BIOCHEM 2B03 Lecture Notes - Lecture 10: Membrane Technology, Ribosomal Rna, Thymidine Triphosphate
6 Sep 2013
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Document Summary
Mrna single stranded and has poly-a tail. Synthesizes dna from mrna in same 5" 3" direction. Cant do first bond synthesis (reason for primers) Produced by eukaryotic retroviruses (eg/ hiv, common cold) dntps (datp, dgtp, dttp, dctp) added by reverse transcriptase to make double stranded rna:dna duplex. Results in ssdna cdna: amplify dna (pcr) Denature separate dsdna by heating (95 c) Dna polymerase must be thermotolerant (taq, pfu, vent etc) Extension time determined by length of desired product. Primers can be synthesized; designed to be complementary to the 3" ends of dna to be amplified. Boundaries of primers determine boundaries of amplified dna (established after multiple cycles) Primers allow addition of extra nucleotides to 5" end useful sequences such as restriction sites and tags. Repeat steps for multiple cycles; subsequent rounds use newly synthesized strands as template. Error prone due to lack of/inefficient 3"5" exonuclease activity: digestion (restriction enzymes)
