleic Acids Res. 1996;24(24):5064–6. doi:l60209 [pii].

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  33. Herman JG, Graff JR, Myohanen 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.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  34. Lo YM, Wong IH, Zhang J, Tein MS, Ng MH, Hjelm NM. Quantitative analysis of aberrant p16 methylation using real-time quantitative methylation-specific polymerase chain reaction. Cancer Res. 1999;59(16):3899–903.

    PubMed  CAS  Google Scholar 

  35. Emanuel BS, Shaikh TH. Segmental duplications: an 'expanding' role in genomic instability and disease. Nat Rev Genet. 2001;2(10):791–800. doi:10.1038/35093500.

    Article  PubMed  CAS  Google Scholar 

  36. Aldred MA, Vijayakrishnan J, James V, Soubrier F, Gomez-Sanchez MA, Martensson G, Galie N, Manes A, Corris P, Simonneau G, Humbert M, Morrell NW, Trembath RC. BMPR2 gene rearrangements account for a significant proportion of mutations in familial and idiopathic pulmonary arterial hypertension. Hum Mutat. 2006;27(2):212–3. doi:10.1002/humu.9398.

    Article  PubMed  Google Scholar 

  37. Aretz S, Stienen D, Uhlhaas S, Stolte M, Entius MM, Loff S, Back W, Kaufmann A, Keller KM, Blaas SH, Siebert R, Vogt S, Spranger S, Holinski-Feder E, Sunde L, Propping P, Friedl W. High proportion of large genomic deletions and a genotype phenotype update in 80 unrelated families with juvenile polyposis syndrome. J Med Genet. 2007;44(11):702–9. doi:10.1136/jmg.2007.052506.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  38. Beetz C, Nygren AO, Schickel J, Auer-Grumbach M, Burk K, Heide G, Kassubek J, Klimpe S, Klopstock T, Kreuz F, Otto S, Schule R, Schols L, Sperfeld AD, Witte OW, Deufel T. High frequency of partial SPAST deletions in autosomal dominant hereditary spastic paraplegia. Neurology. 2006;67(11):1926–30. doi:10.1212/01.wnl.0000244413.49258.f5.

    Article  PubMed  CAS  Google Scholar 

  39. Depienne C, Fedirko E, Forlani S, Cazeneuve C, Ribai P, Feki I, Tallaksen C, Nguyen K, Stankoff B, Ruberg M, Stevanin G, Durr A, Brice A. Exon deletions of SPG4 are a frequent cause of hereditary spastic paraplegia. J Med Genet. 2007;44(4):281–4. doi:10.1136/jmg.2006.046425.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  40. Kanno J, Hutchin T, Kamada F, Narisawa A, Aoki Y, Matsubara Y, Kure S. Genomic deletion within GLDC is a major cause of non-ketotic hyperglycinaemia. J Med Genet. 2007;44(3):e69. doi:10.1136/jmg.2006.043448.

    Article  PubMed Central  PubMed  Google Scholar 

  41. Kluwe L, Nygren AO, Errami A, Heinrich B, Matthies C, Tatagiba M, Mautner V. Screening for large mutations of the NF2 gene. Genes Chromosomes Cancer. 2005;42(4):384–91. doi:10.1002/gcc.20138.

    Article  PubMed  CAS  Google Scholar 

  42. Michils G, Tejpar S, Thoelen R, van Cutsem E, Vermeesch JR, Fryns JP, Legius E, Matthijs G. Large deletions of the APC gene in 15% of mutation-negative patients with classical polyposis (FAP): a Belgian study. Hum Mutat. 2005;25(2):125–34. doi:10.1002/humu.20122.

    Article  PubMed  CAS  Google Scholar 

  43. Redeker EJ, de Visser AS, Bergen AA, Mannens MM. Multiplex ligation-dependent probe amplification (MLPA) enhances the molecular diagnosis of aniridia and related disorders. Mol Vis. 2008;14:836–40.

    PubMed Central  PubMed  CAS  Google Scholar 

  44. Taylor CF, Charlton RS, Burn J, Sheridan E, Taylor GR. Genomic deletions in MSH2 or MLH1 are a frequent cause of hereditary non-polyposis colorectal cancer: identification of novel and recurrent deletions by MLPA. Hum Mutat. 2003;22(6):428–33. doi:10.1002/humu.10291.

