Primer Design for PCR Reactions in Forensic Biology

  • Kelly M. ElkinsEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1275)


The polymerase chain reaction (PCR) is a popular method to copy DNA in vitro. Its invention revolutionized fields ranging from clinical medicine to anthropology, molecular biology, and forensic biology. The method employs one of many available heat-stable DNA polymerases in a reaction that is repeated many times in situ. The DNA polymerase reads a template DNA strand and using the components of the reaction mix, catalyzes the addition of free 2′-deoxynucleotide triphosphate nitrogenous bases to short segment of DNA that forms a complement with the template via Watson–Crick base pairing. This short segment of DNA is referred to as a PCR primer and it is essential to the success of the reaction. The most widely used application of PCR in forensic labs is the amplification of short tandem repeat (STR) loci used in DNA typing. The STRs are routinely evaluated in concert with 16 or more reactions, a multiplex, run in one test tube simultaneously. In a multiplex, it is essential that the primers work specifically and accurately on the intended reactions without hindering the other reactions. The primers, which are very specific, also can be used to probe single nucleotide polymorphisms (SNPs) in a DNA sequence of interest by single base extension. Primers are often designed using one of many available automated software packages. Here the process of manually designing PCR primers for forensic biology using no-cost software is described.

Key words

Molecular biology Nucleic acids/DNA/RNA Hydrogen bonding Oligonucleotide DNA polymerases Polymerase chain reaction PCR primer STR SNP 



Thank you to the editors for inviting me to submit this chapter. This work was supported by start-up funds provided by Towson University (K.M.E.). The author is grateful to Zoë Krohn, Suzanne Gray, and Alison Eychner for testing the protocol as written.


  1. 1.
    Butler JM (2005) Forensic DNA typing, second edition: biology, technology, and genetics of STR markers. Elsevier Academic Press, Burlington, MAGoogle Scholar
  2. 2.
    Elkins KM (2012) Forensic DNA biology: a laboratory manual. Elsevier Academic Press, Waltham, MAGoogle Scholar
  3. 3.
    Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH (1997) Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques 23:504–511PubMedGoogle Scholar
  4. 4.
    Kim TD (2000) PCR primer design: an inquiry-based introduction to bioinformatics on the World Wide Web. Biochem Mol Biol Edu 28:274–276Google Scholar
  5. 5.
    Lima AOS, Garcês SPS (2006) Intrageneric primer design: Bringing bioinformatics tools to the class. Biochem Mol Biol Edu 34:332–337CrossRefGoogle Scholar
  6. 6.
    Thornton B, Basu C (2011) Real-time PCR (qPCR) primer design using free online software. Biochem Mol Biol Educ 39:145–154CrossRefPubMedGoogle Scholar
  7. 7.
    Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden T (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13:1–11CrossRefGoogle Scholar
  8. 8.
    Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana, Totowa, NJ, pp 365–386Google Scholar
  9. 9.
    Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JAM (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35:W71–W74CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Elkins KM (2011) Designing PCR primer multiplexes in the forensic laboratory. J Chem Educ 88:1422–1427CrossRefGoogle Scholar
  11. 11.
    Ruitberg CM, Reeder DJ, Butler JM (2001) STRBase: a short tandem repeat DNA database for the human identity testing community. Nucleic Acids Res 29:320–322CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Papp AC, Pinsonneault JK, Cooke G, Sadée W (2003) Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves. Biotechniques 34:1068–1072PubMedGoogle Scholar
  13. 13.
    Wang J, Chuang K, Ahluwalia M, Patel S, Umblas N, Mirel D, Higuchi R, Germer S (2005) High-throughput SNP genotyping by single-tube PCR with Tm-shift primers. Biotechniques 39:885–893CrossRefPubMedGoogle Scholar
  14. 14.
    Coble MD, Just RS, O’Callaghan JE, Letmanyi IH, Peterson CT, Parsons TJ (2004) Single nucleotide polymorphisms over the entire mtDNA genome that increase the forensic power of testing in Caucasians. Int J Legal Med 118:137–146CrossRefPubMedGoogle Scholar
  15. 15.
    Elkins KM, Kadunc RE (2012) An undergraduate laboratory experiment for upper-level forensic science courses: the use of TPOX single locus primers to amplify human DNA by real-time PCR with SYBR green detection. J Chem Educ 89:784–790CrossRefGoogle Scholar
  16. 16.
    Masibay A, Mozer TJ, Sprecher C (2000) Promega Corporation reveals primer sequences in its testing kits [letter]. J Forensic Sci 45:1360–1362PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Chemistry DepartmentTowson UniversityTowsonUSA

Personalised recommendations