Techniques for DNA Analysis


Molecular biology is a dynamic field with techniques and analytical tools continuously being developed. Many of the fundamental DNA analysis techniques were developed more than 30 years ago and have evolved through various modifications, automation, and computerization. This chapter reviews the basic concepts and techniques in order to understand how the procedures progressed into those used today.


Polymerase Chain Reaction Restriction Fragment Length Polymorphism Bacterial Artificial Chromosome Comparative Genomic Hybridization Methylation Specific Polymerase Chain Reaction 


8. 13Glossary of Terms and Acronyms


radioactive isotope




bacterial artificial chromosome


bisulfite sequencing PCR


comparative genomic hybridization


chemical mismatch cleavage


deoxyadenosine triphosphate


deoxycytidine triphosphate


dideoxyadenosine triphosphate


dideoxycytidine triphosphate


dideoxyguanosine triphosphate


dideoxynucleoside triphosphate


dideoxythymidine triphosphate


denaturing gradient gel electrophoresis


deoxyguanosine triphosphate


deoxyribonucleic acid


DNA methyltransferases


deoxynucleoside triphosphate


double stranded DNA


deoxythymidine triphosphate


deoxyuridine triphosphate


enzyme mismatch cleavage


fluorescence in-situ hybridization


potassium chloride


marker-assisted selection


messenger RNA


methylation specific PCR


sodium hydroxide


polyacrilamide gel electrophoresis


polymerase chain reaction


protein truncation test


quantitative real time polymerase chain reaction


restriction fragment length polymorphism


ribonucleic acid


reverse transcription polymerase chain reaction


sodium dodecyl sulfate polyacrilamide gel electrophoresis


single-stranded conformational polymorphism


temperature gradient gel electrophoresis


transfer RNA




variable number tandem repeats


  1. 1.
    Sambrook J, Russell D. Molecular Cloning: A Laboratory Manual, third edition. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2001.Google Scholar
  2. 2.
    Gerischer U. Direct sequencing of DNA produced in a polymerase chain reaction. Methods Mol Biol 2001; 167:53–61.PubMedGoogle Scholar
  3. 3.
    Graham CA, Hill AJ. Introduction to DNA sequencing. Methods Mol Biol 2001; 167:1–12.PubMedGoogle Scholar
  4. 4.
    Marziali A, Akeson M. New DNA sequencing methods. Annu Rev Biomed Eng 2001; 3:195–223.PubMedCrossRefGoogle Scholar
  5. 5.
    Mitnik L, Novotny M, Felten C, et al. Recent advances in DNA sequencing by capillary and microdevice electrophoresis. Electrophoresis 2001; 22:4104–17.PubMedCrossRefGoogle Scholar
  6. 6.
    Watts D, MacBeath JR. Automated fluorescent DNA sequencing on the ABI PRISM 310 genetic analyzer. Methods Mol Biol 2001; 167:153–70.PubMedGoogle Scholar
  7. 7.
    Zschocke J, Hoffmann GF. Cycle sequencing of polymerase chain reaction-amplified genomic DNA with dye-labeled universal primers. Methods Mol Biol 2001; 167:113–7.PubMedGoogle Scholar
  8. 8.
    Varsha. DNA fingerprinting in the criminal justice system: an overview. DNA Cell Biol 2006; 25:181–8.Google Scholar
  9. 9.
    McClelland M, Welsh J. DNA fingerprinting by arbitrarily primed PCR. PCR Methods Appl 1994; 4:S59–65.PubMedGoogle Scholar
  10. 10.
    Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988; 239:487–91.PubMedCrossRefGoogle Scholar
  11. 11.
    Hagen-Mann K, Mann W. RT-PCR and alternative methods to PCR for in vitro amplification of nucleic acids. Exp Clin Endocrinol Diabetes 1995; 103:150–5.PubMedCrossRefGoogle Scholar
  12. 12.
    Mullis K, Faloona F, Scharf S, et al. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction 1986. Biotechnology 1992; 24:17–27.Google Scholar
  13. 13.
    Mullis KB. The unusual origin of the polymerase chain reaction. Sci Am 1990; 262:56–61, 64–5.Google Scholar
  14. 14.
    Arya M, Shergill IS, Williamson M, et al. Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn 2005; 5: 209–19.PubMedCrossRefGoogle Scholar
  15. 15.
    Rooney PH. Multiplex quantitative real-time PCR of laser microdissected tissue. Methods Mol Biol 2005; 293:27–37.PubMedGoogle Scholar
  16. 16.
    Bustin SA, Benes V, Nolan T, et al. Quantitative real-time RT-PCR – a perspective. J Mol Endocrinol 2005; 34:597–601.PubMedCrossRefGoogle Scholar
  17. 17.
    Wong ML, Medrano JF. Real-time PCR for mRNA quantitation. Biotechniques 2005; 39:75–85.PubMedCrossRefGoogle Scholar
  18. 18.
    Valasek MA, Repa JJ. The power of real-time PCR. Adv Physiol Educ 2005; 29:151–9.PubMedCrossRefGoogle Scholar
  19. 19.
