1. Biotechnology and Genetic Engineering PBIO 450/550 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 probe Note: 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’

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 construction Note: ds cDNAs are typically placed in a cloning vector such as bacteriophage lambda ( ) or a plasmid

8. Bacteriophage cloning system

9. Cloning site Cos sites at the left and right ends

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 and  - 32 P-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 125 I 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=09010.0 4&o=|00510|00610|00520|00530|00540|00560|00 570|00590|00600|00700|00710|00010|00020|000 30|00040|00050|01000|02000|03000|04000|0500 0|06000|07000|08000|09000|10000|11000|12000| 13000|14000|15000|16000|17000|18000|19000|2 0000|21000|22000|23000|99000|&ns=589 http://bcs.whfreeman.com/lodish5e/pages/bcs- main.asp?v=category&s=00010&n=09000&i=09010.0 4&o=|00510|00610|00520|00530|00540|00560|00 570|00590|00600|00700|00710|00010|00020|000 30|00040|00050|01000|02000|03000|04000|0500 0|06000|07000|08000|09000|10000|11000|12000| 13000|14000|15000|16000|17000|18000|19000|2 0000|21000|22000|23000|99000|&ns=589

15. 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 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 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

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 ( ), and cosmids