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Lecture 15

BIOL 5060 Lecture 15: 3:16_RecombinantDNATech_hofman

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Boston College
BIOL 5060

3/16_RecombinantDNATech_Hoffman 3/16/2017 2:01:00 PM Distinguish between mapping and cloning • Cloning gives you physical access to a physical reagent • Library screen by hybridization leads to cloning • Southern blot leads to knowledge • To get physical reagents….  RNA structure analysis • Nesting… o Talked about it with DNA sequencing o You can nest oligos and choose to sequence material by only chewing up that material • 5’ RACE o 1) Isolate total mRNA o 2) reverse transcribe using a gene specific primer o 3) RNase H to degrade RNA o 4) Terminal transferase + dCTP o 5) Second strand synthesis adapter primer o 6) PCR with anchor oligo and GSP2 oligo (nested primer) • 3’ RACE o we need knowledge of the ORF—but we have no idea of the flanking sequences= situation where we would use 3’ RACE o 1) Start by isolating total mRNA (within there is our transcript of interest) o 2) Start with a ply dt oligo (adaptor sequence oligo) ▪ First strand synthesis with poly dt adaptor oligo o 3) treat with RNase H to degrade the RNA o 4) PCR with gene specific primer 1 (GSP1) and the anchor primer ▪ combines second strand synthesis (will produce substrate molecules for the reverse anchor molecule to use as a template) essentially a PCR reaction o 5) product + other products (not so clean because not all steps were selective) ▪ there are a lot of transcripts in there o 6) Do a second PCR with GSP2 (nested) and anchor ▪ at this point, we should get a very clean product • RACE= rapid amplification of cDNA ends Transcriptional and Translational Fusions Transcriptional Fusions • Idea is to join portions of two different genes to make a hybrid RNA molecule; but not a hybrid protein • One fraction is the promoter gene and the other gene (gene 2) is the ORF o They need to be joined together in the same direction/ orientation • Scientific Chain reaction (SCR)= what we studied becomes the tools to study other things in the future o 1) Promoter gene (as a tool)- ▪ a. produce large amounts of proteins (purify) ▪ b. express the protein in a foreign organism or in a different tissue (by transcriptional fusion) ▪
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