    Kaltenboeck B, Wang C. Advances in real-time PCR: application to clinical laboratory diagnostics. Adv Clin Chem 2005; 40: 219–59.PubMedCrossRefGoogle Scholar
  20. 20.
    Pourzand C, Cerutti P. Genotypic mutation analysis by RFLP/PCR. Mutat Res 1993; 288:113–21.PubMedCrossRefGoogle Scholar
  21. 21.
    Woodward SR. RFLP analysis in familial polyposis and Gardner syndrome. Prog Clin Biol Res 1988; 279:305–8.PubMedGoogle Scholar
  22. 22.
    Hammarstrom L, Ghanem N, Smith CI, et al. RFLP of human immunoglobulin genes. Exp Clin Immunogenet 1990; 7:7–19.PubMedGoogle Scholar
  23. 23.
    Permutt MA, Elbein SC. Insulin gene in diabetes. Analysis through RFLP. Diabetes Care 1990; 13:364–74.PubMedCrossRefGoogle Scholar
  24. 24.
    Hayashi K. PCR-SSCP: a simple and sensitive method for detection of mutations in the genomic DNA. PCR Methods Appl 1991; 1:34–8.PubMedGoogle Scholar
  25. 25.
    Hayashi K. PCR-SSCP: a method for detection of mutations. Genet Anal Tech Appl 1992; 9:73–9.PubMedGoogle Scholar
  26. 26.
    Hayashi K, Yandell DW. How sensitive is PCR-SSCP? Hum Mutat 1993; 2:338–46.PubMedCrossRefGoogle Scholar
  27. 27.
    Fan E, Levin DB, Glickman BW, et al. Limitations in the use of SSCP analysis. Mutat Res 1993; 288:85–92.PubMedCrossRefGoogle Scholar
  28. 28.
    Dillon D, Zheng K, Negin B, et al. Detection of Ki-ras and p53 mutations by laser capture microdissection/PCR/SSCP. Methods Mol Biol 2005; 293:57–67.PubMedGoogle Scholar
  29. 29.
    Fodde R, Losekoot M. Mutation detection by denaturing gradient gel electrophoresis (DGGE). Hum Mutat 1994; 3:83–94.PubMedCrossRefGoogle Scholar
  30. 30.
    Glavac D, Dean M. Applications of heteroduplex analysis for mutation detection in disease genes. Hum Mutat 1995; 6: 281–7.PubMedCrossRefGoogle Scholar
  31. 31.
    Nataraj AJ, Olivos-Glander I, Kusukawa N, et al. Single-strand conformation polymorphism and heteroduplex analysis for gel-based mutation detection. Electrophoresis 1999; 20: 1177–85.PubMedCrossRefGoogle Scholar
  32. 32.
    Ellis TP, Humphrey KE, Smith MJ, et al. Chemical cleavage of mismatch: a new look at an established method. Hum Mutat 1998; 11:345–53.PubMedCrossRefGoogle Scholar
  33. 33.
    Den Dunnen JT, Van Ommen GJ. The protein truncation test: A review. Hum Mutat 1999; 14:95–102.CrossRefGoogle Scholar
  34. 34.
    Hauss O, Muller O. The protein truncation test in mutation detection and molecular diagnosis. Methods Mol Biol 2007; 375: 151–64.PubMedCrossRefGoogle Scholar
  35. 35.
    Derks S, Lentjes MH, Hellebrekers DM, et al. Methylation-specific PCR unraveled. Cell Oncol 2004; 26:291–9.PubMedGoogle Scholar
  36. 36.
    Galm O, Herman JG. Methylation-specific polymerase chain reaction. Methods Mol Med 2005; 113:279–91.PubMedGoogle Scholar
  37. 37.
    Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 1996; 93:9821–6.Google Scholar
  38. 38.
    Taylor KH, Kramer RS, Davis JW, et al. Ultradeep bisulfite sequencing analysis of DNA methylation patterns in multiple gene promoters by 454 sequencing. Cancer Res 2007; 67:8511–8.PubMedCrossRefGoogle Scholar
  39. 39.
    Warnecke PM, Stirzaker C, Song J, et al. Identification and resolution of artifacts in bisulfite sequencing. Methods 2002; 27:101–7.PubMedCrossRefGoogle Scholar
  40. 40.
    Shaffer LG, Bejjani BA. Medical applications of array CGH and the transformation of clinical cytogenetics. Cytogenet Genome Res 2006; 115:303–9.PubMedCrossRefGoogle Scholar
  41. 41.
    Gebhart E. Comparative genomic hybridization (CGH): ten years of substantial progress in human solid tumor molecular cytogenetics. Cytogenet Genome Res 2004; 104:352–8.PubMedCrossRefGoogle Scholar
  42. 42.
    Davies JJ, Wilson IM, Lam WL. Array CGH technologies and their applications to cancer genomes. Chromosome Res 2005; 13: 237–48.PubMedCrossRefGoogle Scholar
  43. 43.
    Kallioniemi OP, Kallioniemi A, Sudar D, et al. Comparative genomic hybridization: a rapid new method for detecting and mapping DNA amplification in tumors. Semin Cancer Biol 1993; 4: 41–6.PubMedGoogle Scholar
  44. 44.
    Trask BJ. DNA sequence localization in metaphase and interphase cells by fluorescence in situ hybridization. Methods Cell Biol 1991; 35:3–35.PubMedCrossRefGoogle Scholar
  45. 45.
    Tibiletti MG. Interphase FISH as a new tool in tumor pathology. Cytogenet Genome Res 2007; 118:229–36.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Brain Tumor Biology UnitUniversity of NavarraPamplonaSpain
  2. 2.Department of Health SciencesUniversity of NavarraPamplonaSpain

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