mcb 7200: molecular biology
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MCB 7200: Molecular Biology. Gene libraries cDNA libraries Library screening. Eukaryotic gene organization. enhancers silencers. Genomic library construction. - PowerPoint PPT PresentationTRANSCRIPT
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MCB 7200: Molecular Biology
•Gene libraries•cDNA libraries•Library screening
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Eukaryotic gene organization
enhancerssilencers
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Genomic library
construction
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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
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Production of a (radio-)labeled DNA probe by the random primer method [uses the Klenow fragment of DNA polymerase]
5’
5’5’
3’
3’3’
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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
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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!
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Copyright © 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th EditionLodish et al.
Figure 5.15 A cDNA library contains representative copies of cellular mRNA sequences.
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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
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Bacteriophage cloning system
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Bacteriophage cloning system
Cloningsite
Cos sitesat the leftand rightends
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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)
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Screening a cDNA library using DNA hybridization to a
(radio-)labeled DNA probe
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Screening a cDNA library with a labeled oligonucleotide probe based on a known peptide sequence
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Using polynucleotide kinase and-32P-labeled ATP to radiolabel oligonucleotide probes
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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
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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__
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Plus/min (+/-) or differential
screening
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A cosmid cloning system:another possible cloning vector which can be used
for genomic library but not for cDNA libraries
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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 (), and cosmids