Transcript Biotechnology and Genetic Engineering

MCB 720: Molecular Biology
•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’
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How many genomic clones must be
screened to find your gene?
Theoretically, you will need to screen N clones where
N=ln(1-P)/ln(1-f) where P=the probability of finding your
gene and f=the average size of the cloned genomic
sequence in your vector divided by the total genome
size.
How many clones must you screen to find your gene in a
human gene library packaged in EMBL 3 with 99%
certainty?
N=ln(1-0.99)/ln(1-20kb/2.8 x 106kb)= 6.4 x 105 clones
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
Cos sites
at the left
and right
ends
Cloning
site
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-32P-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 125I
Note: see also MCB Chapter 9 for a related animation
http://bcs.whfreeman.com/lodish5e/pages/bcsmain.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/bcsmain.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/bcsmain.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