Coupled One-Step Reverse Transcription and Polymerase Chain Reaction Procedure for Cloning Large cDNA Fragments

Although Thermus aquaticus (Taq) and Thermus thermophilus (Tth) DNA polymerases have the ability to reverse transcribe RNA to complementary DNA (cDNA) and subsequently amplify the target cDNA, they are not usually the first choices for reverse transcription-polymerase chain reactions (RT-PCR) (1 -4 ). Because they only synthesize short cDNA fragments, their use is not widespread. In general, avian myeloblastosis virus (AMV), or moloney murine leukemia virus (M-MLV) reverse transcriptases (RTs) are used to reverse transcribe RNA to cDNA templates for subsequent PCR. Previous coupled methods are also unable to amplify large cDNA fragments and, thus, they are suitable only for the detection of gene expression (5 -8 ). The onestep RT-PCR procedure presented here was developed to amplify large cDNA fragments suitable for cloning full-length open reading frames (ORFs) encoding rat LH/ CG receptor isoforms (9 -12 ). As we all know, the construction of clones, including library screening and restriction mapping, by conventional cloning methods is very laborious and difficult.