Lecture 17

1. Lecture 17 Working with nucleic acids

2. The Genetic Code

3. Tools for understanding the genetic code/function • Restriction endonucleases • DNA polymerases/enzymes • PCR • Plasmids • Site directed mutagenesis • Northern and Southern blotting • Yeast two-hybrid system • DNA microarrays

4. Restriction endonucleases

5. Restriction enzymes Cleavage site Cleavage site GGCC CCGG GGATCC CCTAGG BamHI HaeIII Cleavage site Cleavage site Produces blunt ends Produces sticky ends =center of symmetry

6. Visualizing DNA fragments Agarose gel electrophoresis Load DNA - Apply electric field Decreasing fragment size Stain with EtBr +

7. Plasmids • Circular DNA (3 kB-15 kB) • Readily taken up by E. coli (Transformation) • Contains an antibiotic resistance cassette • (for selection) • Contain polylinker region for easy insertion • of genes • Gene coding for protein is inducible Transformation will be discussed in lab next week.

8. How to clone a gene into a plasmid

9. Cloning into an expression vector 1. Find restriction enzyme sites at end of gene 2. Isolate gene fragment 3. Cleave plasmid with same restriction enzymes 4. Isolate plasmid (without linker) 5. Ligate gene fragment and plasmid 6.Transform into E. coli, select for antibiotic resistance

10. PCR(Polymerase chain reaction) Amplifies small amounts of DNA Taq (or pfu) DNA polymerase allows multiple rounds without the addition of more enzyme

11. Genomic Libraries cDNA library Genomic library All mRNA from a cell Chromosomal DNA Reverse transcriptase Cleave with restriction enzyme DNA fragments DNA fragments Put into cloning vectors Put into cloning vectors

12. Isolating a gene in a library Replicate on nitrocellulose probe Treat with NaOH ssDNA bound to nitrocellulose Plate transformed with plasmid library

13. Northern blotting Detects amount of a gene transcribed (probe for specific sequence) Radiolabeled oligonucleotide mRNA with complementary sequence Run agarose gel on cell extract. Many mRNA’s , transfer to nitrocellulose

14. Southern blotting (probe for specific sequence) Radiolabeled oligonucleotide DNA with complementary sequence Run agarose gel on cell extract. Transfer to nitrocellulose (Single stranded DNA)

15. E. coli Most common organism used for genetic engineering • Advantages • Fast growing (doubling time ~30 min.) • Well-characterized transcription processes • Plasmids (or phages) are easily incorporated • Disadvantages • Lacks capability of mRNA processing • Lack posttranslation processing steps • Introduced protein may be toxic to the cell

16. Protein overexpression • Requires inducer • Lac operon I P O Z Y A Control sites Code for protein Regulatory gene

17. Protein overexpression • In absence of inducer, regulatory protein binds to operator and prevents synthesis of protein I P O X Control sites No protein produced Repressor protein • In presence of inducer, regulatory protein cannot bind • to operator, protein is produced I P O X IPTG Control sites Protein produced Repressor protein

18. E.coli expression vector

19. Yeast two-hybrid system

20. DNA Microarrays