GENETICALLY ALTERED RTS PREPARE BETTER CDNA LIBRARIES

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date9/30/979/29/98

Funding

  • National Cancer Institute

ASJC

  • Immunology and Allergy

Fingerprint Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.