Lec5 - Lecture 5 Molecular Biology Methods 2 (Lodish,6th...

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Unformatted text preview: Lecture 5 Molecular Biology Methods 2 (Lodish,6th ed. Chapters 5.2-5.3) Cloning and sequencing mRNAs / cDNAs Use of Reverse transcriptase an RNA-dependent DNA polymerase that copies RNA into complementary DNA (cDNA) Howard Temin & David Baltimore In 1970: they independently discover reverse transcriptase an enzyme that transcribes RNA into DNA and that is required for replication of retroviruses (RNA viruses, e.g. HIV). 1975 Nobel Prize in Medicine or Physiology. DNA RNA Reverse transcriptase RNA polymerases Life cycle of retroviruses- In contrast to most retroviruses HIV eventually kills its host cell- Some retroviruses contain cancer-causing genes ( oncogenes ) L4-49 Model for reverse transcription of retroviral genomic RNA into DNA RT RNase H RT RNase H RT RT L6-14 Construction of a cDNA library Reverse transcriptase (Terminal transferase) Isolation of total cellular mRNA RNA-DNA hybrid (DNA polymerase) (T4 DNA ligase) Clone into EcoRI site of favorite vector (e.g. lambda) Plaques...
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Lec5 - Lecture 5 Molecular Biology Methods 2 (Lodish,6th...

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