A key problem in the generation of representational, full length cDNA libraries is the poor processivity of the copying enzyme, reverse transcriptase (RT). We propose to utilize mutants of human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (MoMuLV) RT that display improved processivity and/or better polymerase fidelity in the construction of representational, full-length cDNA libraries. We will compare selected HIV-1 RT mutants to identify the RT that displays the greatest increase in processivity and introduce point mutations that are known to increase the fidelity of DNA synthesis. Using procedures that we will develop to evaluate the representativeness of the DNA products, we will test a variety of conditions that will augment the representativeness of the cDNAs synthesized. These will include the use of two different RTs to saturate binding to mRNAs, addition of viral nucleocapsid protein to further increase the processivity as well as the use of a 5'-m7G cap-affinity procedure to enrich full-length mRNAs. Following the identification of the best RT enzyme and the optimization of conditions for processive synthesis, we will attempt to construct full-length representational cDNA libraries from transformed cell lines.
|Effective start/end date
|9/30/97 → 9/29/99
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