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Biotechnology and Genetic Engineering PBIO 4500/5500

Biotechnology and Genetic Engineering PBIO 4500/5500. Gene libraries cDNA libraries Library screening. Eukaryotic gene organization. enhancers silencers. Genomic library construction.

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Biotechnology and Genetic Engineering PBIO 4500/5500

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  1. Biotechnology and Genetic EngineeringPBIO 4500/5500 Gene libraries cDNA libraries Library screening

  2. Eukaryotic gene organization enhancers silencers

  3. Genomic library construction

  4. Screening a genomic library using DNA hybridization to a (radio-)labeled DNA probeNote: a cDNA is commonly (radio-)labeled and used as a DNA probe to screen a genomic library

  5. 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’

  6. 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!

  7. Complementary DNA or cDNA cloning:cDNA library constructionNote: ds cDNAs are typically placed in a cloning vector such as bacteriophage lambda (l) or a plasmid

  8. Bacteriophage lcloning system

  9. Bacteriophage l cloning system Cos sites at the left and right ends Cloning site

  10. 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)

  11. Screening a cDNA library using DNA hybridization to a (radio-)labeled DNA probe

  12. Screening a cDNA library with a labeled oligonucleotide probe based on a known peptide sequence

  13. Using polynucleotide kinase andg-32P-labeled ATP to radiolabel oligonucleotide probes

  14. 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

  15. 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__

  16. Plus/min (+/-) or differential screening

  17. A cosmid cloning system:another possible cloning vector which can be used for genomic library but not for cDNA libraries

  18. 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

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