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Lecture 15

BIOL 5060 Lecture Notes - Lecture 15: Myc, Polyadenylation, Reading Frame

5 pages62 viewsSpring 2017

Department
Biology
Course Code
BIOL 5060
Professor
Hoffman
Lecture
15

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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 ORFbut 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
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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)
c. regulate expression of a protein
o 2) ORF (as a tool)-
a. allows easy monitoring of gene expression from a
promoter of interest
transcriptional fusions- reporters can be used as a tool
to study expression of ORF (GFP, luciferase, etc.)
Translational Fusions- combining parts of two or more genes; goal is to
produce hybrid proteins (extra challenge to making a translational fusion=
don’t only worry that they are in the same direction BUT ALSO… need to
maintain the same reading frame between the two open reading frames)
Supply promoter and additional sequence to help with production,
detection and purification
OR on the other hand, could be providing sequence to help with
detection and/ or purification= new thing about translational fusions
o Rather than simply detecting level of transfusion, it’s a
reflection of the level of transcription
o Can tell where in the cell its localized and it can allow for
purification
o Allows us to study promoter and protein product
So what are these sequences…?
o ORF (as a tool)
A. used to help with detection and purification
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