Class Notes (1,000,085)
US (390,059)
BC (3,835)
BIOL (487)
BIOL 5060 (27)
Hoffman (27)
Lecture 15

BIOL 5060 Lecture 15: 3:16_RecombinantDNATech_hofman

5 Pages
59 Views
Spring 2017

Department
Biology
Course Code
BIOL 5060
Professor
Hoffman
Lecture
15

This preview shows pages 1-2. Sign up to view the full 5 pages of the document.
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
find more resources at oneclass.com
find more resources at oneclass.com
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
find more resources at oneclass.com
find more resources at oneclass.com

Loved by over 2.2 million students

Over 90% improved by at least one letter grade.

Leah — University of Toronto

OneClass has been such a huge help in my studies at UofT especially since I am a transfer student. OneClass is the study buddy I never had before and definitely gives me the extra push to get from a B to an A!

Leah — University of Toronto
Saarim — University of Michigan

Balancing social life With academics can be difficult, that is why I'm so glad that OneClass is out there where I can find the top notes for all of my classes. Now I can be the all-star student I want to be.

Saarim — University of Michigan
Jenna — University of Wisconsin

As a college student living on a college budget, I love how easy it is to earn gift cards just by submitting my notes.

Jenna — University of Wisconsin
Anne — University of California

OneClass has allowed me to catch up with my most difficult course! #lifesaver

Anne — University of California
Description
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) ▪
More Less
Unlock Document

Only pages 1-2 are available for preview. Some parts have been intentionally blurred.

Unlock Document
You're Reading a Preview

Unlock to view full version

Unlock Document

You've reached the limit of 4 previews this month

Create an account for unlimited previews.

Already have an account?

Log In


OR

Don't have an account?

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


OR

By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.


Submit