Fig. 20-11 TECHNIQUE Nitrocellulose membrane (blot) Alkaline solution DNA transfer (blotting) Sponge Gel Heavy weight Paper towels Preparation of restriction fragments Gel electrophoresis I II III I II III I II III Radioactively labeled probe for CD4 gene RNA macrophage B cells T cells Film over blot Probe detection Hybridization with radioactive probe Probe base-pairs with mRNA Nitrocellulose blot 1 4 5 3 2 Northern Blot (to detect CD4 mRNA)
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Fig. 20-11 TECHNIQUE Nitrocellulose membrane (blot) Alkaline solution DNA transfer (blotting) Sponge Gel Heavy weight Paper towels Preparation of restriction.
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Fig. 20-11TECHNIQUE
Nitrocellulosemembrane (blot)
Alkalinesolution
DNA transfer (blotting)
Sponge
Gel
Heavyweight
Papertowels
Preparation of restriction fragments Gel electrophoresis
I II III
I II IIII II III
Radioactively labeledprobe for CD4 gene
RNA
macrophageB cellsT cells
Film overblot
Probe detectionHybridization with radioactive probe
Probe base-pairswith mRNA
Nitrocellulose blot
1
4 5
32
Northern Blot (to detect CD4 mRNA)
Fig. 20-13
TECHNIQUE
RESULTS
Gel electrophoresis
cDNAs
CD4 mRNA
PCR amplification
Different cell types
Primers
1 2 3 4 5 6
mRNAscDNA synthesis 1
2
3
Reverse Transcriptase PCR (RT-PCR) to detect CD4 mRNA
Fig. 20-15
TECHNIQUE
Isolate mRNA.
Make cDNA by reversetranscription, usingfluorescently labelednucleotides.
Apply the cDNA mixture to amicroarray, a different gene ineach spot. The cDNA hybridizeswith any complementary DNA onthe microarray.
Rinse off excess cDNA; scanmicroarray for fluorescence.Each fluorescent spot represents agene expressed in the tissue sample.
Tissue sample
mRNA molecules
Labeled cDNA molecules(single strands)
DNA fragmentsrepresentingspecific genes
DNA microarraywith 2,400human genes
DNA microarray
1
2
3
4
Microarrays to detect many (or all) mRNAs at once
WT
dif1
∆ dif1
myb98
∆ myb98
genes
Example of array data
Table 21-1
Human Genome Project (Multinational Consortium)1990-2003
Entire 3 x 10^9 nucleotide sequence of a composite haploid human genome
~$500 million - $1 billion
Celera Genomics (Private Company)1998-2003
Shotgun sequencing approach
~ $300 million
“Divide and conquer” approach
Fig. 20-12a
DNA(template strand)
TECHNIQUE
DNA polymerase
Primer Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)
dATP
dCTP
dTTP
dGTP
ddATP
ddCTP
ddTTP
ddGTP
How can we sequence DNA? (Sanger dideoxy method)
Fig. 20-12bTECHNIQUE
RESULTS
DNA (template strand)
Shortest
Labeled strands
Longest
Shortest labeled strand
Longest labeled strand
Laser
Directionof movementof strands
Detector
Last baseof longest
labeledstrand
Last baseof shortest
labeledstrand
Linkage mapping1
2
3
Geneticmarkers
Physical mapping
Overlappingfragments
DNA sequencing
How can we sequence an entire genome?
Linkage mapping1
2
3
Geneticmarkers
Physical mapping
Overlappingfragments
DNA sequencing
How can we sequence an entire genome?
Genome sequencing:Divide and conquer approach
-Ordered, large fragments of chromosomes are cloned