Download What would we like to know about DNA and how do we obtain that

Quiz
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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
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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
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Pymol Tutorial
• Selection of bases and displaying them
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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
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What would we like to know
about DNA/RNA?
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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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PCR –
explained
again
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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
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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)
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Gel Electrophoresis
Molecules migrate according to size
and shape
What contributes to the size and shape
of nucleic acids?
Or staining with Ethidium Bromide
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Sequencing
PCR, with the addition of
ddNTP at low concnetrations
ddNTPs are additionally modified
with a fluorescence probe (unique
to each nucleotide)
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Sequencing –
Explained again
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Sequencing Automation
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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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Restriction fragment length polymorphism (RFLP)
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PCR as an alternative to RFLP assays
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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
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Does a protein bind to a particular DNA/RNA
sequence? Gel mobility shift assay
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Where does the protein bind? DNA footprinting
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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)
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X-ray crystallography
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NOESY spectrum
1H
chemical shift (ppm)
1H
NMR
Provides distances between atoms
chemical shift (ppm)
Put all the distances together
and calculate a structure
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Genomics
• Sequencing an organisms entire genome
• Why would we want to do this?
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Phylogeny
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Functional Characterization
• We need to know what the genes are in
order to functionally characterize the gene
product.
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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
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