Skip to main content
SpringerLink
Log in

Techniques Involved in DNA Fingerprinting: Isolation, Quantification, PCR, Genotyping, and Analysis

  • Chapter
  • First Online:
DNA Fingerprinting: Advancements and Future Endeavors

  • 1356 Accesses

Abstract

The primary objective of forensic DNA fingerprinting is to establish the relatedness between the questioned samples and reference samples with absolute certainty beyond any reasonable doubt. This sophisticated and robust technique however requires a series of methodologies and procedures which are to be followed by the forensic scientists to obtain an accurate result. The procedures of DNA fingerprinting have accelerated through a series of major stages of technological advancement. It began with an initial methodology called “restriction fragment length polymorphism (RFLP)” crossing the stages of “minisatellites” or VNTR analysis and currently advancing through the stages of “microsatellite” or STRs and SNPs. Similarly the instrumentation processes involved also changed from horizontal gel to vertical gel system to automated analyzers consisting of sensitive microcapillary arrays with laser-based detection system. Quality softwares are now available for data analysis and generation of user-friendly reports. Even though the techniques involved in DNA fingerprinting vary greatly from laboratory to laboratory throughout the world, the basic concept remains the same and is represented here.

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

Access this chapter

Log in via an institution
eBook
USD 16.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 179.99
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. Ahmed HA, MacLeod ET, Hide G, Welburn SC, Picozzi K (2011) The best practice for preparation of samples from FTA® cards for diagnosis of blood borne infections using African trypanosomes as a model system. Parasit Vectors 4:68

    Article  Google Scholar 

  2. Akbari M, Hansen MD, Halgunset J, Skorpen F, Krokan HE (2005) Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner. J Mol Diagn 7:36–39

    Article  CAS  Google Scholar 

  3. Alonso A, Martin P, Albarrán C, Garcia P, Fernandez de Simon L, JesúsIturralde M, Fernández-Rodriguez A, Atienza I, Capilla J, García-Hirschfeld J, Martinez P, Vallejo G, García O, García E, Real P, Alvarez D, León A, Sancho M (2005) Challenges of DNA profiling in mass disaster investigations. Croat Med J 46:540–548

    PubMed  Google Scholar 

  4. Al-Soud WA, Rådström P (1998) Capacity of nine thermostable DNA polymerases to mediate DNA amplification in the presence of PCR-inhibiting samples. Appl Environ Microbiol 64:3748–3753

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Arnold T, Linke D (2008) The use of detergents to purify membrane proteins. Curr Protoc Protein Sci 4:4.8.1–4.8.30

    Article  Google Scholar 

  6. Billard A, Laval V, Fillinger S, Leroux P, Lachaise H, Beffa R, Debieu D (2012) The allele-specific probe and primer amplification assay, a new real-time PCR method for fine quantification of single-nucleotide polymorphisms in pooled DNA. Appl Environ Microbiol 78:1063–1068

    Article  CAS  Google Scholar 

  7. Brownie J, Shawcross S, Theaker J, Whitcombe D, Ferrie R, Newton C, Little S (1997) The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res 25:3235–3241

    Article  CAS  Google Scholar 

  8. Butler JM, Buel E, Crivellente F, McCord BR (2004) Forensic DNA typing by capillary electrophoresis using the ABI prism 310 and 3100 genetic analyzers for STR analysis. Electrophoresis 25:1397–1412

    Article  CAS  Google Scholar 

  9. Cha RS, Thilly WG (1993) Specificity, efficiency, and Fidelity of PCR. Cold Spring Harbor Laboratory ISSN 1054-9805/93

    Google Scholar 

  10. Christoph Heller C, Pakleza C, Viovy JL (1995) DNA separation with field inversion capillary electrophoresis. Electrophoresis 16:1423–1428

    Article  Google Scholar 

  11. Deepak SA, Kottapalli KR, Rakwal R, Oros G, Rangappa KS, Iwahashi H, Masuo Y, Agrawal GK (2007) Real-time PCR: revolutionizing detection and expression analysis of genes. Curr Genomics 8:234–251

    Article  CAS  Google Scholar 

  12. Dell’Anno A, Fabiano M, Duineveld GCA, Kok A, Danovaro R (1998) Nucleic acid (DNA, RNA) quantification and RNA/DNA ratio determination in marine sediments: comparison of spectrophotometric, fluorometric, and high performance liquid chromatography methods and estimation of detrital DNA. Appl Environ Microbiol 64:3238–3245

    PubMed  PubMed Central  Google Scholar 

  13. Dobnik D, Štebih D, Blejec A, Morisset D, Žel J (2016) Multiplex quantification of four DNA targets in one reaction with bio-rad droplet digital PCR system for GMO detection. Sci Rep 6:35451

    Article  CAS  Google Scholar 

  14. Durney BC, Crihfield CL, Holland LA (2015) Capillary electrophoresis applied to DNA: determining and harnessing sequence and structure to advance bioanalyses (2009–2014). Anal Bioanal Chem 407:6923–6938