    Article  PubMed  CAS  Google Scholar 

  45. 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(12):e57.

    Article  PubMed Central  PubMed  Google Scholar 

  46. 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(23):e153.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  47. 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(14):e128. doi:10.1093/nar/gni127.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  48. Bittel DC, Kibiryeva N, Butler MG. Methylation-specific multiplex ligation-dependent probe amplification analysis of subjects with chromosome 15 abnormalities. Genet Test. 2007;11(4):467–75. doi:10.1089/gte.2007.0061.

    Article  PubMed  CAS  Google Scholar 

  49. Dikow N, Nygren AO, Schouten JP, Hartmann C, Kramer N, Janssen B, Zschocke J. Quantification of the methylation status of the PWS/AS imprinted region: comparison of two approaches based on bisulfite sequencing and methylation-sensitive MLPA. Mol Cell Probes. 2007;21(3):208–15. doi:10.1016/j.mcp.2006.12.002.

    Article  PubMed  CAS  Google Scholar 

  50. Procter M, Chou LS, Tang W, Jama M, Mao R. Molecular diagnosis of Prader-Willi and Angelman syndromes by methylation-specific melting analysis and methylation-specific multiplex ligation-dependent probe amplification. Clin Chem. 2006;52(7):1276–83. doi:10.1373/clinchem.2006.067603.

    Article  PubMed  CAS  Google Scholar 

  51. Hess CJ, Errami A, Berkhof J, Denkers F, Ossenkoppele GJ, Nygren AO, Schuurhuis GJ, Waisfisz Q. Concurrent methylation of promoters from tumor associated genes predicts outcome in acute myeloid leukemia. Leuk Lymphoma. 2008;49(6):1132–41. doi:10.1080/10428190802035990.

    Article  PubMed  CAS  Google Scholar 

  52. Jeuken J, Cornelissen S, Boots-Sprenger S, Gijsen S, Wesseling P. Multiplex ligation-dependent probe amplification: a diagnostic tool for simultaneous identification of different genetic markers in glial tumors. J Mol Diagn. 2006;8(4):433–43. doi:10.2353/jmoldx.2006.060012.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  53. Fodde R, Losekoot M. Mutation detection by denaturing gradient gel electrophoresis (DGGE). Hum Mutat. 1994;3(2):83–94. doi:10.1002/humu.1380030202.

    Article  PubMed  CAS  Google Scholar 

  54. Lerman LS, Beldjord C. Comprehensive mutation detection with denaturing gradient gel electrophoresis. In: Cotton RGH, Edkins E, Forrest S, editors. Mutation detection: a practical approach. Oxford: Oxford University Press; 1998. p. 35–9.

    Google Scholar 

  55. Riesner D, Steger G, Zimmat R, Owens RA, Wagenhofer M, Hillen W, Vollbach S, Henco K. Temperature-gradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions. Electrophoresis. 1989;10(5–6):377–89. doi:10.1002/elps.1150100516.

    Article  PubMed  CAS  Google Scholar 

  56. Rosenbaum V, Riesner D. Temperature-gradient gel electrophoresis. Thermodynamic analysis of nucleic acids and proteins in purified form and in cellular extracts. Biophys Chem. 1987;26(2–3):235–46. doi:0301-4622(87)80026-1 [pii].

    Article  PubMed  CAS  Google Scholar 

  57. van der Luijt RB, Khan PM, Vasen HF, Tops CM, van Leeuwen-Cornelisse IS, Wijnen JT, van der Klift HM, Plug RJ, Griffioen G, Fodde R. Molecular analysis of the APC gene in 105 Dutch kindreds with familial adenomatous polyposis: 67 germline mutations identified by DGGE, PTT, and southern analysis. Hum Mutat. 1997;9(1):7–16. doi:10.1002/(SICI)1098-1004(1997)9:1<7::AID-HUMU2>3.0.CO;2-8 [pii] 10.1002/(SICI)1098-1004(1997)9:1<7::AID-HUMU2>3.0.CO;2-8.

    Article  PubMed  Google Scholar 

  58. De Braekeleer M, Mari C, Verlingue C, Allard C, Leblanc JP, Simard F, Aubin G, Ferec C. Complete identification of cystic fibrosis transmembrane conductance regulator mutations in the CF population of Saguenay Lac-Saint-Jean (Quebec, Canada). Clin Genet. 1998;53(1):44–6.

