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Quiz 1 Class Business • Friday seminars – I will not be able to award extra credit for seminars – I will give EC for one seminar for anyone who did not yet do so • Wed Office Hours will be at 2PM • Review and Paper Sessions – Gilmer 190 – I have loaded a guide to reading scientific papers onto Toolkit (Handouts and Reading Material directory) • The Double Helix on Thursday – Write down three topics of discussion 2 More Class Business • RNAi blurb in new edition on pages 402-403 • Additional reading material on DNA miroarrays and riboswitches on Toolkit (Handout directory) • Writing Center – They give 1 hour long tutoring. – They are located in 314 Bryan Hall. – You can stop by or call (924-6678) to make an appointment 3 Pymol Tutorial • Selection of bases and displaying them 4 Review • DNA Damage – Deamination of cytosine – Thiamine dimers • Nucleases • Function and structure of RNA – Stabilizing forces for 3D structure – The different types of RNA – Catalysis acid base mechanism – General concepts of RNA World 5 What would we like to know about DNA/RNA? 6 PCR relies on components of DNA Replication Each strand of the helix must be copied in complementary fashion by DNA polymerase •Each strand is a template for copying •DNA polymerase requires template and primer •Primer: an oligonucleotide that pairs with the end of the template molecule to form dsDNA •DNA polymerases add nucleotides in 5'-3' direction 7 PCR Amplification Steps Reactants: Primers dNTPs Template DNA Heat stable DNA Polymerase 8 PCR – explained again 9 Applications of PCR/DNA Methods Application Example of method Typing genetic markers Restriction fragment length polymorphism (RFLP) Detecting point mutations PCR (RT-PCR) Genomic DNA cloning PCR from single cell or isolated genomic DNA DNA sequencing PCR with ddNTPs In vitro mutagenesis PCR with primers with incorporated mutation Gene expression studies RT-PCR 10 Examples of Inherited Disorders Detectable by PCR Disease Affected Gene Adenosine deaminase deficiency Adenosine deaminase (ADA) Lesch-Nyhan syndrome Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) α-1-Antitrypsin deficiency α-1-Antitrypsin Cystic fibrosis cystic fibrosis transmembrane conductance (CFTR) protein Fabry disease α-Galactosidase Gaucher's disease Glucocerebrosidase Sandhoff-Jatzkewitz disease Hexosaminidase A and B Tay-Sachs disease Hexosaminidase A Familial hypercholesterolemia (FH) LDL receptor Glucose-6-phosphate dehydrogenase deficiency Glucose-6-phosphate dehydrogenase Maple syrup urine disease branched-chainα-keto acid dehydrogenase Phenylketonuria (PKU) Phenylalanine hydroxylase Ornithine transcarbamylase deficiency Ornithine transcarbamylase Retinoblastoma (Rb) RB gene product, pRB Sickle-cell anemia point mutation in β-globin β-Thalassemia mutations in β-globin gene that result in loss of synthesis of protein Hemophilia A Factor VIII Hemophilia B Factor IX von Willebrand disease von Willebrand factor (vWF) 11 Gel Electrophoresis Molecules migrate according to size and shape What contributes to the size and shape of nucleic acids? Or staining with Ethidium Bromide 12 Sequencing PCR, with the addition of ddNTP at low concnetrations ddNTPs are additionally modified with a fluorescence probe (unique to each nucleotide) 13 Sequencing – Explained again 14 Sequencing Automation 15 Hybridization • Using a probe on a complimentary strand to identify the nucleic acid of interest 16 Does an organism have a particular DNA/RNA sequence (alternatives to PCR)? PCR is one way using two specific primers – another technique is the Southern Blot (DNA) and nothern blot (RNA) 17 Restriction fragment length polymorphism (RFLP) 18 PCR as an alternative to RFLP assays 19 Applications to Forensic Science A non specific probe that hybridizes to multiple sites (CAG)5 This is one way to solve paternity cases Or identify DNA in trace evidence 20 Does a protein bind to a particular DNA/RNA sequence? Gel mobility shift assay 21 Where does the protein bind? DNA footprinting 22 Structure Determination Two techniques used to obtain atomic resolution structures such as those that you are looking at (pdb files): X – ray crystallography requires crystallization of biomolecule no molecular weight limit Nuclear Magnetic Spectroscopy in solution molecular weight limit (standard methods limited to ≈30 kD) 23 X-ray crystallography 24 NOESY spectrum 1H chemical shift (ppm) 1H NMR Provides distances between atoms chemical shift (ppm) Put all the distances together and calculate a structure 25 Genomics • Sequencing an organisms entire genome • Why would we want to do this? 26 Phylogeny 27 Functional Characterization • We need to know what the genes are in order to functionally characterize the gene product. 28 Genomics Databases • GenBank – http://www.ncbi.nlm.nih.gov/Genbank/ – Sequence database • TIGR – http://www.tigr.org/ • JGI – http://genome.jgi-psf.org/ • Blast – http://www.ncbi.nlm.nih.gov/BLAST/ – Sequence comparison 29