Allele specific amplification by tetra-primer PCR

摘要: Genotyping a known point mutation or polymorphism can be achieved by restriction endonuclease digestion allele specific oligonucleotide (ASO) hybridization. Recently, amplification (ASA) the refractory system (ARMS) has been introduced, avoiding use of enzymes and radioisotopes (1). This method requires two separate polymerase chain reactions (PCR) to amplify each using different primers. We propose protocol accomplish ASA in single PCR, annealing temperatures (2) four In contrast ARMS, which mismatched 3'-residue mismatch tetra-primer PCR is middle internal A pair flanking primers (1 & 2) (3 4), conjunction with temperature programs, are required this (Figure 1). The Tms (4(G + C) 2(A T)) should at least 10°C higher than those (3), usually designing longer shorter Primer 3 completely complementary sense strand 1 primer 4 antisense 2. therefore on 2, because its unstable. also applies 1. first block cycles performed subsequent lower temperature. program, DNA fragment produced utilising only too high for anneal. During second acts as concentrated template facilitates low Tm anneal specifically. Two fragments generated heterozygote, one from 3, another 2 4; but amplified an homozygote (from 3) 4). Because not centered primers, created program unequal length, allowing them distinguished agarose gel electrophoresis. applied genotype Haein /3 fibrinogen gene number individuals 2a). bands products were unambiguous genotypes supported results ASO hybridization 2b). have employed distinguish base dilution mutant 40 normal molecules 8 fold greater amount (data shown). Tetra-primer easily applicable genotyping diallelic polymorphisms where may wider applications. ACKNOWLEDGEMENTS