    Article  CAS  Google Scholar 

  15. Edwards MC, Gibbs RA (1994) Multiplex PCR: advantages, development, and applications. Genome Res 3:S65–S75

    Article  CAS  Google Scholar 

  16. Fan A, Byrnes S, Klapperich C (2013) Purification of DNA/RNA in a microfluidic device. Methods Mol Biol 949:403–411

    Article  CAS  Google Scholar 

  17. Ferri F (1997) Use of a charge coupled device camera for low-angle elastic light scattering. Rev Sci Instrum 68:2265–2274

    Article  CAS  Google Scholar 

  18. Flock S, Labarbe R, Houssier C (1996) Dielectric constant and ionic strength effects on DNA precipitation. Biophys J 70:1456–1465

    Article  CAS  Google Scholar 

  19. Ghatak S, Muthukumaran RB, Nachimuthu SK (2013) A simple method of genomic DNA extraction from human samples for PCR-RFLP analysis. J Biomol Tech 24:224–231

    PubMed  PubMed Central  Google Scholar 

  20. Gong R, Li S (2014) Extraction of human genomic DNA from whole blood using a magnetic microsphere method. Int J Nanomedicine 9:3781–3789

    Article  CAS  Google Scholar 

  21. Greco M, Sáez CA, Brown MT, Bitonti MB (2014) A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpussiliculosus, the model Brown alga. PLoS One 9:e96470

    Article  Google Scholar 

  22. Gudnason H, Dufva M, Bang DD, Wolff A (2007) Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic Acids Res 35:e127

    Article  Google Scholar 

  23. Gupta S, Charakana C, Sreelakshmi Y, Sharma R (2011) Fluorescent dye labeled DNA size standards for molecular mass detection in visible/infrared range. BMC Res Notes 4:12

    Article  CAS  Google Scholar 

  24. Hares DR (2015) Selection and implementation of expanded CODIS core loci in the United States. Forensic Sci Int Genet 17:33–34

    Article  CAS  Google Scholar 

  25. Hobbie RK, Roth BJ (2007) Intermediate physics for medicine and biology. Springer Science & Business Media, New York, p 616

    Google Scholar 

  26. Houck MM (2005) DNA and the criminal justice system the technology of justice. J Clin Invest 1115:1398–1398

    Article  Google Scholar 

  27. Housley DJE, Zalewski ZA, Beckett SE, Venta PJ (2006) Design factors that influence PCR amplification success of cross-species primers among 1147 mammalian primer pairs. BMC Genomics 7:253

    Article  Google Scholar 

  28. Iqbal M, Tao Y, Xie S, Zhu Y, Chen D, Wang X, Huang L, Peng D, Sattar A, Shabbir MAB, Hussain HI, Ahmed S, Yuan Z (2016) Aqueous two-phase system (ATPS): an overview and advances in its applications. Biol Proced Online 18:18

    Article  Google Scholar 

  29. Ishii K, Fukui M (2001) Optimization of annealing temperature to reduce bias caused by a primer mismatch in multitemplate PCR. Appl Environ Microbiol 67:3753–3755

    Article  CAS  Google Scholar 

  30. Kebelmann-Betzing C, Seeger K, Dragon S, Schmitt G, Möricke A, Schild TA, Henze G, Beyermann B (1998) Advantages of a new Taq DNA polymerase in multiplex PCR and time-release PCR. BioTechniques 24:154–158

    Article  CAS  Google Scholar 

  31. Koshy L, Anju AL, Harikrishnan S, Kutty VR, Jissa VT, Kurikesu I, Jayachandran P, Jayakumaran Nair A, Gangaprasad A, Nair GM, Sudhakaran PR (2017) Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays. Mol Biol Rep 44:97–108

    Article  CAS  Google Scholar 

  32. Lebedev AV, Paul N, Yee J, Timoshchuk VA, Shum J, Miyagi K, Kellum J, Hogrefe RI, Zon G (2008) Hot start PCR with heat-activatable primers: a novel approach for improved PCR performance. Nucleic Acids Res 36:e131

    Article  Google Scholar 

  33. Lee PY, Costumbrado J, Hsu CY, Kim YH (2012) Agarose gel electrophoresis for the separation of DNA fragments. J Vis Exp (62):3923

    Google Scholar 

  34. Liu D, Daubendiek SL, Zillman MA, Ryan K, Kool ET (1996) Rolling circle DNA synthesis: small circular oligonucleotides as efficient templates for DNA polymerases. J Am Chem Soc 118:1587–1594

    Article  CAS  Google Scholar 

  35. Lorenz TC (2012) Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J Vis Exp (63):3998

    Google Scholar 

  36. Ma TS (1995) Applications and limitations of polymerase chain reaction amplification. Chest J 108:1393–1404

    Article  CAS  Google Scholar 

  37. Marino MA, Devaney JM, Davis PA, Girard JE (1999) Optimization of intercalation dye concentration for short tandem repeat allele genotyping using capillary electrophoresis with laser-induced fluorescence detection. J Chromatogr B Biomed Sci Appl 732:365–374

    Article  CAS  Google Scholar 

  38. Markoulatos P, Siafakas N, Moncany M (2012) Multiplex polymerase chain reaction: a practical approach. J Clin Lab Anal 16:47–51

