Fig. 20-11 TECHNIQUE Nitrocellulose membrane (blot) Alkaline solution DNA transfer (blotting) Sponge...
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Transcript of Fig. 20-11 TECHNIQUE Nitrocellulose membrane (blot) Alkaline solution DNA transfer (blotting) Sponge...
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
Fig. 20-4-4
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant plasmid
Bacteria carryingplasmids
RESULTS
Colony carrying non-recombinant plasmidwith intact lacZ gene
One of manybacterialclones
Colony carrying recombinant plasmid with disrupted lacZ gene
Linkage mapping1
2
3
Geneticmarkers
Physical mapping
Overlappingfragments
DNA sequencing
Genome sequencing:Divide and conquer approach
- Ordered, large fragments of chromosomes are cloned
-Each fragment is sequenced
How can we sequence an entire genome?
Cut the DNAinto overlappingfragments short enoughfor sequencing
1
2
3
4
Clone the fragmentsin plasmid or phagevectors.
Sequence eachfragment.
Order thesequences intoone overallsequencewith computer software.
“Shotgun” sequencing approach
TTATTTCCCATTTTTCTTAAAAAGGAAGAACAAACTGTGCCCTAGGGTTTACTGTGTCAGAACAGAGTGTGCCGATTGTGGTCAGGACTCCATAGCATTTCACCATTGAGTTATTTCCGCCCCCTTACGTGTCTCTCTTCAGCGGTCTATTATCTCCAAGAGGGCATAAAACACTGAGTAAACAGCTCTTTTATATGTGTTTCCTGGATGAGCCTTCTTTTAATTAATTTTGTTAAGGGATTTCCTCTAGGGCCACTGCACGTCATGGGGAGTCACCCCCAGACACTCCCAATTGGCCCCTTGTCACCCAGGGGCACATTTCAGCTATTTGTAAAACCTGAAATCACTAGAAAGGAATGTCTAGTGACTTGTGGGGGCCAAGGCCCTTGTTATGGGGATGAAGGCTCTTAGGTGGTAGCCCTCCAAGAGAATAGATGGTGAATGTCTCTTTTCAGACATTAAAGGTGTCAGACTCTCAGTTAATCTCTCCTAGATCCAGGAAAGGCCTAGAAAAGGAAGGCCTGACTGCATTAATGGAGATTCTCTCCATGTGCAAAATTTCCTCCACAAAAGAAATCCTTGCAGGGCCATTTTAATGTGTTGGCCCTGTGACAGCCATTTCAAAATATGTCAAAAAATATATTTTGGAGTAAAATACTTTCATTTTCCTTCAGAGTCTGCTGTCGTATGATGCCATACCAGAGTCAGGTTGGAAAGTAAGCCACATTATACAGCGTTAACCTAAAAAAACAAAAAACTGTCTAACAAGATTTTATGGTTTATAGAGCATGATTCCCCGGACACATTAGATAGAAATCTGGGCAAGAGAAGAAAAAAAGGTCAGAGTTTAATCCTCATTCCTAAGTTATGTAAACCAAAAATAAAATTCTGAAGATGTCCTGATCATCTGAATGGACCCTTCCTCTGGACCAGGGCATTCCAAAGTTAACCTGAAAATTGGTTTGGGCCATGATGGGAAGGGAGGTTTGGATATGCCTCATTATGCCCTCTTCCCTTTCAGAATTCAGGAAAAGCCAACCAGCATTAACATCAACACAGATTTTCAGATCTTAGGTTTCTTTCCGATCTATTCTCTCTGAACCCTGCTACCTGGAGGCTTCATCTGCATAATAAAACTTTAGTCTCCACAACCCCTTATCTTACCCCAGACATTCCTTTCTATTGATAATAACTCTTTCAACCAATTGCCAATCAGGGTATGTTTAAATCTACCTATGACCTGGAAGCCCCCACTTTGCACCCTGAGATCAAACCAGTGCAAATCTTATATGTATTGATTTGTCAATGAAAACAGTCAAAGCCAGTCAGGCACAGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGTAGATCACCTGAGGTCAGGAGTTCGACACCAGCCTGGCCAACATGGTGAAACCCCGTCCCTACTAAAATACAAAAATTAGCCCAGCTTGGTGGTGGGCACCTGTAATCTTAGCTACTGCAGAGACTGAGGCAGGAGAATCGCTTGAACCCAGGAGGTGGAGGTTGCAGTGACCTGAGATTTTGCCATTGCACTCCAGCCTGGGCAACAGAGCAAGACTCTATCTCAAAAAACAAACAAACAAACAAACAAACAAACAAACTGTCAAAATCTGTACAGTATGTGAAGAGATTTGTTCTGAACCAAATATGAATGACCATGGTCCATGACACAGCCCTCAGAAGACCCTGAGAACATGTGCCCAAGGTGGTCACAGTGCATCTTAGTTTTGTACATTTTAGGGAGATATGAGACTTCAGTCAAATACATTTTTAAAAAATACATTGGTTTTGTCCAGAAAGCCAGAACCACTCAAAGCAGGGGTTTCCAGGTTATAAGTAGATTTAAAATTTTTCTGATTGACAATTGGTTGAAAGAGTTGTCAATAGAAAGGAATGTCTGCATTGTGACAAGAGGTTGTGGAGACCAAGTTTCTGTCATGCAGATGAAGCCTTCAGGTAGCAGGCTTCCAAGATAACAGGTTGTAAATAGTTCTTATCAGACTTAAGTTCTGTGGAGACGTAAAATGAGGCATATCTGACCTCCACTTCCAAAAACATCTGAGACAGGTCTCAGTTAATTAAGAAAGTTTGTTCTGCCTAGTTTAAGGACATGCCCATGACACTGCCTCAGGAGGTCCTGACAGCATGTGCCCAAGGTGGTCAGGATACAGCTTGCTTCTATATATTTTAGGGAGAAAATACATCAGCCTGTAAACAAAAAATTAAATTCTAAGGTCCCTGAACCATCTGAATGGGCTTTCTTCTAGGCCAGGGCACTCTAAAATTGAAGAACCTGAACATTCCTTTCTATTGATAATACTTTCAGCCAGTTGAGCCCATTCAGACCACAGCAAGGTGCCAGGCCAGGCAAGGGCTGACTTGAGATACCTGCCAGATGAGTCACTGGCAAAAGGTGCTGCTCCCTGGTGAGGGAGAAACACCAGGGGCTGGGAGAGGCCCAGAAGGCTCTGAAGGAGTTTTGGTTTGGCTGGCCATGTGTGCAATTAGCGTGATGAGCTCTGACATGGCCTTGCATGGACGGATTGGGCAGGACACCCCAGCTGAGGAGGATGGCAGGAGTGATGGCACAGGGGAAAGGGTGGCATACCCAGGTGACAGCTCCCCACTACCTCCACTCTGTGCTGCAGCTCAGGGGCTGGGTCTTCTGCTGCAACTCAGCCCCTCTGTACCAGCCCTGGCCTCATTCCCTTGGTTCCAGGACACCCAGCTGACAAAAGGGACTTGCCTGTACCCCTGCACCTGGTCCTACACCTGGCTCCTGGGTTGTCAGCAGGTGTTTGTTGGGCCAACGAGTGCATGGATGGAAACACAGACAGAAGGACAGATGGAGAGATGGTGGGTGGCCAGACAAAGGAGTAACTTGGTGAGGAATGTGCATTAGGAAATCACAGAAGAGCAGAAACTGTTTGAAAATTCCAAGTGGGGAAAGTGAGGAGGTGAAGCAGGGCTGAAGGGCCTCCCTCAGAGCCTTCTCCCACTCTGTGGTGTCCACATCCCCTTGGTCGTCCTTGTGGGAGGCACTCACCTTTTGCTCAGCCTATTGTGGCTACAGCCCAGCAGGTCCCAGGTGGCACCAGCCAAGATGAAGGTGGCATTGAGGGCTGAAGTCTCCCTCACCATGAAGGGATGATGTATAGTGGGTGGGGCCTCAGGAGGAAGAGGGCCACCAACCCTACCTGGCCCCTAACCTGCTGCCTGGAGTAGGCAGGTACCAGAGGCATGGGGTGAGGCATGTTGCAGGTCGAGGACCAGGGCCATCTCACTGCCTGAGCCCATGGACTGGCTCAGGGGTCTGTCAGATGATTCTAGAGCTGAGTTGGAGGTAAGGGCAGGGGGTTTGTTCCTGGGTTCAAGACCATGGAAGGAAGGGGTAGAGAAGGAGGCCAACAAGTGAGGAGGCAAATTACAGTGGCTGGCAGAAGGAGAGAGAAGCCAGGACAGGTGGCTGTGGCCCTGTCCCTGCAGGCAGACCCAGGAAGGAGCTCAGAGACAGGATTCATGCCAAGCCTGCCTACCCAGCACATCTCTCCTCATGGACATGAGAGAAAACCCTCCAGCTTGGCCCTCACATCTGTGAAACCCACAGTAATGGGGCTGACATCCTCTGCCCTATGCAAGAGAGGTTTCCCAAGCACTTGCAGCAAGTGAGACTGCACAGGATGGCGAATCCACAAAGAACACGTTGTTCTCATGCTCTTTGGAAGCACCAATTTACATTCTG
The Human Genome (partial sequence….)
Table 21-1
Fig. 21-7Exons (regions of genes coding for protein
or giving rise to rRNA or tRNA) (1.5%)
RepetitiveDNA thatincludestransposableelementsand relatedsequences(44%)
Introns andregulatorysequences(24%)
UniquenoncodingDNA (15%)
RepetitiveDNAunrelated totransposableelements (15%)
L1sequences(17%)
Alu elements(10%)
Simple sequenceDNA (3%)
Large-segmentduplications (5–6%)
Comparison of gene organization in different species (centered on region containing RNA polymerase gene)
Fig. 21-9a
TransposonNew copy oftransposon
DNA ofgenome Transposon
is copiedInsertion
Mobile transposon
(a) Transposon movement (“copy-and-paste” mechanism)
Fig. 21-9b
RetrotransposonNew copy of
retrotransposon
Reversetranscriptase
Insertion
RNA
(b) Retrotransposon movement
TECHNIQUE
Gel electrophoresis
cDNAs
PCR amplificationPrimers
mRNAscDNA synthesis 1
2
3
Reverse Transcriptase PCR (RT-PCR)
Large scale sequencing of cDNA fragments
Sequence large numbers (millions) of cDNA fragments
No UV(3 samples)
UV(3 samples)
Large scale sequencing of cDNA fragments
Fragments matching rad51