Molecular & Genomic Surgery

Eric M. Wilson 1/5/10

I am by no means an expert on this subject. If you have questions, I suggest

you ask Tom Theruvath or look it up.

DISCLAIMER

Molecular Biology

DNA

DNA Replication

Gene Expression

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

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

Gene Expression

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

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

Apoptosis

Signal Transduction Pathways

TGF-β

Gene Therapy

-Immunotherapy- Herceptin

-Chemotherapy- Gleevac

-Gene Therapy

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

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

Polymerase Chain Reaction

Immunoblotting (Western)

Immunopreciptation

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.

Cell Culture

Transgenic Mice

Gene Knockout in Mice