Biotechnology and Genetic Engineering PBIO 4500/5500 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’ 3’ 5’ 3’ 3’ 5’

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

Animations for two related uses of expression vectors Expression cloning of receptor proteins-see MCB Chapter 5 http://bcs.whfreeman.com/lodish7e/#800911__812046__ Looking for protein-protein interactions with the yeast two hybrid system-see MCB Chapter 7 http://bcs.whfreeman.com/lodish7e/#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 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