Biobanking pp 385-402 | Cite as

An Overview of DNA Analytical Methods

  • Valerie A. Arboleda
  • Rena R. Xian
Part of the Methods in Molecular Biology book series (MIMB, volume 1897)


The development of rapid parallel sequencing in the last 20 years has begun a revolution in the field of genetics that is changing nearly all disciplines within biology and medicine. Genomic sequencing has become crucial to the diagnosis and clinical management of patients with constitutional diseases and cancer and has quickly become an integral part of the new era of personalized and precision medicine. The precision medicine initiative, released by the NIH in 2015, has catapulted genomic technologies to the forefront of the practice of medicine and biomedical research.

This chapter focuses on the core technologies driving the genomic revolution from first generation (Sanger) sequencing to microarray-based technologies, to second, commonly referred to as next-generation sequencing (NGS) methods, and finally to the emerging third generation technologies capable of performing single-molecule and long-read sequencing. The goal of the chapter is to provide a broad overview of these methods of DNA analysis and highlight their strengths and weaknesses. Furthermore, with a knowledge of the different mutation types, we seek to provide the basis for understanding how these technologies work, and can be adopted, to explore other type of nucleic acids and epigenetic changes.

Key words

Sequencing Next-generation sequencing (NGS) DNA RNA Microarray Illumina Ion torrent Nanopore Bioinformatics 