    Article  PubMed  Google Scholar 

  59. Alkan S, Cosar E, Ergin M, Hsi E. Detection of T-cell receptor-gamma gene rearrangement in lymphoproliferative disorders by temperature gradient gel electrophoresis. Arch Pathol Lab Med. 2001;125(2):202–7. doi:10.1043/0003-9985(2001)125<0202:DOTCRG>2.0.CO;2.

    PubMed  CAS  Google Scholar 

  60. Nagamine CM, Chan K, Lau YF. A PCR artifact: generation of heteroduplexes. Am J Hum Genet. 1989;45(2):337–9.

    PubMed Central  PubMed  CAS  Google Scholar 

  61. Bhattacharyya A, Lilley DM. The contrasting structures of mismatched DNA sequences containing looped-out bases (bulges) and multiple mismatches (bubbles). Nucleic Acids Res. 1989;17(17):6821–40.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  62. Hayashi K. PCR-SSCP: a simple and sensitive method for detection of mutations in the genomic DNA. PCR Methods Appl. 1991;1(1):34–8.

    Article  PubMed  CAS  Google Scholar 

  63. Liu Q, Feng J, Buzin C, Wen C, Nozari G, Mengos A, Nguyen V, Liu J, Crawford L, Fujimura FK, Sommer SS. Detection of virtually all mutations-SSCP (DOVAM-S): a rapid method for mutation scanning with virtually 100% sensitivity. Biotechniques. 1999;26(5):932. 936–8, 940–2.

    PubMed  CAS  Google Scholar 

  64. Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci U S A. 1989;86(8):2766–70.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  65. Widjojoatmodjo MN, Fluit AC, Verhoef J. Molecular identification of bacteria by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene. J Clin Microbiol. 1995;33(10):2601–6.

    PubMed Central  PubMed  CAS  Google Scholar 

  66. Liu W, Smith DI, Rechtzigel KJ, Thibodeau SN, James CD. Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations. Nucleic Acids Res. 1998;26(6):1396–400. doi:gkb270 [pii].

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  67. O’Donovan MC, Oefner PJ, Roberts SC, Austin J, Hoogendoorn B, Guy C, Speight G, Upadhyaya M, Sommer SS, McGuffin P. Blind analysis of denaturing high-performance liquid chromatography as a tool for mutation detection. Genomics. 1998;52(1):44–9. doi:10.1006/geno.1998.5411. S0888-7543(98)95411-8 [pii].

    Article  PubMed  Google Scholar 

  68. Wagner T, Stoppa-Lyonnet D, Fleischmann E, Muhr D, Pages S, Sandberg T, Caux V, Moeslinger R, Langbauer G, Borg A, Oefner P. Denaturing high-performance liquid chromatography detects reliably BRCA1 and BRCA2 mutations. Genomics. 1999;62(3):369–76. doi:10.1006/geno.1999.6026. S0888-7543(99)96026-3 [pii].

    Article  PubMed  CAS  Google Scholar 

  69. Roest PA, Roberts RG, Sugino S, van Ommen GJ, den Dunnen JT. Protein truncation test (PTT) for rapid detection of translation-terminating mutations. Hum Mol Genet. 1993;2(10):1719–21.

    Article  PubMed  CAS  Google Scholar 

  70. Pohlreich P, Stribrna J, Kleibl Z, Zikan M, Kalbacova R, Petruzelka L, Konopasek B. Mutations of the BRCA1 gene in hereditary breast and ovarian cancer in the Czech Republic. Med Princ Pract. 2003;12(1):23–9. doi:10.1159/00006816368163 [pii].

    Article  PubMed  CAS  Google Scholar 

  71. Zajac V, Kovac M, Kirchhoff T, Stevurkova V, Tomka M. The most frequent APC mutations among Slovak familial adenomatous polyposis patients. Adenomatous polyposis coli. Neoplasma. 2002;49(6):356–61.

    PubMed  CAS  Google Scholar 

  72. McKinney JT, Longo N, Hahn SH, Matern D, Rinaldo P, Strauss AW, Dobrowolski SF. Rapid, comprehensive screening of the human medium chain acyl-CoA dehydrogenase gene. Mol Genet Metab. 2004;82(2):112–20. doi:10.1016/j.ymgme.2004.04.004.

