Advertisement

In Silico PCR

  • Babajan Banaganapalli
  • Noor Ahmad Shaik
  • Omran M. Rashidi
  • Bassam Jamalalail
  • Rawabi Bahattab
  • Hifaa A. Bokhari
  • Faten Alqahtani
  • Mohammed Kaleemuddin
  • Jumana Y. Al-Aama
  • Ramu ElangoEmail author
Chapter

Abstract

A successful polymerase chain reaction (PCR) is the consequence of efficient primer designing and selective amplification of the target genetic region. The advancement of computational algorithms has allowed us to calculate the theoretical possibility of a successful PCR by designing highly specific and sensitive primers before starting expensive laboratory assays. Variety of web servers freely available for designing primer sequences and computational optimization of the PCR conditions. In the current chapter, we discuss and demonstrate how to design the primer sequences using “Primer-BLAST” program and validate those test sequences by simple primer evaluation methods. This in silico PCR method considers different primer-quality influencing factors like GC content, primer length, and melting temperature to design the five most suitable primer sets for the target gene sequence. The selection of best PCR primer set depends on how good the primer properties are and also its coverage area of the target gene. The short-listed primer sets are finally validated for future possibility of GC clamp, self-annealing, and hairpin formation using “PCR Primer Stat” program.

Keywords

Amplification Gene PCR Primer Stat Primer-BLAST GC clamp 

References

  1. Abd-Elsalam KA (2003) Bioinformatic tools and guideline for PCR primer design. Afr J Biotechnol 2(5):91–95Google Scholar
  2. Breslauer KJ, Frank R, Blöcker H, Marky LA (1986) Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A 83(11):3746–3750PubMedPubMedCentralGoogle Scholar
  3. Chen H, Zhu G (1997) Computer program for calculating the melting temperature of degenerate oligonucleotides used in PCR or hybridization. Biotechniques 22(6):1158–1160Google Scholar
  4. Dieffenbach CW, Lowe TM, Dveksler GS (1993) General concepts for PCR primer design. Genome Res 3(3):S30–S37Google Scholar
  5. Gaudet M, Fara AG, Beritognolo I, Sabatti M (2009) Allele-Specific PCR in SNP Genotyping. In: Komar A (eds) Single Nucleotide Polymorphisms. Methods in Molecular Biology™ (Methods and Protocols), vol 578. Humana Press, Totowa, NJGoogle Scholar
  6. He Q, Marjamaki M, Soini H, Mertsola J, Viljanen MK (1994) Primers are decisive for sensitivity of PCR. BioTechniques 17(1):82, 84, 86–82, 84, 87Google Scholar
  7. Kwok S, Kellogg DE, McKinney N, Spasic D, Goda L, Levenson C, Sninsky JJ (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res 18(4):999–1005PubMedPubMedCentralGoogle Scholar
  8. Rychlik W, Rhoads RE (1989) A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Res 17(21):8543–8551PubMedPubMedCentralGoogle Scholar
  9. Rychlik W, Spencer WJ, Rhoads RE (1990) Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res 18(21):6409–6412PubMedPubMedCentralGoogle Scholar
  10. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT et al (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239(4839):487–491Google Scholar
  11. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230(4732):1350–1354PubMedPubMedCentralGoogle Scholar
  12. Sheffield VC, Cox DR, Lerman LS, Myers RM (1989) Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci U S A 86(1):232–236PubMedPubMedCentralGoogle Scholar
  13. Tabchoury CP, Sousa MC, Arthur RA, Mattos-Graner RO, Del Bel Cury AA, CURY JA (2008) Evaluation of genotypic diversity of Streptococcus mutans using distinct arbitrary primers. J Appl Oral Sci 16:403–407Google Scholar
  14. Wallace RB, Shaffer J, Murphy RF, Bonner J, Hirose T, Itakura K (1979) Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch. Nucleic Acids Res 6(11):3543–3557PubMedPubMedCentralGoogle Scholar
  15. Wu DY, Ugozzoli L, Pal BK, Qian J, Wallace RB (1991) The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction. DNA Cell Biol 10(3):233–238.  https://doi.org/10.1089/dna.1991.10.233CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Babajan Banaganapalli
    • 1
  • Noor Ahmad Shaik
    • 2
  • Omran M. Rashidi
    • 3
  • Bassam Jamalalail
    • 2
  • Rawabi Bahattab
    • 2
  • Hifaa A. Bokhari
    • 2
  • Faten Alqahtani
    • 2
  • Mohammed Kaleemuddin
    • 2
  • Jumana Y. Al-Aama
    • 1
  • Ramu Elango
    • 1
    Email author
  1. 1.Princess Al-Jawhara Center of Excellence in Research of Hereditary Disorders, Department of Genetic Medicine, Faculty of MedicineKing Abdulaziz UniversityJeddahSaudi Arabia
  2. 2.Department of Genetic Medicine, Faculty of MedicineKing Abdulaziz UniversityJeddahSaudi Arabia
  3. 3.Princess Al-Jawhara Center of Excellence in Research of Hereditary DisordersKing Abdulaziz UniversityJeddahSaudi Arabia

Personalised recommendations