Identification of Alternatively Spliced mRNAs and Localization of 5' Ends by Polymerase Chain Reaction Amplification

Abstract

Messenger RNAs of higher eucaryotes are usually modified posttran-scriptionally to contain 5' caps and nucleotide methylation, 3' polyadenylation, and one or more internal splices to remove introns and join exon segments into the mature protein-coding sequences. With the abilities of retroviral reverse transcriptases to create complementary DNA (cDNA) copies of RNA and of thermostable DNA polymerases to amplify specific DNA segments by repeated cycles of denaturation of duplex templates, annealing of complementary oligonucleotide primers, and strand elongation, it is becoming increasingly popular to use this highly sensitive polymerase chain reaction (PCR) to isolate partial cDNA sequences for the purpose of identifying RNA splice sites and inferring the coding capacity. The splice junction information from the partial cDNAs, together with additional biophysical or biochemical information, can then be employed to map the 5' ends of the mRNAs and assign the AUG protein initiation codon and open reading frame to the message.