    Article  PubMed  CAS  Google Scholar 

  73. Burczak JD, Ching S, Hu H-Y, Lee HH. Ligase chain reaction for the detection of infectious agents. In: Wiedbrauk DL, Farkas DH, editors. Molecular methods for virus detection. San Diego: Academic; 1995. p. 315–28.

    Chapter  Google Scholar 

  74. Laffler TG, Carrino JJ, Marshall RL. The ligase chain reaction in DNA-based diagnosis. Ann Biol Clin (Paris). 1993;51(9):821–6.

    CAS  Google Scholar 

  75. Mahony J, Chong S, Jang D, Luinstra K, Faught M, Dalby D, Sellors J, Chernesky M. Urine specimens from pregnant and nonpregnant women inhibitory to amplification of Chlamydia trachomatis nucleic acid by PCR, ligase chain reaction, and transcription-mediated amplification: identification of urinary substances associated with inhibition and removal of inhibitory activity. J Clin Microbiol. 1998;36(11):3122–6.

    PubMed Central  PubMed  CAS  Google Scholar 

  76. Gorrin G, Friesenhahn M, Lin P, Sanders M, Pollner R, Eguchi B, Pham J, Roma G, Spidle J, Nicol S, Wong C, Bhade S, Comanor L. Performance evaluation of the VERSANT HCV RNA qualitative assay by using transcription-mediated amplification. J Clin Microbiol. 2003;41(1):310–7.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  77. Walker GT, Little MC, Nadeau JG, Shank DD. Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. Proc Natl Acad Sci U S A. 1992;89(1):392–6.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  78. Westin L, Xu X, Miller C, Wang L, Edman CF, Nerenberg M. Anchored multiplex amplification on a microelectronic chip array. Nat Biotechnol. 2000;18(2):199–204. doi:10.1038/72658.

    Article  PubMed  CAS  Google Scholar 

  79. Sooknanan R, van Gemen B, Malek LT. Nucleic acid sequence-based amplification. In: Wiedbrauk DL, Farkas DH, editors. Molecular methods for virus detection. San Diego: Academic; 1995. p. 261–85.

    Chapter  Google Scholar 

  80. Spadoro JP, Dragon EA. Quality control of the polymerase chase reaction. In: Farkas DH, editor. Molecular biology and pathology: a guidebook for quality control. San Diego: Academic; 1993. p. 149–58.

    Google Scholar 

  81. Nolte FS. Branched DNA signal amplification for direct quantitation of nucleic acid sequences in clinical specimens. Adv Clin Chem. 1998;33:201–35.

    Article  PubMed  CAS  Google Scholar 

  82. Wilber JC. Branched DNA for quantification of viral load. Immunol Invest. 1997;26(1–2):9–13.

    Article  PubMed  CAS  Google Scholar 

  83. Elbeik T, Alvord WG, Trichavaroj R, de Souza M, Dewar R, Brown A, Chernoff D, Michael NL, Nassos P, Hadley K, Ng VL. Comparative analysis of HIV-1 viral load assays on subtype quantification: Bayer Versant HIV-1 RNA 3.0 versus Roche Amplicor HIV-1 Monitor version 1.5. J Acquir Immune Defic Syndr. 2002;29(4):330–9.

    Article  PubMed  CAS  Google Scholar 

  84. Gleaves CA, Welle J, Campbell M, Elbeik T, Ng V, Taylor PE, Kuramoto K, Aceituno S, Lewalski E, Joppa B, Sawyer L, Schaper C, McNairn D, Quinn T. Multicenter evaluation of the Bayer VERSANT HIV-1 RNA 3.0 assay: analytical and clinical performance. J Clin Virol. 2002;25(2):205–16.

    Article  PubMed  CAS  Google Scholar 

  85. Hann HW, Fontana RJ, Wright T, Everson G, Baker A, Schiff ER, Riely C, Anschuetz G, Gardner SD, Brown N, Griffiths D, United States Lamivudine Compassionate Use Study Group. A United States compassionate use study of lamivudine treatment in nontransplantation candidates with decompensated hepatitis B virus-related cirrhosis. Liver Transpl. 2003;9(1):49–56. doi:10.1053/jlts.2003.50005.

