Current problems in generating full-length cDNAs are due to a poor processivity of reverse transcriptas (RT). We propose to develop novel RT variants with enhanced processivity and fidelity, develop conditions that favor synthesis of long cDNA products and construct cDNA libraries using mRNAs selected to be the full-length copies. (1) First, we will develop novel RTs for processive cDNA synthesis both by in vitro evolution as well as use previously designed mutants. Combining DNA-shuffling with functional screening for RTs that can melt secondary structures and can perform at higher temperatures, new RTs with desired properties will be identified. Work from this and other laboratories has shown that both processivity and fidelity of HIV-1 RT can be further improved by specific mutations introduced into HIV-1 RT. Recombinant HIV-1 RT variants containing mutations that increase polymerase processivity have already been generated in our laboratory. (2) Next, with such new and improved RTs, we will develop procedures to evaluate the size and the representativeness of the cDNA products made. This will be accomplished by testing the ability of these RTs to copy long in vitro transcripts, RT-PCR of long mRNAs using gene-specific primers and ability to detect a greater number of genes in a microarray hybridization. We will also develop procedures that enhance accessibility of all mRNAs to the RT by dilution method, by removal of the mRNAs bound to RTs and by using two different RTs. As the viral nucleocapsid (NC) protein increases RT processivity and reduces pausing, we will study the influence of HIV-1 NCp7 on the ability of wild type and variant HIV-1 RTs to generate fuller length copies. (3) Finally, we will employ the highly processive RTs identified above with the optimal conditions to be developed to prepare representational cDNA libraries consisting full-length cDNAs. We will isolate mRNAs that are purified via 5'-m7G-cap affinity and via oligodT-affinity sequentially to enrich for full-length polyadenylated mRNAs. With the full-length mRNA thus isolated as templates, we will prepare double stranded cDNAs enriched for full-length cDNAs using eIF-4 and construct a cDNA library in lambda Zap II. Up to 1000 cDNA clones will be analyzed for insert size and 5'-terminal sequence to evaluate whether novel, unreported 5'-end sequences were obtained by our approach.
|Effective start/end date||9/30/99 → 9/29/00|