Eukaryotic gene organization

enhancers
silencers
 Genomic
library
constructio
n
 Screening a
genomic library
using DNA
hybridization to a
(radio-)labeled
DNA probe
Production of a (radio-)labeled DNA probe by the random
primer method [uses the Klenow fragment of DNA
polymerase]
5 3

5 3
3 5
 The first step in
making a cDNA
library: Purification
of polyadenylated
mRNA using
oligo(dT)-cellulose
 Figure 5.15 A cDNA library contains representative copies of cellular mRNA sequences.

Molecular Cell Biology, 7th Edition

Copyright 2013 by W. H. Freeman and Company
Lodish et al.
Complementary DNA
or cDNA cloning:
cDNA library
construction

Note: ds cDNAs are

typically placed in a
cloning vector such as
bacteriophage lambda
() or a plasmid
Bacteriophage
cloning system
 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)
 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
-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
 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