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Reverse_Transcription - Reverse Transcription Reverse...

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Unformatted text preview: Reverse Transcription Reverse Transcription Central Dogma Central Dogma Normal transcription involves synthesis of RNA from DNA. Reverse transcription is the transcription of single stranded RNA into double stranded DNA With the help of the enzyme Reverse Transcriptase. Reverse Transcriptase also known as RNA directed DNA Polymerase ­ DNA Nucleotidyl transferase (RNA directed) ­ Revertase Reverse Transcriptase was discovered by Howard Temin and Baltimore in 1970 independently ­ shared Nobel Prize in Physiology or Medicine in 1975 for their discovery. Reverse transcriptase common in Retrovirus. ­ HIV M­MLV (Moloney Murine Leukemia Virus) AMV (Avian Myeloblastosis Virus) Reverse Transcriptase enzyme includes two activity: DNA polymerase and RNase H Retrovirus Replication Cycle Retrovirus Replication Cycle ds DNA host DNA Packaging into virus ; budding Provirus Antiviral drug Antiviral drug Zidovudine Lamivudine Tenfovir Typical retrovirus has three or four genes. Gag­pol­ env Retrovirus are called + strand because viral RNA itself code for protein products. Reverse transcriptase enzyme code for proteins are called – strand R : Redundant or repeating seq – 10­80 U5: 80 – 100 U3: 170­1350 Like DNA polymerase, Reverse transcriptase requires primers. tRNA of the host is Primer MECHANSIM OF RETROVIRUS REPLICATION 1. A Retrovirus specific cellular tRNA hybridizes with a complementary region called PBS (Primer Binding Sites) 2. Reverse Transcriptase (RT) starts at this binding site and copies RNA into a single strand of complementary DNA. A DNA segment is extended from tRNA based on the sequence of the retroviral genomic RNA 3. The viral R and U5 sequences are removed by RNase H. LTR – Long Terminal Repeat Left LTR ­ Redundant sequence [R]+ 5’ untranslated region (U5) Right LTR ­ Redundant sequence [R]+ 3’ untranslated region (U3) 4. First jump: DNA hybridizes with the remaining R sequence at the 3' end. 5. A DNA strand is extended from the 3' end. 6. Most viral RNA is removed by RNase H 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. 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 M­MLV cDNA 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. This powerpoint was kindly donated to www.worldofteaching.com http://www.worldofteaching.com Is home to well over a thousand powerpoints submitted by teachers. This a free site. Please visit and I hope it will help in your teaching ...
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