Inhibition of PCR amplification by a point mutation downstream of a primer

Qiang Liu, Erik C. Thorland, Steve S. Sommer

Research output: Contribution to journalArticle

36 Scopus citations

Abstract

A T → C point mutation is shown to specifically inhibit PCR amplification when compared to wild-type controls in exon H of the factor IX gene. Multiple primers of different lengths and locations were designed to examine this phenomenon. The experiments suggest that poor annealing and/or extension from the downstream primer are responsible for the observed inhibition and that the mutation can exert an inhibitory effect upon PCR amplification at a distance of at least 84 bp. The inhibition was not alleviated when amplification conditions such as annealing temperature, time of extension, type of DNA polymerase or concentration of DNA template, primer or DNA polymerase were varied. The inhibitory factor(s) are likely to be contained within the amplified segment itself because neither the use of a previously amplified PCR product as template for nested PCRs nor the restriction enzyme digestion of that previously amplified product relieved the inhibition of PCR amplification in the mutant sample. Computer analyses with the FOLDRNA and FOLDDNA programs did not reveal the mechanism of inhibition. Although dramatic inhibition, as shown here, may be frequent. Documentation of differential amplification caused by a single-base substitution in template sequence has implications for certain commonly used PCR-based methods such as quantitive PCR, differential display and DNA fingerprinting. In addition, heterozygous single-base pair mutations downstream of a primer may be missed if the PCR is inhibited; alternatively, the mutation may appear to be homozygous if amplification of the mutated allele is selectively enhanced.

Original languageEnglish (US)
Pages (from-to)292-300
Number of pages9
JournalBioTechniques
Volume22
Issue number2
DOIs
StatePublished - Feb 1997

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

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