  1. 1.
    Watson JD, Crick FH (1953) Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature 171(4356):737–738CrossRefGoogle Scholar
  2. 2.
    Saiki RK et al (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239(4839):487–491CrossRefGoogle Scholar
  3. 3.
    Shampo MA, Kyle RA (2002) Kary B. Mullis—Nobel Laureate for procedure to replicate DNA. Mayo Clin Proc 77(7):606CrossRefGoogle Scholar
  4. 4.
    Lander ES et al (2001) Initial sequencing and analysis of the human genome. Nature 409(6822):860–921CrossRefGoogle Scholar
  5. 5.
    Sachidanandam R et al (2001) A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 409(6822):928–933CrossRefGoogle Scholar
  6. 6.
    Venter JC et al (2001) The sequence of the human genome. Science 291(5507):1304–1351CrossRefGoogle Scholar
  7. 7.
    Harrison MJ, Murphy BM, Plant BJ (2013) Ivacaftor in a G551D homozygote with cystic fibrosis. N Engl J Med 369(13):1280–1282CrossRefGoogle Scholar
  8. 8.
    Wainwright CE et al (2015) Lumacaftor-Ivacaftor in patients with cystic fibrosis homozygous for Phe508del CFTR. N Engl J Med 373(3):220–231CrossRefGoogle Scholar
  9. 9.
    Holland PM et al (1991) Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 88(16):7276–7280CrossRefGoogle Scholar
  10. 10.
    Yau SC et al (1996) Accurate diagnosis of carriers of deletions and duplications in Duchenne/Becker muscular dystrophy by fluorescent dosage analysis. J Med Genet 33(7):550–558CrossRefGoogle Scholar
  11. 11.
    Procter M et al (2006) Molecular diagnosis of Prader-Willi and Angelman syndromes by methylation-specific melting analysis and methylation-specific multiplex ligation-dependent probe amplification. Clin Chem 52(7):1276–1283CrossRefGoogle Scholar
  12. 12.
    Schouten JP et al (2002) Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 30(12):e57CrossRefGoogle Scholar
  13. 13.
    Sanger F, Coulson AR (1975) A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol 94(3):441–448CrossRefGoogle Scholar
  14. 14.
    Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74(12):5463–5467CrossRefGoogle Scholar
  15. 15.
    Lyon E et al (2010) A simple, high-throughput assay for Fragile X expanded alleles using triple repeat primed PCR and capillary electrophoresis. J Mol Diagn 12(4):505–511CrossRefGoogle Scholar
  16. 16.
    Ronaghi M, Uhlen M, Nyren P (1998) A sequencing method based on real-time pyrophosphate. Science 281(5375):363, 365Google Scholar
  17. 17.
    Nyrén P (2007) The history of Pyrosequencing®. In: Walker J, Marsh S (eds) Pyrosequencing® protocols. Humana Press, New York, pp 1–13Google Scholar
  18. 18.
    Steemers FJ et al (2006) Whole-genome genotyping with the single-base extension assay. Nat Methods 3(1):31–33CrossRefGoogle Scholar
  19. 19.
    Johansen P et al (2013) Evaluation of the iPLEX(R) Sample ID Plus Panel designed for the Sequenom MassARRAY(R) system. A SNP typing assay developed for human identification and sample tracking based on the SNPforID panel. Forensic Sci Int Genet 7(5):482–487CrossRefGoogle Scholar
  20. 20.
    Ross P et al (1998) High level multiplex genotyping by MALDI-TOF mass spectrometry. Nat Biotechnol 16(13):1347–1351CrossRefGoogle Scholar
  21. 21.
    Pinkel D et al (1998) High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nat Genet 20(2):207–211CrossRefGoogle Scholar
  22. 22.
    Kallioniemi A et al (1992) Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258(5083):818–821CrossRefGoogle Scholar
  23. 23.
    Pinkel D, Albertson DG (2005) Comparative genomic hybridization. Annu Rev Genomics Hum Genet 6:331–354CrossRefGoogle Scholar
  24. 24.
    Mei R et al (2000) Genome-wide detection of allelic imbalance using human SNPs and high-density DNA arrays. Genome Res 10(8):1126–1137CrossRefGoogle Scholar
  25. 25.
    Sellick GS et al (2004) Genomewide linkage searches for Mendelian disease loci can be efficiently conducted using high-density SNP genotyping arrays. Nucleic Acids Res 32(20):e164CrossRefGoogle Scholar
  26. 26.
    Nakano M et al (2003) Single-molecule PCR using water-in-oil emulsion. J Biotechnol 102(2):117–124CrossRefGoogle Scholar
  27. 27.
    Kojima T, Zhu B, Nakano H (2015) Construction of a DNA library on microbeads using whole genome amplification. Methods Mol Biol 1347:87–100CrossRefGoogle Scholar
  28. 28.
    Metzker ML (2010) Sequencing technologies—the next generation. Nat Rev Genet 11(1):31–46CrossRefGoogle Scholar
  29. 29.
    Rothberg JM et al (2011) An integrated semiconductor device enabling non-optical genome sequencing. Nature 475(7356):348–352CrossRefGoogle Scholar
  30. 30.
    Rosenstein J (2014) The promise of nanopore technology: nanopore DNA sequencing represents a fundamental change in the way that genomic information is read, with potentially big savings. IEEE Pulse 5(4):52–54CrossRefGoogle Scholar
  31. 31.
    Loomis EW et al (2013) Sequencing the unsequenceable: expanded CGG-repeat alleles of the fragile X gene. Genome Res 23(1):121–128CrossRefGoogle Scholar
  32. 32.
    Smith CC et al (2012) Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia. Nature 485(7397):260–263CrossRefGoogle Scholar
  33. 33.
    Carneiro MO et al (2012) Pacific biosciences sequencing technology for genotyping and variation discovery in human data. BMC Genomics 13:375CrossRefGoogle Scholar
  34. 34.
    Stoddart D et al (2009) Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore. Proc Natl Acad Sci U S A 106(19):7702–7707CrossRefGoogle Scholar
  35. 35.
    Clarke J et al (2009) Continuous base identification for single-molecule nanopore DNA sequencing. Nat Nanotechnol 4(4):265–270CrossRefGoogle Scholar
  36. 36.
    Brownstein CA et al (2014) An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge. Genome Biol 15(3):R53CrossRefGoogle Scholar
  37. 37.
    Oliver GR, Hart SN, Klee EW (2015) Bioinformatics for clinical next-generation sequencing. Clin Chem 61(1):124–135CrossRefGoogle Scholar
  38. 38.
    Van der Auwera GA et al (2013) From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr Protoc Bioinformatics 11(1110):11.10.1–11.10.33Google Scholar
  39. 39.
    Barutcu AR et al (2016) C-ing the genome: a compendium of chromosome conformation capture methods to study higher-order chromatin organization. J Cell Physiol 231(1):31–35CrossRefGoogle Scholar
  40. 40.
    Jin F et al (2013) A high-resolution map of the three-dimensional chromatin interactome in human cells. Nature 503(7475):290–294CrossRefGoogle Scholar
  41. 41.
    Nagano T et al (2013) Single-cell Hi-C reveals cell-to-cell variability in chromosome structure. Nature 502(7469):59–64CrossRefGoogle Scholar
  42. 42.
    Mundade R et al (2014) Role of ChIP-seq in the discovery of transcription factor binding sites, differential gene regulation mechanism, epigenetic marks and beyond. Cell Cycle 13(18):2847–2852CrossRefGoogle Scholar
  43. 43.
    Bowman SK (2015) Discovering enhancers by mapping chromatin features in primary tissue. Genomics 106(3):140–144CrossRefGoogle Scholar
  44. 44.
    Winter DR, Amit I (2014) The role of chromatin dynamics in immune cell development. Immunol Rev 261(1):9–22CrossRefGoogle Scholar
  45. 45.
    Fanelli M et al (2011) Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue. Nat Protoc 6(12):1905–1919CrossRefGoogle Scholar
  46. 46.
    Fanelli M et al (2010) Pathology tissue-chromatin immunoprecipitation, coupled with high-throughput sequencing, allows the epigenetic profiling of patient samples. Proc Natl Acad Sci U S A 107(50):21535–21540CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Pathology and Laboratory MedicineDavid Geffen School of Medicine at UCLALos AngelesUSA
  2. 2.Department of PathologyJohns Hopkins University School of MedicineBaltimoreUSA

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