MCB 7200 Molecular Biology Gene libraries c DNA
MCB 7200: Molecular Biology • Gene libraries • c. DNA 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 c. DNA 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’ 3’ 3’ 5’
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 -20 kb/2. 8 x 106 kb)= 6. 4 x 105 clones
The first step in making a c. DNA library: Purification of polyadenylated m. RNA using oligo(d. T)-cellulose Note: selection of the proper source (organ, tissue) of the RNA is critical here!
Figure 5. 15 A c. DNA library contains representative copies of cellular m. RNA sequences. Molecular Cell Biology, 7 th Edition Lodish et al. Copyright © 2013 by W. H. Freeman and Company
Complementary DNA or c. DNA cloning: c. DNA library construction Note: ds c. DNAs 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 c. DNA library • Using a DNA probe with a homologous sequence (e. g. , a homologous c. DNA 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 c. DNA library using DNA hybridization to a (radio-)labeled DNA probe
Screening a c. DNA 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 c. DNA 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
Animations for two related uses of expression vectors • Expression cloning of receptor proteins-see MCB Chapter 5 • http: //bcs. whfreeman. com/lodish 7 e/#800911__812046__ • Looking for protein-protein interactions with the yeast two hybrid system-see MCB Chapter 7 • http: //bcs. whfreeman. com/lodish 7 e/#800911__812055__
Plus/min (+/-) or differential screening
A cosmid cloning system: another possible cloning vector which can be used for genomic library but not for c. DNA libraries
In summary, you have seen: • How to make and screen gene libraries • How to make and screen c. DNA libraries • Several different cloning vectors including plasmids, bacteriophage lambda (l), and cosmids
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