Download Real-time Quantitative PCR

PHYSICAL MAPPING AND
POSITIONAL CLONING
• Linkage mapping – Flanking markers identified
– 1cM, for example
• Probably ~ 1 MB or more in humans
• Need very many families to get closer than
this in human, or very large populations
What to Do Next?
• Identify genes in this region
• Then determine what is the gene of
interest
What to do next?
• In 1990, clone region around markers, make
physical map(s), look for genes experimentally
• In 2000, use better physical maps, at least in
some organisms
• In 2010, use bioinformatic knowledge,
experimental proof still necessary
Physical Mapping
A physical map is primarily based on the
locations of landmarks along a DNA
molecule and units of distance are
expressed in base pairs.
Low Resolution Physical Mapping
• Cytogenetic map
• In situ hybridization
Chromosome Sorting
• Flow cytometry
• Used in library
construction
• Also for chromosome
paints
Pulsed Field Gel Electrophoresis
Polymerase Chain Reaction (PCR)
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Ability to generate identical high copy number DNAs made possible
in the 1970s by recombinant DNA technology (i.e., cloning).
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Cloning DNA is time consuming and expensive (>>$15/sample).
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Probing libraries can be like hunting for a needle in a haystack.
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PCR, “discovered” in 1983 by Kary Mullis, enables the amplification
(or duplication) of millions of copies of any DNA sequence with
known flanking sequences.
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Requires only simple, inexpensive ingredients and a couple hours.
DNA template
Primers (anneal to flanking sequences)
DNA polymerase
dNTPs
Mg2+
Buffer
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Can be performed by hand or in a machine called a thermal cycler.
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1993: Nobel Prize for Chemistry
Fig. 9.3
Denature
Anneal PCR Primers
Extend PCR Primers
w/Taq
Repeat…
Somatic Cell Genetics in Mammals
Radiation Hybrid Mapping
Fluorescent Dye Dideoxy-sequencing
Top-down mapping
Bottom-up Mapping
(Contig Maps, Mapping With Ordered Clones)
Whose genome did Celera sequence?
Supposedly:
African-American Asian-Chinese
Hispanic-Mexican
Caucasian
Caucasian
Actually:
Celera’s genome is Craig Venter’s
Science v. 291, pp 1304-1351
Cloning Vectors
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Plasmid
Phagemid
Cosmid, fosmid
YACs
BACs
PACs
BAC Construction Scheme
 BAC vector (pIndigo 536)
T7
Insert
Insert size ; 50-300Kb
( mean 110kb)
SP6
Application Of BAC library
Chromosome
walking &
contig assembly
Whole genome
sequencing
Fluorescent in situ
hybridization
BAC library
Fine mapping
for interest
gene
Positional
cloning
Construction of
integrated
genetic and
physical map
“virtual autorad” - real-time DNA sequence output from ABI 377
1.
Trace files (dye signals) are analyzed and bases
called to create chromatograms.
2.
Chromatograms from opposite strands are
reconciled with software to create doublestranded sequence data.
Maximum pyrosequencing read lengths currently are 300-500 nt.
Commercial applications:
454 Life Sciences Genome Sequencer FLX
Generate 400 million nt in 10 hours
$5-7K USD per run
$1M for mammalian genome
Zoo Blot
CpG islands
Northern Blots
Real-time Quantitative PCR:
Measures the abundance of DNA as it is amplified.
Useful for quantitatively measuring the levels of mRNA in a sample.
Uses reverse transcriptase to generate cDNA for the template.
Can also be used to quantitatively estimate fraction of DNA from
various organisms in a heterogenous sample (e.g, can be used to
measure abundance of different microbes in soil sample).
Fluorescent dye, SYBR Green, is incorporated into PCR reaction.
SYBR Green fluoresces strongly when bound to DNA, but emits
little fluorescence when not bound to DNA.
SYBR Green fluorescence is proportional to the amount of DNA
amplified, detected with a laser or other device.
Experimental samples are compared to control sample with
known concentration of cDNA.
Fig. 10.9
SYBR Green binds
to double-stranded DNA
and fluoresces
Real-time Quantitative PCR amplification plot:
1d
12h
8h
4h
2h
1h
30’
15’
NA
1d
12h
8h
4h
2h
1h
30’
15’
NA
P. trifoliata
C. paradisi
PtCBF
CORc115
Use trapped exons to identify candidate
genes from cDNAs
Sequence Conservation
• Homology searches
Array Technology
Gene Complementation
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Mutants
Overexpression
“Knockouts”
RNAi
Reporter assays