    Article  PubMed  Google Scholar 

  86. Hendricks DA, Stowe BJ, Hoo BS, Kolberg J, Irvine BD, Neuwald PD, Urdea MS, Perrillo RP. Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay. Am J Clin Pathol. 1995;104(5):537–46.

    PubMed  CAS  Google Scholar 

  87. Martinot-Peignoux M, Boyer N, Colombat M, Akremi R, Pham BN, Ollivier S, Castelnau C, Valla D, Degott C, Marcellin P. Serum hepatitis B virus DNA levels and liver histology in inactive HBsAg carriers. J Hepatol. 2002;36(4):543–6.

    Article  PubMed  CAS  Google Scholar 

  88. Pawlotsky JM, Bastie A, Hezode C, Lonjon I, Darthuy F, Remire J, Dhumeaux D. Routine detection and quantification of hepatitis B virus DNA in clinical laboratories: performance of three commercial assays. J Virol Methods. 2000;85(1–2):11–21.

    Article  PubMed  CAS  Google Scholar 

  89. Beld M, Sentjens R, Rebers S, Weegink C, Weel J, Sol C, Boom R. Performance of the New Bayer VERSANT HCV RNA 3.0 assay for quantitation of hepatitis C virus RNA in plasma and serum: conversion to international units and comparison with the Roche COBAS Amplicor HCV Monitor, Version 2.0, assay. J Clin Microbiol. 2002;40(3):788–93.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  90. Trimoulet P, Halfon P, Pohier E, Khiri H, Chene G, Fleury H. Evaluation of the VERSANT HCV RNA 3.0 assay for quantification of hepatitis C virus RNA in serum. J Clin Microbiol. 2002;40(6):2031–6.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  91. Lorincz A, Anthony J. Hybrid capture: a system for nucelic acid detection by signal amplification technology. In: Van Dyke C, Woodfork K, editors. Luminescence biotechnology: instruments and applications. Boca Raton, FL: CRC Press; 2002. p. 149–58.

    Google Scholar 

  92. Lorincz AT. Hybrid Capture method for detection of human papillomavirus DNA in clinical specimens: a tool for clinical management of equivocal Pap smears and for population screening. J Obstet Gynaecol Res. 1996;22(6):629–36.

    Article  PubMed  CAS  Google Scholar 

  93. Mazzulli T, Drew LW, Yen-Lieberman B, Jekic-McMullen D, Kohn DJ, Isada C, Moussa G, Chua R, Walmsley S. Multicenter comparison of the digene hybrid capture CMV DNA assay (version 2.0), the pp 65 antigenemia assay, and cell culture for detection of cytomegalovirus viremia. J Clin Microbiol. 1999;37(4):958–63.

    PubMed Central  PubMed  CAS  Google Scholar 

  94. Schachter J, Hook 3rd EW, McCormack WM, Quinn TC, Chernesky M, Chong S, Girdner JI, Dixon PB, DeMeo L, Williams E, Cullen A, Lorincz A. Ability of the digene hybrid capture II test to identify Chlamydia trachomatis and Neisseria gonorrhoeae in cervical specimens. J Clin Microbiol. 1999;37(11):3668–71.

    PubMed Central  PubMed  CAS  Google Scholar 

  95. Kessler HH, Pierer K, Dragon E, Lackner H, Santner B, Stunzner D, Stelzl E, Waitzl B, Marth E. Evaluation of a new assay for HBV DNA quantitation in patients with chronic hepatitis B. Clin Diagn Virol. 1998;9(1):37–43.

    Article  PubMed  CAS  Google Scholar 

  96. Dyanov HM, Dzitoeva SG. Method for attachment of microscopic preparations on glass for in situ hybridization, PRINS and in situ PCR studies. Biotechniques. 1995;18(5):822–4. 826.

    PubMed  CAS  Google Scholar 

  97. Guan XY, Zhang H, Bittner M, Jiang Y, Meltzer P, Trent J. Chromosome arm painting probes. Nat Genet. 1996;12(1):10–1. doi:10.1038/ng0196-10.

    Article  PubMed  CAS  Google Scholar 

  98. Schrock E, du Manoir S, Veldman T, Schoell B, Wienberg J, Ferguson-Smith MA, Ning Y, Ledbetter DH, Bar-Am I, Soenksen D, Garini Y, Ried T. Multicolor spectral karyotyping of human chromosomes. Science. 1996;273(5274):494–7.

