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)

© 2006 Jones and Bartlett Publishers

Table 10. 1. Some examples of genomic sequencing

© 2006 Jones and Bartlett Publishers

Table 10. 1. Some examples of genomic sequencing

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. 10.13. Gene expression microarrays

© 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

Fig 10.15

© 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]

RT-PCR

RT-PCR

reverse transcriptase PCRmRNA in low #’s

Real-Time PCRquantitative

q

© 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’