Coupled reverse transcription-polymerase chain reaction (RT-PCR) as a sensitive and rapid method for isozyme genotyping.
We have developed a highly sensitive and rapid coupled reverse transcription-polymerase chain reaction (RT-PCR) technique for detection of alpha-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 (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 x 10(3)-fold more sensitive than Northern blotting in detecting AMY gene transcripts in human pancreatic total RNA.
Mocharla
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Department of Medical Genetics, School of Medicine, Indiana University, Indianapolis 46202-5251.
Mocharla
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Hodes
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D000681:Amylases / Q000235:genetics*; D005091:Exons; D006801:Humans; D007527:Isoenzymes / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000737:chemistry / Q000145:classification; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D012469:Salivary Glands / Q000201:enzymology; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D007527:Isoenzymes; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D000681:Amylases