Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Complementary DNA (cDNA) copies of the common (OM) and Cowpea (Cc) strains of tobacco mosaic virus (TMV) RNA polyadenylated in vitro have been synthesized with AMV reverse transcriptase and oligo (dT)10 as primer. The cDNA was converted to a double-stranded form by E. coli DNA polymerase I followed by S1 nuclease digestion. The double-stranded cDNA copies were cloned in pBR322 at the PstI site using the oligo(dC)-oligo(dG) tailing method. With the common strain, several clones containing inserts covering different parts of genomic RNA were selected using partially reconstituted RNA and partially stripped virus RNA as hybridization probes. Restriction mapping of three clones and their overlaps showed that cloned sequences covered about 4000 nucleotides of the common strain RNA from the 3' end. With the Cc strain, clones containing the 3' portion were selected by Southern hybridization using coat protein mRNA isolated from short particles as a probe. One cloned recombinant plasmid was found by restriction analysis and R-loop mapping to carry about a 1700 nucleotide sequence of Cc strain RNA from the 3' end. A restriction map of the OM strain was very similar to the map of the vulgare strain, as predicted from its nucleotide sequence, but completely different from that of Cc strain.
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PMID:Molecular cloning of the complementary DNA copies of the common and cowpea strains of tobacco mosaic virus RNA. 1863 28

The use of avian myeloblastosis virus reverse transcriptase (AMV RTase) to produce DNA copies of mRNA templates is a common and well-documented method (1-3). Briefly, the method involves synthesis of a complementary DNA strand to the mRNA from a short double-stranded region, usually provided by using an oligo(dT) primer on poly(A)(+)RNA. The enzyme does not always produce full length transcripts, but all the complementary strands are finished off with a short hairpin loop. This provides a ready-made primer for second strand synthesis, useful whether this is to be performed by more reverse transcriptase or by E. coli DNA polymerase 1 (pol 1). An idealized picture is shown in Fig. 1. Before the double-stranded cDNA (ds cDNA) copy can be cloned it is necessary to remove this hairpin loop using the single-strand specific nuclease S1. Fig. 1. Stages in the production of double-stranded cDNA from poly(A)(+)mRNA. The original RNA is represented by a solid line, while the cDNA is represented by a dashed line. Note that this diagram is not intended as an accurate representation of the enzymatic processes involved, but as a general guide to the principles of cDNA synthesis.
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PMID:Synthesis of Double-Stranded Complementary DNA from Poly(A)(+)mRNA. 2137 88


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