Rapid cycle allele-specific amplification: Studies with the cystic fibrosis ΔF508 locus

C. T. Wittwer, B. C. Marshall, G. H. Reed, J. L. Cherry

Research output: Contribution to journalArticlepeer-review

Abstract

Rapid cycle DNA amplification is a polymerase chain reaction technique with improved product specificity and cycle times of 20-60 s, allowing complete 30-cycle reactions in 10-30 min. The presence or absence of the ΔF508 deletion and wild-type allele was determined in 104 cystic fibrosis patients by rapid cycle DNA amplification. In separate allele-specific assays, sequences on both sides of the ΔF508 locus were amplified with the 3' end of a discriminating primer at the ΔF508 locus, with either a 3-bp or a 1-bp mismatch. With rapid cycling (35-s cycles), single-base discrimination was achieved over a broad range of annealing temperatures (50 °C or lower); with conventional cycling and 'hot starts' (160-s cycles), only annealing temperatures of 61-62 °C sufficiently discriminated between alleles. With rapid cycling, genotype could still be assessed with annealing temperatures as low as 25 °C. We conclude that faster temperature cycling can improve the results of allele-specific amplification.

Original languageEnglish (US)
Pages (from-to)804-809
Number of pages6
JournalClinical chemistry
Volume39
Issue number5
StatePublished - Jan 1 1993
Externally publishedYes

Keywords

  • DNA probes
  • heritable disorders
  • polymerase chain reaction
  • screening

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

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