Research Project
6337791
DESCRIPTION (Applicant's abstract): In this application we will discuss the rationale for the development of a novel method for chemically tagging a synthetic oligonucleotide to the 5'-end of full-length mRNAs using the cap structure (7-methyl guanosine triphosphate, m7Gppp). There are two major technical limitations in full-length cDNA library construction. The first is reduced efficiency of the reverse transcriptase reaction and the second limitation is the inability to efficiently select only full-length cDNA. This is compounded by the need to use RNase H to generate RNA primers for second strand synthesis, which biases against the isolation full-length cDNAs. Current RACE methods (Rapid Amplification of cDNA ends) and commercial kits to isolate full-length cDNAs are technically dificule whith variable at best. In this proposal we have designed a procedure which will address some of the problems inherent with current protocols. The technique can be used as either a 5'-RACE method or a full-length cDNA library construction method. By attaching a ribo-oligonucleotide through the morpholino-nucleoside residue to the 5'-capped end of mRNA, the reverse transcriptase can read through the cap structure, and using the attached ribo-oligonucleotide as a template, continuing systhesis of first strand cDNA to the end of this oligo. The complementary sequence to the ribooligonucleotide becomes attached to the 3'-end of the cDNA, presenting a known priming site for second strand synthesis to generate full-length double strand cDNA, This method is especially advantageous in that it does not require an RNase H step to generate primers for second strand synthesis, which in itself biases against 5'-end identification. PROPOSED COMMERCIAL APPLICATION: The ultimate goal will be to generate an efficient method for specifically tagging the 5'-cap structure of full-length cDNA. This technology will hopefully supercede the current technologies, which are technically difficult and inefficient. Quality full-length cDNA libraries and 5'-EST libraries will be developed. These libraries will result in the identification of novel 5' -untranslated regions and longer and rarer genes.