    Article  PubMed  CAS  Google Scholar 

  99. du Manoir S, Speicher MR, Joos S, Schrock E, Popp S, Dohner H, Kovacs G, Robert-Nicoud M, Lichter P, Cremer T. Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization. Hum Genet. 1993;90(6):590–610.

    Article  PubMed  Google Scholar 

  100. Hachitanda Y, Toyoshima S, Akazawa K, Tsuneyoshi M. N-myc gene amplification in rhabdomyosarcoma detected by fluorescence in situ hybridization: its correlation with histologic features. Mod Pathol. 1998;11(12):1222–7.

    PubMed  CAS  Google Scholar 

  101. Kallioniemi A, Kallioniemi OP, Sudar D, Rutovitz D, Gray JW, Waldman F, Pinkel D. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science. 1992;258(5083):818–21.

    Article  PubMed  CAS  Google Scholar 

  102. Tagawa H, Karnan S, Suzuki R, Matsuo K, Zhang X, Ota A, Morishima Y, Nakamura S, Seto M. Genome-wide array-based CGH for mantle cell lymphoma: identification of homozygous deletions of the proapoptotic gene BIM. Oncogene. 2005;24(8):1348–58. doi:10.1038/sj.onc.1208300.

    Article  PubMed  CAS  Google Scholar 

  103. Sharp AJ, Hansen S, Selzer RR, Cheng Z, Regan R, Hurst JA, Stewart H, Price SM, Blair E, Hennekam RC, Fitzpatrick CA, Segraves R, Richmond TA, Guiver C, Albertson DG, Pinkel D, Eis PS, Schwartz S, Knight SJ, Eichler EE. Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome. Nat Genet. 2006;38(9):1038–42. doi:10.1038/ng1862.

    Article  PubMed  CAS  Google Scholar 

  104. Shaw-Smith C, Pittman AM, Willatt L, Martin H, Rickman L, Gribble S, Curley R, Cumming S, Dunn C, Kalaitzopoulos D, Porter K, Prigmore E, Krepischi-Santos AC, Varela MC, Koiffmann CP, Lees AJ, Rosenberg C, Firth HV, de Silva R, Carter NP. Microdeletion encompassing MAPT at chromosome 17q21.3 is associated with developmental delay and learning disability. Nat Genet. 2006;38(9):1032–7. doi:10.1038/ng1858.

    Article  PubMed  CAS  Google Scholar 

  105. Tagawa H, Suguro M, Tsuzuki S, Matsuo K, Karnan S, Ohshima K, Okamoto M, Morishima Y, Nakamura S, Seto M. Comparison of genome profiles for identification of distinct subgroups of diffuse large B-cell lymphoma. Blood. 2005;106(5):1770–7. doi:10.1182/blood-2005-02-0542.

    Article  PubMed  CAS  Google Scholar 

  106. Dhami P, Coffey AJ, Abbs S, Vermeesch JR, Dumanski JP, Woodward KJ, Andrews RM, Langford C, Vetrie D. Exon array CGH: detection of copy-number changes at the resolution of individual exons in the human genome. Am J Hum Genet. 2005;76(5):750–62. doi:10.1086/429588.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  107. Bovolenta M, Neri M, Fini S, Fabris M, Trabanelli C, Venturoli A, Martoni E, Bassi E, Spitali P, Brioschi S, Falzarano MS, Rimessi P, Ciccone R, Ashton E, McCauley J, Yau S, Abbs S, Muntoni F, Merlini L, Gualandi F, Ferlini A. A novel custom high density-comparative genomic hybridization array detects common rearrangements as well as deep intronic mutations in dystrophinopathies. BMC Genomics. 2008;9:572. doi:10.1186/1471-2164-9-572.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  108. del Gaudio D, Yang Y, Boggs BA, Schmitt ES, Lee JA, Sahoo T, Pham HT, Wiszniewska J, Chinault AC, Beaudet al, Eng CM. Molecular diagnosis of Duchenne/Becker muscular dystrophy: enhanced detection of dystrophin gene rearrangements by oligonucleotide array-comparative genomic hybridization. Hum Mutat. 2008;29(9):1100–7. doi:10.1002/humu.20841.

