Overlapping PCR for bidirectional PCR amplification of specific alleles: a rapid one-tube method for simultaneously differentiating homozygotes and heterozygotes.
Rapid detection of single-base changes is fundamental to molecular medicine. PASA (PCR Amplification of Specific 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.
Liu
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Q
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, Minnesota 55905, USA.
Thorland
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EC
Heit
J A
JA
Sommer
S S
SS
D000483:Alleles; D002394:Catechol O-Methyltransferase / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics*; D005165:Factor V / Q000235:genetics; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D017931:DNA Primers; D005165:Factor V; D002394:Catechol O-Methyltransferase