Literature reviews revised is due4/11 (Friday) turn in together: revised paper (with bibliography) and peer review and 1st draft.
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Literature reviews
revised is due 4/11 (Friday)
turn in together:revised paper
(with bibliography)and peer reviewand 1st draft
€
h2 =M ' − M
M* − M
available fo
r the te
st
€
H 2 =σ g
2
σ p2
=σ g
2
σ g2 + σ e
2
mean
variance
€
χ =∑ f ix i
N
€
s2 =∑ f i x i − x ( )
2
N −1
€
X 2 = ∑(observed − expected)2
expectedChi-square
€
σ p2 = σ g
2 + σ e2 (eq. 15.3)
Mendel 1860’s
studied “genetics”
inheritance and variability
Sanger 1977
first genome sequenced (phage)
genomics -study of entire genome
Human Genome project:
Proposed in 1986Funded in 1989
Preliminary report in 2001 (94%)Completed in 2003
(completed many others too)
Genomics comparative and functional
similarities and relationshipssimple vs. complexgene familiesgene functions
……
Proteomics study the protein content of an organism
Normal functionDisease processesRepair/drug interaction…..
GenomicsProteomics
huge amounts of information
computers
used to storestudy
comparebioinformatics
use of computers to manage and interpret biological information
Ivers 218 4 pm today
© 2006 Jones and Bartlett Publishers
Fig. 10.12. Genes in the genome of Mycoplasma genitalium classified by function. [Data from C. M. Fraser, et al. 1995. Science 270: 397.]
Genome results
1. 95% of human DNA is non-coding (not genes)2. Fewer genes found than expected (35,000)3. Many genes have unknown functions4. Only 1% of our genes are unique
(similar to 46% of genes in yeast)5. 200 genes like bacteria6. mutation rates differ in different parts of genome7. Many sites (15) for variability
(each individual is genetically unique)
2n 8 8,388,60823 = 223 =
Even though we know the genetic “information” we are still a long way from understanding how it all functions as an integrated package
How would you go about studying?
When do genes get turned “on?”
When do genes get turned “off?”
What happens when genes are changed?
What genes affect other genes?
microarrays
modifed Southern blot
modifed Southern blot
Fig. 6.27. Southern blot
being able to correlate the location of the DNA and its binding to your “probe”
modifed Southern blot
DNA-1 DNA-2 DNA-3 …
DNA sources cDNAgemonic DNAoligonucleotides
being able to correlate the location of the DNA and its binding to your “probe”
microarrays
Expression analysis
Comparative Genomic Hybridization(CGH)
Mutation/Polymorphism analysis
http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html
look at differential expression
look for chromosome rearrangements
look for mutations/polymorphisms
microarrays
Expression analysis
Comparative Genomic Hybridization(CGH)
Mutation/Polymorphism analysis
http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html
bound DNA is cDNA from mRNA
pieces of chromosomes
oligos (variations on normal genes)
© 2006 Jones and Bartlett Publishers
Fig. 10.14. Small part of a yeast DNA chip. The color of each spot indicates the relative level of gene expression in experimental and control samples. [Courtesy of Dr. Jason Kang/National Cancer Institute]
microarrays
http://learn.genetics.utah.edu/units/biotech/microarray/
Analysis of information
What kind of things can be learned?
examples:
genetic control of early development
protein interactions
© 2006 Jones and Bartlett Publishers
Fig. 10.16. Two-hybrid analysis by means of a GAL4 protein
Fig. 10.15
© 2006 Jones and Bartlett Publishers
Fig. 10.17. Physical interactions among nuclear proteins in yeast. [Adapted from S. Maslov and K. Sneppen, 2002. Science 296: 910. © AAAS]
© 2006 Jones and Bartlett Publishers
Real-Time PCR
Method of PCR in which the amount of amplicon generated is measured each cycle
Quantitative (relative or absolute)
Allows comparison of several samples in a single run (experiment) or across multiple runs
Uses much less RNA than Northern Blot
Amount of amplicon is measured using fluorescence:
probe
intercalating dye
molecular beacon
Techniques used in Genomics
© 2006 Jones and Bartlett Publishers
Real-Time PCR
•Probe Method:– 5’ and 3’ primers
Techniques used in Genomics
5’ 3’– Probe with reporter dye on 5’
end and quencher on 3’ end
– Close proximity of quencher keeps reporter from fluorescing
– Taq polyermase adds dNTPs to extend complementary sequence
– Taq polymerase 5’ → 3’ nuclease activity cleaves the reporter dye
– Reporter dye fluoresces
– Each round of PCR will result in more released reporter dye
– Thermocycler records amount of fluorescence each round of PCR
© 2006 Jones and Bartlett Publishers
Real-Time PCR
• SYBR Green Method:
– 5’ and 3’ primers
Techniques used in Genomics
– SYBR Green intercalates double-stranded DNA and fluoresces
– Each round of PCR results in more SYBR Green binding of double-stranded DNA
– Thermocycler records amount of fluorescence each round
© 2006 Jones and Bartlett Publishers
Real-Time PCR
•Molecular Beacon Method:
Techniques used in Genomics
5’ 3’
– Beacon with reporter dye on 5’ end and quencher on 3’ end
– Close proximity of quencher keeps reporter from fluorescing
– Taq polyermase adds dNTPs to extend complementary sequence
– Beacon hybridizes to complementary sequence
– Reporter dye far enough away from quencher to fluoresce
– Each round of PCR will result in more hybridized beacon
– Thermocycler records amount of fluorescence each round of PCR
– In second round of PCR, the copied sequences & beacon are denatured
5’3’
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