    Google Scholar 

  109. Hegde MR, Chin EL, Mulle JG, Okou DT, Warren ST, Zwick ME. Microarray-based mutation detection in the dystrophin gene. Hum Mutat. 2008;29(9):1091–9. doi:10.1002/humu.20831.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  110. Miller DT, Adam MP, Aradhya S, Biesecker LG, Brothman AR, Carter NP, Church DM, Crolla JA, Eichler EE, Epstein CJ, Faucett WA, Feuk L, Friedman JM, Hamosh A, Jackson L, Kaminsky EB, Kok K, Krantz ID, Kuhn RM, Lee C, Ostell JM, Rosenberg C, Scherer SW, Spinner NB, Stavropoulos DJ, Tepperberg JH, Thorland EC, Vermeesch JR, Waggoner DJ, Watson MS, Martin CL, Ledbetter DH. Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. Am J Hum Genet. 2010;86(5):749–64. doi:10.1016/j.ajhg.2010.04.006.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  111. Chen P, Lepikhova T, Hu Y, Monni O, Hautaniemi S. Comprehensive exon array data processing method for quantitative analysis of alternative spliced variants. Nucleic Acids Res. 2011;39(18):e123. doi:10.1093/nar/gkr513.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  112. Schutte M, Elstrodt F, Bralten LB, Nagel JH, Duijm E, Hollestelle A, Vuerhard MJ, Wasielewski M, Peeters JK, van der Spek P, Sillevis Smitt PA, French PJ. Exon expression arrays as a tool to identify new cancer genes. PLoS One. 2008;3(8):e3007. doi:10.1371/journal.pone.0003007.

    Article  PubMed Central  CAS  Google Scholar 

  113. Wesolowski R, Ramaswamy B. Gene expression profiling: changing face of breast cancer classification and management. Gene Expr. 2011;15(3):105–15.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  114. Holt RA, Jones SJ. The new paradigm of flow cell sequencing. Genome Res. 2008;18(6):839–46. doi:10.1101/gr.073262.107.

    Article  PubMed  CAS  Google Scholar 

  115. Marguerat S, Wilhelm BT, Bahler J. Next-generation sequencing: applications beyond genomes. Biochem Soc Trans. 2008;36(Pt 5):1091–6. doi:10.1042/BST0361091.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  116. Tucker T, Marra M, Friedman JM. Massively parallel sequencing: the next big thing in genetic medicine. Am J Hum Genet. 2009;85(2):142–54. doi:10.1016/j.ajhg.2009.06.022.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  117. Meder B, Haas J, Keller A, Heid C, Just S, Borries A, Boisguerin V, Scharfenberger-Schmeer M, Stahler P, Beier M, Weichenhan D, Strom TM, Pfeufer A, Korn B, Katus HA, Rottbauer W. Targeted next-generation sequencing for the molecular genetic diagnostics of cardiomyopathies. Circ Cardiovasc Genet. 2011;4(2):110–22. doi:10.1161/CIRCGENETICS.110.958322.

    Article  PubMed  CAS  Google Scholar 

  118. Hu H, Wrogemann K, Kalscheuer V, Tzschach A, Richard H, Haas SA, Menzel C, Bienek M, Froyen G, Raynaud M, Van Bokhoven H, Chelly J, Ropers H, Chen W. Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing. Hugo J. 2009;3(1–4):41–9. doi:10.1007/s11568-010-9137-y.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  119. Raffan E, Semple RK. Next generation sequencing–implications for clinical practice. Br Med Bull. 2011;99:53–71. doi:10.1093/bmb/ldr029.

    Article  PubMed  CAS  Google Scholar 

  120. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, Dewell SB, Du L, Fierro JM, Gomes XV, Godwin BC, He W, Helgesen S, Ho CH, Irzyk GP, Jando SC, Alenquer ML, Jarvie TP, Jirage KB, Kim JB, Knight JR, Lanza JR, Leamon JH, Lefkowitz SM, Lei M, Li J, Lohman KL, Lu H, Makhijani VB, McDade KE, McKenna MP, Myers EW, Nickerson E, Nobile JR, Plant R, Puc BP, Ronan MT, Roth GT, Sarkis GJ, Simons JF, Simpson JW, Srinivasan M, Tartaro KR, Tomasz A, Vogt KA, Volkmer GA, Wang SH, Wang Y, Weiner MP, Yu P, Begley RF, Rothberg JM. Genome sequencing in microfabricated high-density picolitre reactors. Nature. 2005;437(7057):376–80. doi:10.1038/nature03959.

