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

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.

Early modification of sickle cell disease clinical course by UDP-glucuronosyltransferase 1A1 gene promoter polymorphism

“…Genomic DNA was extracted from peripheral blood leukocytes by a salting-out procedure (Miller et al 1988). The homozygosity for the SCD mutation was confirmed by a bidirectional allele-specific polymerase chain reaction (PCR) as described (Liu et al 1997). The number of (TA) repeats in the UGT1A1 promoter was analysed by a PCR-based methodology using the primers described by Monaghan et al (1996) in which the sense primer was 5-carboxyfluorescein (FAM) fluorescent-tagged.…”

Study of disease-relevant polymorphisms in the TLR4 and TLR9 genes: A novel method applied to the analysis of the Portuguese population

“…1). Primers for Bi-PASA were designed following the guidelines proposed by Liu et al [26], taking into account both the melting temperature of both the primers and the largest PCR segment (PQ). The primers used in this study are shown in Table 2.…”

“…However, most of the genotyping techniques currently available are still either time-consuming and laborious or require expensive equipment [23][24][25]. In this sense, we adapted a simple and rapid method, allowing genotype determination in a single reaction based in the bidirectional PCR amplification of specific alleles (Bi-PASA) [26] in both the TLR4 (Asp299Gly, Thr399Ile) and TLR9 (T-1237C) genes, a methodology amenable to large scale analysis, using technology available in most scientific laboratories.…”

Prognostic significance of genetic variants in the IL-23/Th17 pathway for the outcome of T cell-depleted allogeneic stem cell transplantation

“…Allelic variants were genotyped using bidirectional PCR amplification of specific alleles, as previously described. 31,32 Regarding quality control, each PCR set comprised randomly selected replicates of previously typed samples from the study and two negative controls. Agreement for quality control and duplicates was X99% for all assays.…”