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’ 3’ 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(1-20kb/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/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

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