    Article  Google Scholar 

  39. Mohammadi Z, Shalavi S, Jafarzadeh H (2013) Ethylenediaminetetraacetic acid in endodontics. Eur J Dent 7:S135–S142

    Article  Google Scholar 

  40. Nigam PS (2013) Microbial enzymes with special characteristics for biotechnological applications. Biomol Ther 3:597–611

    Google Scholar 

  41. Pamidimarri DVNS, Meenakshi SR, Boricha G, Reddy MP (2009) A simplified method for extraction of high quality genomic DNA from Jatrophacurcas for genetic diversity and molecular marker studies. Indian J Biotechnol 8:187–192

    CAS  Google Scholar 

  42. Pereira R, Phillips C, Alves C, Amorim A, Carracedo A, Gusmão L (2009) A new multiplex for human identification using insertion/deletion polymorphisms. Electrophoresis 30:3682–3690

    Article  CAS  Google Scholar 

  43. Ponche F, Toomes C, Bransfield K, Leong FT, Douglas SH, Field SL, Bell SM, Combaret V, Puisieux A, Mighell AJ, Robinson PA, Inglehearn CF, Isaacs JD, Markham AF (2003) Real-time PCR based on SYBR-green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC Biotechnol 3:18

    Article  Google Scholar 

  44. Puers C, Hammond HA, Jin L, Caskey CT, Schumm JW (1993) Identification of repeat sequence heterogeneity at the polymorphic short tandem repeat locus HUMTH01 [AATG]n and reassignment of alleles in population analysis by using a locus-specific allelic ladder. Am J Hum Genet 53:953–958

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Puskás LG, Bottka S (1995) Reduction of mispriming in amplification reactions with restricted PCR. Genome Res 5:309–311

    Article  Google Scholar 

  46. Romsos EL, Vallone PM (2015) Rapid PCR of STR markers: applications to human identification. Forensic Sci Int Genet 18:90–99

    Article  CAS  Google Scholar 

  47. Rutledge RG, Stewart D (2008) Critical evaluation of methods used to determine amplification efficiency refutes the exponential character of real-time PCR. BMC Mol Biol 9:96

    Article  Google Scholar 

  48. Rychlik W, Spencer WJ, Rhoads RE (1990) Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res 18:6409–6412

    Article  CAS  Google Scholar 

  49. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  50. Tan SC, Yiap BC (2009) DNA, RNA, and protein extraction: the past and the present. J Biomed Biotechnol 2009:574398

    Article  Google Scholar 

  51. Templeton NS (1992) The polymerase chain reaction. History, methods, and applications. Diagn Mol Pathol 1:58–72

    Article  CAS  Google Scholar 

  52. Tweedie JW, Stowell KM (2005) Quantification of DNA by agarose gel electrophoresis and analysis of the topoisomers of plasmid and M13 DNA following treatment with a restriction endonuclease or DNA topoisomerase I. Biochem Mol Biol Educ 33:28–33

    Article  CAS  Google Scholar 

  53. Walsh PS, Metzger DA, Higuchi R (1991) Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10:506–513

    CAS  PubMed  Google Scholar 

  54. Wang X, Lim HJ, Son A (2014) Characterization of denaturation and renaturation of DNA for DNA hybridization. Environ Health Toxicol 29:e2014007

    Article  Google Scholar 

  55. Zhang S, Tian H, Wu J, Zhao S, Li C (2013) A new multiplex assay of 17 autosomal STRs and amelogenin for forensic application. PLoS One 8:e57471

    Article  CAS  Google Scholar 

  56. Zhu M, Hansen DL, Burd S, Gannon F (1989) Factors affecting free zone electrophoresis and isoelectric focusing in capillary electrophoresis. J Chromatogr A 480:311–319

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology and DNA Fingerprinting Unit, Central Forensic Science Laboratory (CBI), New Delhi, Delhi, India

    Braja Kishore Mohapatra

Authors
  1. Braja Kishore Mohapatra
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. DNA Fingerprinting Unit, State Forensic Science Laboratory, Sagar, Madhya Pradesh, India

    Hirak Ranjan Dash

  2. DNA Fingerprinting Unit, State Forensic Science Laboratory, Sagar, Madhya Pradesh, India

    Pankaj Shrivastava

  3. Department of Biology and DNA Fingerprinting Unit, Central Forensic Science Laboratory, New Delhi, Delhi, India

    Braja Kishore Mohapatra

  4. Department of Life Science, National Institute of Technology, Rourkela, Odisha, India

    Surajit Das

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Nature Singapore Pte Ltd.

About this chapter

Check for updates. Verify currency and authenticity via CrossMark

Cite this chapter

Mohapatra, B.K. (2018). Techniques Involved in DNA Fingerprinting: Isolation, Quantification, PCR, Genotyping, and Analysis. In: Dash, H., Shrivastava, P., Mohapatra, B., Das, S. (eds) DNA Fingerprinting: Advancements and Future Endeavors. Springer, Singapore. https://doi.org/10.1007/978-981-13-1583-1_3

Download citation

Publish with us

Policies and ethics

Search

Navigation