Molecular & Genomic Surgery

1. Molecular & Genomic Surgery Eric M. Wilson 1/5/10

2. DISCLAIMER I am by no means an expert on this subject. If you have questions, I suggest you ask Tom Theruvath or look it up.

3. Molecular Biology

4. DNA

5. DNA Replication

6. Gene Expression

7. Transcription • DNA strand is used as a template by RNA polymerase for synthesis of a mRNA strand • Occurs in nucleus • 3 RNA polymerases • -responsible for reading the gene, and building the mRNA strand. It reads only the 3' to 5' strand • Transcription Factors: bind DNA & help transcription of genes • Initiation Factors: bind RNA polymerase & initiate transcription http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a2.html

8. Translation • mRNA used as a template by ribosomes for synthesis of proteins • Occurs in cytoplasm • Ribosomes have small & large subunits that read mRNA, then bind appropriate tRNAs that have amino acids & eventually make proteins • ç http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a3.html

9. Gene Expression

10. Terminology • Genome: collective term for all genes present in one organism • DNA sequences of 3 billion base-pairs, carried by 23 pairs of chromosomes • 25,000 to 30,000 genes, and overall it is 99.9% identical in all people • Genomics: study of genetic material • Proteomics: study of the structure and expression of proteins as well as the interactions among proteins encoded by a human genome

11. Cell Cycle • G1: most variable, determines cell cycle length • S: most radioresistant • Growth factors affect cell during G1 • Mitosis • Prophase: centromere attachment • Nucleus disappears • Metaphase: csome alignment • Anaphase: csome pulled apart • Telephase: separate nucleus reforms around each set of csomes

12. Apoptosis

13. Signal Transduction Pathways

14. TGF-β

15. Gene Therapy • Immunotherapy • Herceptin • Chemotherapy • Gleevac • Gene Therapy

16. Southern Blotting • Technique of transferring DNA fragments from an electrophoresis gel to a membrane support, and the subsequent analysis of the fragments by hybridization with a radioactively labeled probe • Enables reliable and efficient analysis of size-fractionated DNA fragments in an immobilized membrane support • Obtain information about the physical organization of single and multicopy sequences in complex genomes

17. Northern Blotting • Technique of size fractionation of RNA in a gel and the transferring of an RNA sample to a solid support (membrane) in such a manner that the relative positions of the RNA molecules are maintained • Resulting membrane then is hybridized with a labeled probe complementary to the mRNA of interest • Signals generated from detection of the membrane can be used to determine the size and abundance of the target RNA • Differs from Southern blot: uses mRNA instead of DNA • Only method that provides information regarding mRNA size and has remained a standard method for detection and quantitation of mRNA

18. Polymerase Chain Reaction

19. Immunoblotting (Western)

20. Immunopreciptation

21. DNA Microarray There are two major application forms for the technology: identification of sequence (gene/gene mutation) and determination of expression level (abundance) of genes.

22. Cell Culture

23. Transgenic Mice

24. Gene Knockout in Mice