Coupled reverse transcription-polymerase chain reaction (RT-PCR) as a sensitive and rapid method for isozyme genotyping

Abstract

We have developed a highly sensitive and rapid coupled reverse transcription-polymerase chain reaction (RT-PCR) technique for detection of α-amylase-encoding gene transcripts and for distinguishing between the human salivary AMY1 and pancreatic (AMY2) gene transcripts. The two genes are 93–94% homologous. However, the AMY1 gene has an additional exon known as exon S, and an extra 32 bp in exon 1. Genotyping of the different AMYs by RT-PCR was based on this unique feature of the AMY1 mRNA sequence. Detection of AMY gene and ( AMY1 and AMY2) transcripts in cellular RNA was achieved with a set of primers common to both human AMY1 and AMY2 genes and derived from the exon 3–4 regions. In contrast, AMY1 gene transcripts were distinguished from the pancreatic AMY2 gene transcripts by use of primers specific to the exon S-1 regions of the AMY1 gene. To distinguish AMY1 transcripts from a mixture of AMY1 and AMY2, use was made of the differences in the ethidium bromide-stained agarose gel patterns obtained after digestion of the amplified exon 3–4 fragments with TaqI. AMY gene transcripts were detectable by autoradiography in RT-PCR amplified DNA obtained from as little as 5 pg of human pancreatic or parotid total RNA. A comparison of sensitivity of Northern blotting vs. RT-PCR suggested that the RT-PCR method is about 3–6 × 10 3-fold more sensitive than Northern blotting in detecting AMY gene transcripts in human pacreatic total RNA.