โ Scribed by R. J. Crawford; J. R. E. Wells
No coin nor oath required. For personal study only.
DNA, complementary to chicken globin mRNA was synthesized using either Avian Myeloblastosis virus reverse transcriptase, or E. coli DNA polymerase I. Transcriptase cDNA sediments at 9 S on sucrose gradients, and is 620 nucleotides in length, representing a complete copy of globin mRNA template. In contrast, Polymerase I eDNA sediments at 4 S, is 100 to 200 nucleotides in length, and is a copy of a small region at the 3' (poly A) end of globin mRNA.
Similarly, Transcriptase eDNA and Polymerase I eDNA hybridize to globin mRNA template with characteristic, individual Crot y2 values. The Crotn value for Transcriptase eDNA hybridization is 7 โข 10 -4 mol s F ~ , and that for Polymerase I cDNA is 5 โข 10 -a .
This work shows that Avian Myeloblastosis virus reverse transcriptase can use Polymerase I cDNA to prime further eDNA synthesis along the mRNA template. The product of extended eDNA synthesis is identical in length and hybridization properties to oligo (dT) primed transcriptase eDNA.
Abbreviations. eDNA, complementary DNA; Transcriptase, Avian Myeloblastosis virus reverse transcriptase; Polymerase I, E. coli DNA Polymerase I; Cro t, product of initial RNA concentration (moles nucleotide, litre -1 ) and time (see).
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A sensitive assay for quantitating DNA damage within individual genes would be a valuable tool for identifying the molecular mechanisms of disease and the sites of action of various carcinogens and anticancer drugs. This report describes a competitive PCR assay that was used to quantitate DNA damage