STRUCTURE: 1000-1000000 bases/gene, human genome 3 billion bases, 20,000 genes, 23 pairs of chromosomes; on chromo, p = small, q = big REPLICATION exonuclease = repair enzyme discontinuous - leading vs. lagging (Okazaki) 1953 Watson, Crick, Wilkins, Franklin = structure GENETICS meiosis 2 ÷ , 4 haploids, indep assort X linked (sex) IN MALES: traits colorblind, hemophilia, ALD, MD, male pattern baldness Karyotype - nondisjunction nondisjunction disorders: Klinefelters(XXY) Klinefelters(XXY) Turner (Xo) Down (tri21) diploid, monosomy, trisomy GENE EXPRESSION operons - lac/trp Jacob and Monod 1. regulator gene gene - producing the repressor molecule molecule 2. operator gene gene - repressor binds here. Repressible it binds, binds, Inducible it releases. RNA polymerase binds binds here 3. structural gene is gene is responsible for the production of enzymes used enzymes used in biochemical pathways 4. repressor molecule is molecule is responsible for combining to either lactose or the the operator gene. gene. DETECTION of gene expression - BLOTTING SNoW DRoP MUTATIONS - chromo # chromo # - nondisjunction, chromos size - add/sub/invert. genes genes - frame shift = add/sub, point = substitution, SEQUENCING - Sanger ddNTP are NOT elongating. elongating. End growth, run on gel RFLP-Restriction fragment length polymorphism-exploits variations in homologous DNA sequences; DNA broken into pieces by restriction enzymes --> restriction fragments are fragments are separated by length by gel electrophoresis Plasmid selection and isolation - mini, midi, maxi prep (culture volume --> plasmid yeild) an extraction w Basic buffer selection w drug (amp^r) PCR - amplify DNA copies exponentially, exponentially, region between primers, for ID or detection of disease, Taq DNA polymerase, Thermocycler (denature, anneal, extend by temp 95, 50, 72o) Kary Mullis 1983 Nobel Polymerase chain reaction (PCR) o Copy a gene many times to generate sufficient amount of material for experiments o Describe PCR cycle: denaturation, annealing of primers, primer extension o Reagents: DNA polymerase, free nucleotides o Number of DNA copies doubles in each cycle (chain reaction) ELISA – Enzyme Linked ImmunoSorbent Assay – identify antibody/antigen with the other, color changing enzyme linked to 2ndary antibody GENE THERAPY - supplement or alter genes to treat disease. disease. Replace a mutated gene, viral vectors, vectors, Cystic Fibrosis - place CFTR gene which regulates salts into the Cold virus or liposome. In genetics, and operon is a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter. lac - metabolism of lactose in bacteria, esp. E. coli; trp - codes for tryptophan production The lac operon operon consists of three structural genes, and a promoter, a terminator, regulator, and an operator. The three structural genes are: lacZ , lacY , and lacA. lacA. -lacZ encodes encodes β-galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose. -lacY encodes encodes β-galactoside permease (LacY), a membrane-bound transport protein that pumps lactose into the cell. -lacA encodes -lacA encodes β-galactoside transacetylase (LacA), an enzyme that transfers an acetyl group from acetyl-CoA to
β-galactosides. Only lacZ and lacY appear to be necessary for lactose catabolism. TRP: negative repressive feedback mechanism. The repressor for the trp operon produced upstream by trpR gene, which is continually expressed at a low level. Creates monomers, which associate into tetramers. These tetramers are inactive,are dissolved in nucleoplasm. When tryptophan is present, these tryptophan repressor tetramers bind to tryptophan, cause change in conformation (in repressor), allows repressor to the operator. This prevents RNA polymerase from binding to and transcribing the operon, so tryptophan is not produced from its precursor. When tryptophan is not present, the repressor is in its inactive conformation and cannot bind the operator region, so transcription is not inhibited by the repressor.
Autoradiography: The process of detection of radioactively labeled molecules by exposure of an X-ray sensitive film. Hybridization: annealing of a single stranded DNA to its complimentary region on another single stranded DNA Short tandem repeats or STRs are non-coding DNA that consist of 2 – 5 nucleotide sequences repeated many times (in tandem). Their number and arrangement in human chromosomes is unique to each individual. DNA Sequencing o Dideoxy DNA sequencing (Frederick Sanger): Determine nucleotide sequence from sequencing gel Standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) -Chain-terminating dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP) lack the 3’-OH group -The DNA iinto four separate sequencing reactions, containing all four of the standard deoxynucleotides and DNA poly. To each reaction is added only one of the four dideoxynucleotides.. Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis. This is frequently performed using a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes (lanes A, T, G, C). The DNA bands may then be visualized by autoradiography or UV light and the DNA sequence can be directly read off the X-ray film or gel image.
