REVERSE TRANSCRIPTASE

Description

Enzyme possessed by retroviruses that allows transcription of DNA from an RNA template. Such an enzyme has never been isolated from other sources, but speculation about its possible occurrence continues. Such an enzyme is widely used experimentally in genetic engineering to allow the formation of complementary DNA (cDNA) from purified RNA. The discovery of this enzyme faulted the widely held central dogma, which stated that the information flow in nature was always from DNA to RNA.

Uses

Synthesizes a complementary DNA strand using single-stranded RNA or DNA in the presence of a primer

Uses

Reverse Transcriptase AMV is suitable for:

• First- and second-strand cDNA synthesis and synthesis of first strand cDNA for use in subsequent amplification reactions (RT-PCR)
• Dideoxy DNA sequencing
• Primer extension
• RNA sequencing
• 3′-end labeling of DNA fragments
• Generation of single-stranded probes for genomic footprint experiments

Uses

M-MLV Reverse Transcriptase has been used:

• for the preparation of cDNA libraries or for first strand cDNA synthesis
• for use in a 2-step RT-PCR assay
• for the synthesis of cDNA that is further used in cloning

General Description

M-MLV (Moloney murine leukemia virus) reverse transcriptase enzyme is isolated from E. coli expressing a portion of the pol gene of M-MLV on a plasmid. MoMLV RT is made up of 671 amino acid residues. It is a DNA polymerase that uses single-stranded RNA, DNA, or an RNA-DNA hybrid (using a primer) to synthesize a complementary DNA strand.

Purification Methods

This enzyme produces the complementary DNA from the RNA (as template). These are purified by solubilising the virus with non-ionic detergents. Lysed virions are adsorbed on DEAE-cellulose or DEAE-Sephadex columns, and the enzymes are eluted with a salt gradient, then chromatographed on a phosphocellulose column and fractions with enzyme activity are eluted in a salt gradient. They are also purified from other viral proteins by affinity chromatography on a pyran-Sepharose column. [Verna Biochim Biophys Acta 473 1 1977, Smith Methods Enzymol 65 560 1980; see commercial catalogues for other transcriptases.]