Functional Genomics

how genome functions to orchestrate biological processes

microarray

how to increase stringency

do what to temp

what to salt concentrations

method for assying entire genome in one experiment

increase temp

decrease salt

decreases hybridization of probes to seqs with less than 100% complementarity

how are DNA microarrays the inverse of southern and northern blots?

in southern and northern blot, you immobilize DNA/RNA on plate and then add labeled probe

in DNA microarrays, the probe is immobilized on the plate and you add labeled sample DNA/RNA

what does Southern blot tell you

and what are the steps

given a known sequence, where does it occur in the genome

1. fragment sample DNA

2. run on gel

3.denature into single strands

4. immobilize on plate

5. incubate with many copies of labeled probe

6.remove unbound probe

7. visualize location with audoradiography

what does northern blot tel you?

what are the steps

given a known coding sequence, what is the level of gene expression?

quantitative: level of signal correlates with mRNA level

1. extract and purify mRNA

2. run on gel

3. immobilize on plate

5. add labeled probe

6. remove unbound probe

7. visualize with blot development

DNA microarrays

difference between spotted/printed arrays (give three examples)

and on array synthesis (give two examples)

Which can hold more features?

spotted / printed arrays

make probe first then immobilize on surface

cDNA or BAC clones

PCR product

oligonucleotide

on array synthesis* holds more features

synthesize probe on array

lithographic (e.g. sequential masks)

ink jet

DNA microarrays

different uses

allows to detect and compare different isoforms, fusion transcripts

genotype mutant genomes (Detect SNPs)

by measuring changes in expression levels, make inferences about what gene function and correlation with other genes

see patterns of gene regulation (what transcription factor binding)

DNA microarray

key parameters

probe length

probe density

varies depending on platform

range in probe length DNA microarray

25bp (oligonucleotide) - 1MB (e.g. spotting with BAC clones)

probe or sample labels

in southern

in northern

in microarrays

in southern: probe labeled

in northern: probe labeled

in microarray: sample labeled

in northern blot, signal depends on what

level of mRNAs

in microarray, signal depends on what

and what does that in turn depend on

strength of hybridization

which in turn depends on

1.# of paired bases

2. hybridization conditions (e.g. temp)

3. washing after hybridization

feature

each cluster of millions of identical probes on a microarray plate

DNA hybridization

is what type of reaction

between what

relies on what

bimolecular reaction

btw probe and sample

relies on Watson & Crick base pairing

A-T

Watson Crick Base Pair

G-C

Watson Crick Base Pair

Stringency

At higher stringency you have lower what

sensitivity in hybridization

a more efficient detection of the genes under study

ability to id rarely expressed transcripts in a complex background

higher sensitivity: lower number of false negatives

specificity in hybridization

ability to discern between different family members

higher specificity: lower false positive

four factors effecting DNA hybridization

1. probe length

short: higher sensitivity, decreased specificity

long: decreased sensitivity, increased specificity

2. base composition

higher GCs,lower stringency

3. salt concentration

higher salt, lower stringency

4. denaturant concentration

higher conc of denaturants, higher stringency

how does salt concentration effect hybridization?

higher salt, lower stringency

salt neutralizes DNA backbone repulsion

makes double strands more stable

more likely to stay together even if they are not 100% homologous

 how does concentration of denaturants effect hybridization?

reduce tolerance for mismatched bases

lowers melting temp

increase stability of single-strandedness

so is ok being single--will only form duplex if very homologous

the lower the melting temp,

(in terms of stringency and annealing rate)

what lowers melting temp

higher the stringency

lower the annealing rate

what lowers melting temp:

high concentration of denaturants

high AT content (low GC content)

smaller probe

define melting temp

point at which 50% of DNA is in duplex state

diff types of microarray samples

and what can they tell us

DNA --comparative hybridization (analysis of copy number changes--gains losses in DNA content often in tumor cells)

RNA--expression levels

protein bound DNA or RNA (regulation or epigenetic studies)

relative quantitation

microarrays

spots fluroresce in intensity that corresponds to extent of hybridization

not interested in absolute intensity

but in ratio btw intenstities of experimental sample and reference sample, or btw two samples in different conditions

ratio expression in microarray

what does change in log by value of 1 mean

log₂(test/reference)

change in log value by 1 means you are either doubling or halfing the DNA/RNA

why log₂ values for microarray analysis

allows for symmetrical distribution of data

makes easy to conceptualize 2 fold changes

hierarchical clustering

similarity tree based on pairwise distances

two levels

genes w similar expression patterns grouped together (calculate distance btw two expression vectors); first two closest then move to all others

then connect groups in series of branches (using linkage methods--e.g. average distance btw all members of one cluster with those of another)

what can clustering genes tell you?

allows you to infer functional relationships btw genes

id genes that may be in same biochemical pathway

group clusters into higher level systems

how can clustering aid in classifying disease?

using microarrays with exons probes

are you looking for gene expression levels?

looking at different isoforms

no abundance ot presense of exon

microarray assessment of transcription factor binding

direct and indirect

indirect: look at mRNAs with similar expression levels during certain stage of growth for example

then go to genes that encode them and look for similar motifs in promoters

direct: ChIP on CHIP

chromatin immunoprecipitation steps

1. isolate (lyse cells and nuclei)

2.cross link

3. sheer

4. immunoprecipitate with antibodies

5. reverse crosslink

6. isolate DNA

7. PCR

8. hybridization assay

ChIP chip steps

1. isolate cells you want and DNA--lyse cells and nuclei

2. crosslink

3. fragment DNA

4. immunoprecipitate with antibodies

5. reverse cross link

6. PCR and label

7. hybridize to tiling array

tiling array

subtype of microarray with overlapping probes that cover entire genome

probe density and hybridization in microarrays

higher probe density, higher the stringency (lower the hybridization)

because high probe densities effectively cluster large number of negatively charged DNA together in the spot.

shorter probe, higher

e.g. can spot shorter probes in higher densiter across array

higher stringency, reduced what

stringency

in mRNA microarrays,

why reverse transcribe samples into cDNA?

cluster analysis

used to id what

hierarchical clustering

attempts to group genes into small clusters

group clusters into higher level systems

what to put on microarray for ChIP chip?

ideally intergenic regions

coding regions for transcription

two uses for ChIP chip

1. functions of transcription factors

2. help id their target genes

make a replacement gene with regions homologous to gene of interest upstream and downstream

pcr it up

add to cells which contrain your target gene

will be incorporated by homologous recombination

why would you perform genome-wide mutant screens?