    PubMed Central  PubMed  CAS  Google Scholar 

  121. Mir KU, Qi H, Salata O, Scozzafava G. Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template. Nucleic Acids Res. 2009;37(1):e5. doi:10.1093/nar/gkn906.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  122. Mamanova L, Coffey AJ, Scott CE, Kozarewa I, Turner EH, Kumar A, Howard E, Shendure J, Turner DJ. Target-enrichment strategies for next-generation sequencing. Nat Methods. 2010;7(2):111–8. doi:10.1038/nmeth.1419.

    Article  PubMed  CAS  Google Scholar 

  123. Ng SB, Bigham AW, Buckingham KJ, Hannibal MC, McMillin MJ, Gildersleeve HI, Beck AE, Tabor HK, Cooper GM, Mefford HC, Lee C, Turner EH, Smith JD, Rieder MJ, Yoshiura K, Matsumoto N, Ohta T, Niikawa N, Nickerson DA, Bamshad MJ, Shendure J. Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome. Nat Genet. 2010;42(9):790–3. doi:10.1038/ng.646.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  124. Mao C, Evans C, Jensen RV, Sobral BW. Identification of new genes in Sinorhizobium meliloti using the Genome Sequencer FLX system. BMC Microbiol. 2008;8:72. doi:10.1186/1471-2180-8-72.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  125. Fujimoto A, Nakagawa H, Hosono N, Nakano K, Abe T, Boroevich KA, Nagasaki M, Yamaguchi R, Shibuya T, Kubo M, Miyano S, Nakamura Y, Tsunoda T. Whole-genome sequencing and comprehensive variant analysis of a Japanese individual using massively parallel sequencing. Nat Genet. 2010;42(11):931–6. doi:10.1038/ng.691.

    Article  PubMed  CAS  Google Scholar 

  126. Drmanac R. The advent of personal genome sequencing. Genet Med. 2011;13(3):188–90. doi:10.1097/GIM.0b013e31820f16e6.

    Article  PubMed  Google Scholar 

  127. Green ED, Guyer MS, National Human Genome Research Institute. Charting a course for genomic medicine from base pairs to bedside. Nature. 2011;470(7333):204–13. doi:10.1038/nature09764.

    Article  PubMed  CAS  Google Scholar 

  128. Haas J, Katus HA, Meder B. Next-generation sequencing entering the clinical arena. Mol Cell Probes. 2011;25(5–6):206–11. doi:10.1016/j.mcp.2011.08.005.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA

    D. Hunter Best Ph.D. & Rong Mao M.D.

  2. ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA

    D. Hunter Best Ph.D., Shale A. Dames M.S., Whitney Wooderchak-Donahue Ph.D., Tracey Lewis Ph.D., Kelli L. Sumner B.S., Cecily P. Vaughn M.S., Rong Mao M.D. & Daniel H. Farkas Ph.D.

Authors
  1. D. Hunter Best Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shale A. Dames M.S.
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Whitney Wooderchak-Donahue Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tracey Lewis Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kelli L. Sumner B.S.
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Cecily P. Vaughn M.S.
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rong Mao M.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Daniel H. Farkas Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to D. Hunter Best Ph.D. .

Editor information

Editors and Affiliations

  1. Pathology and Laboratory Medicine, University of Vermont College of Medicine and University of Vermont Medical Center, Burlington, Vermont, USA

    Debra G.B. Leonard

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer International Publishing Switzerland

About this chapter

Cite this chapter

Best, D.H. et al. (2016). Molecular Pathology Methods. In: Leonard, D. (eds) Molecular Pathology in Clinical Practice. Springer, Cham. https://doi.org/10.1007/978-3-319-19674-9_2

Download citation

  • DOI: https://doi.org/10.1007/978-3-319-19674-9_2

  • Publisher Name: Springer, Cham

  • Print ISBN: 978-3-319-19673-2

  • Online ISBN: 978-3-319-19674-9

  • eBook Packages: MedicineMedicine (R0)

Publish with us

Policies and ethics

Search

Navigation