What would we like to know about DNA and how do we obtain that knowledge?Class Business •
Friday seminars –
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Review and Paper Sessions –
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Write down three topics of discussion
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and riboswitches
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They are located in 314 Bryan Hall. –
You can stop by or call (924-6678) to make an appointment
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Pymol
Tutorial
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Review •
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Catalysis acid base mechanism
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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
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PCR Amplification Steps Reactants: Primers dNTPs Template DNA Heat stable DNA Polymerase
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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
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
Fabry disease α-Galactosidase
Gaucher's disease Glucocerebrosidase
Tay-Sachs disease Hexosaminidase A
Maple syrup urine disease branched-chainα-keto acid dehydrogenase
Phenylketonuria (PKU) Phenylalanine hydroxylase
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
Gel Electrophoresis
Molecules migrate according to size and shape What contributes to the size and shape of nucleic acids?
Or staining with Ethidium
ddNTPs
are additionally modified with a fluorescence probe (unique to each nucleotide)
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Hybridization •
Using a probe on a complimentary strand to identify the nucleic acid of interest
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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)
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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
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Does a protein bind to a particular DNA/RNA sequence? Gel mobility shift assay
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as those that you are looking at (pdb
files):
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Genomics
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Phylogeny
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We need to know what the genes are in order to functionally characterize the gene product.
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Slide Number 7
PCR Amplification Steps
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Hybridization
Does an organism have a particular DNA/RNA sequence (alternatives to PCR)?
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Applications to Forensic Science
Does a protein bind to a particular DNA/RNA sequence? Gel mobility shift assay
Where does the protein bind? DNA footprinting
Structure Determination
X-ray crystallography