Reverse Transcription

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5. A DNA strand is extended from the 3' end.

6. Most viral RNA is removed by RNase H

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7. A second DNA strand is extended from the viral RNA.

8. Both tRNA and the remaining viral RNA are removed by RNase H.

9. Second jump: The PBS region of the second strand hybridizes with the PBS region of the first strand.

10. Extension on both DNA strands.

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Reverse transcriptase has a high error rate when transcribing RNA into DNA as unlike DNA Polymerase, since it has no proof reading ability

This high error rate allows mutations to accumulate

The commercially available RT produced by Promega are quoted high error rates in range of one in 17,000 bp for AMV and one in 30,000 bases for M-MLV

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Cdna

Reverse Transcriptase can use single stranded RNA into DS DNA – cDNA.

cDNA library: A set of clones representing as many as possible of the mRNAs in a given cell type at given time.

In the preparation cDNA library, mRNA is extracted, purified, and treated with the enzyme reverse transcriptase.

Complementary DNA (cDNA) analogs of the isolated mRNA are thereby obtained.

Since mature mRNA contains no introns or regulatory regions, a cDNA library composed of coding regions.