Overlapping PCR for Bidirectional PCR Amplification of Specific Alleles: A Rapid One-Tube Method for Simultaneously Differentiating Homozygotes and Heterozygotes

  1. Qiang Liu1,3,
  2. Erik C. Thorland1,
  3. John A. Heit2, and
  4. Steve S. Sommer1,3,4
  1. 1Department of Biochemistry and Molecular Biology and 2Department of Internal Medicine, Mayo Clinic/Foundation, Rochester, Minnesota 55905

Abstract

Rapid detection of single-base changes is fundamental to molecular medicine. PASA (PCR amplification ofspecific alleles) is a rapid method of genotyping single-base changes, but one reaction is required for each allele. Bidirectional PASA (Bi-PASA) was developed to distinguish between homozygotes and heterozygotes in one PCR reaction by utilizing novel primer design with appropriate cycling conditions. In Bi-PASA, one of the alleles is amplified by a PASA reaction in one direction while the second allele is amplified by a PASA reaction in the opposite direction. Two outer (P and Q) and two inner allele-specific (A and B) primers are required. In heterozygotes, three segments are amplified: a segment of size AQ resulting from one allele, another segment of size PB resulting from the second allele, and a combined segment of size PQ. In homozygotes, segment PQ and either segments AQ or PB amplify. The two inner primers (A and B) contain a relatively short complementary region and a 10-nucleotide G+C-rich 5′ tail. The inner primers “switch” from low-efficiency to high-efficiency amplification when genomic DNA is replaced by previously amplified template DNA. In addition, the 5′ tails prevent “megapriming”. The parameters for optimizing Bi-PASA were investigated in detail for common mutations in the human factor V and catechol–O-methyltransferase genes. Guidelines for optimization of Bi-PASA also were developed and tested in a prospective study. Three additional Bi-PASA assays were optimized rapidly by utilizing these guidelines. In conclusion, Bi-PASA is a simple and rapid method for detecting the zygosity of known mutations in a single PCR reaction.

Footnotes

  • 3 Present address: City of Hope, Beckman Research Institute, Duarte, California 91010 USA.

  • 4 Corresponding author.

  • E-MAIL ssommer{at}smtplink.coh.org; FAX (818) 301-8142.

    • Received November 22, 1996.
    • Accepted February 19, 1997.
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