Biotechnology and Genetic Engineering-PBIO 450/550

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12 Δεκ 2012 (πριν από 4 χρόνια και 6 μήνες)

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Biotechnology and Genetic Engineering

PBIO 450/550


Gene libraries


cDNA

libraries


Library screening


Eukaryotic gene organization


enhancers

silencers

Genomic
library
construction

Screening a genomic
library using DNA
hybridization to a
(radio
-
)labeled
DNA
probe



Note: a cDNA is commonly
(radio
-
)labeled and used as
a DNA probe to screen a
genomic library

Production of a (radio
-
)labeled
DNA probe

by the random primer
method [uses the Klenow fragment of DNA polymerase]

5’

5’

5’

3’

3’

3’

The first step in making a
cDNA library:
Purification
of polyadenylated mRNA
using oligo(dT)
-
cellulose



Note: selection of the
proper source (organ,
tissue) of the RNA is
critical here!



Complementary DNA or
cDNA cloning:

cDNA library construction



Note: ds cDNAs are typically
placed in a cloning vector such
as bacteriophage lambda (
l
)
or a plasmid

Bacteriophage
l

cloning system

Bacteriophage
l

cloning system

Cloning

site

Cos sites

at the left

and right

ends

There are several possible ways to
screen a
cDNA

library


Using a DNA probe with a homologous sequence
(e.g., a homologous
cDNA

or gene clone from a
related species)


Using an
oligonucleotide

probe based on a known
amino acid sequence (requires purification of the
protein and some peptide sequencing)


Using an antibody against the protein of interest
(note: this requires use of an expression vector)


Plus/minus or differential screening (the least
specific way)


Screening a cDNA
library using DNA
hybridization to a
(radio
-
)labeled
DNA probe

Screening a cDNA library with a labeled
oligonucleotide probe

based on a known peptide sequence

Using polynucleotide kinase and

g
-
32
P
-
labeled ATP to radiolabel oligonucleotide probes

Immunological screening of an
expression

cDNA library with a
primary antibody and labeled
secondary antibody; note the
label is often an enzyme label
like alkaline phosphatase or
horseradish peroxidase, but it
can also be
125
I


Note: see also MCB Chapter 9 for a related animation
http://bcs.whfreeman.com/lodish5e/pages/bcs
-
main.asp?v=category&s=00010&n=09000&i=09010.0
4&o=|00510|00610|00520|00530|00540|00560|00
570|00590|00600|00700|00710|00010|00020|000
30|00040|00050|01000|02000|03000|04000|0500
0|06000|07000|08000|09000|10000|11000|12000|
13000|14000|15000|16000|17000|18000|19000|2
0000|21000|22000|23000|99000|&ns=589

Animations for two related uses of
expression vectors


Expression cloning of receptor proteins
-
see MCB Chapter 9


http://bcs.whfreeman.com/lodish5e/pages/bcs
-
main.asp?v=category&s=00010&n=09000&i=09010.04&o=|00510|00610|00520|00530|00540|
00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|
03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|
16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=589


Looking for protein
-
protein interactions with the yeast two
hybrid system
-
see MCB Chapter 11


http://bcs.whfreeman.com/lodish5e/pages/bcs
-
main.asp?s=00010&n=11000&i=11010.01&v=category&o=|00510|00610|00520|00530|00540|
00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|
03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|
16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=798&t=&uid=0&rau=0

Plus/min (+/
-
)
or differential
screening

A
cosmid

cloning system:

another possible cloning
vector which can be used
for genomic library but
not for cDNA libraries

In summary, you have seen:


How to make and screen gene libraries


How to make and screen
cDNA

libraries


Several different cloning vectors including
plasmids,
bacteriophage

lambda (
l
), and
cosmids