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Southern blotting
A: yes!
Q: is the Beta-globin gene expressed? (= is the mRNA present in my total RNA sample?)
Northern blotting
Total RNA
Hybridize with
labeled DNA
probe
A: yes!
And its
expression
increases
over time
1. halogen
tungsten lamp
3.
intensifier
2b. emission
filters
2a. excitation
filters
4. sample plate
5. CCD
chip
AMOUNT OF DNA
1400000000
AMOUNT OF DNA
1
2
4
8
16
32
64
128
256
512
1,024
2,048
4,096
8,192
16,384
32,768
65,536
131,072
262,144
524,288
1,048,576
2,097,152
4,194,304
8,388,608
16,777,216
33,554,432
67,108,864
134,217,728
268,435,456
536,870,912
1,073,741,824
1,400,000,000
1,500,000,000
1,550,000,000
1,580,000,000
1200000000
1000000000
800000000
600000000
400000000
200000000
0
0
10
15
20
25
30
35
A MOU N T OF D N A
CYCLE NUMBER
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
10000000000
1000000000
100000000
10000000
1000000
100000
10000
1000
100
10
1
0
10
15
20
25
30
35
Cloning
Cloning
Recombinant DNA technology enables to produce large numbers of identical DNA molecules (DNAcloning)
Clones are typically generated by placing a DNA fragment of interest into a vector DNA molecule,
which can replicate in bacterial host cells
When a single vector containing a single DNA fragment is introduced into a host cell, large numbers
of the fragment are reproduced (cloned) along with the vector
Two common vectors are E. coli plasmid vectors and bacteriophage vectors
Cloning
Cloning
Cloning
Cloning
Cloning
Recombinant DNA:
Cloning
Cloning
Cloning
Sequencing
Sequencing
Sequencing
Technologies/Platforms:
Roche/454 FLX: 2004
Illumina Solexa Genome Analyzer: 2006
Applied Biosystems SOLiD System: 2007
Helicos Heliscope: 2010
Pacific Biosciencies SMRT: 2010
LifeTechnologies Ion Torrent: 2011
Parameters:
Cost (device, cost/Mb)
Read length
Speed
Accuracy
Preperation time
Manipulation steps (amplification needed)
Sequencing
Illumina
SOLiD
Chemistry
Pyrosequencing
Polymerase-based Ligation-based
Amplification
Emulsion PCR
Bridge Amp
Emulsion PCR
Paired ends/sep
Yes/3kb
Yes/200 bp
Yes/3 kb
Mb/run
100 Mb
1300 Mb
3000 Mb
Time/run
7h
4 days
5 days
Read length
250 bp
32-40 bp
35 bp
$8439
$8950
$17447
Cost per Mb
$84.39
$5.97
$5.81
Sequencing
Sequencing
Illumina (Solexa)
Workflow
Sequencing
Sequencing
Ion Torrent
Sequencing
Applications
Genomes
ResequencingHumanExons(Microarraycapture/amplification)
small(includingmiRNA)andlongRNAprofiling(includingsplicing)
ChIPSeq:
TranscriptionFactors
HistoneModifications
EffectorProteins
DNAMethylation
PolysomalRNA
OriginsofReplication/ReplicatingDNA
WholeGenomeAssociation(rare,highimpactSNPs)
CopyNumber/StructuralVariationinDNA
ChIAPET:TranscriptionFactorLoopingInteractions
The$1000genome
Current bottle neck: Data management!!!
a
a
a
a
Bacterial colonies
Yeast colonies
Replica plating
a Loss of function
`Knock-out mice
`siRNA
a Gain of function
`Transgenic mouse
a Flavours
`Conditional (inducible system)
`Tissue-specific
a The two copies of each gene may be the same or the copies may
be different. Different forms of each gene are termed alleles
(allele= naturally occuring mutation)
a Alleles may be dominant or recessive
Advantage:
Effect on specific
tissue / process can
be studied
Embryonal lethal
knock-outs can be
studied in tissues
where the knock out
is not lethal
Knock-out can be
induced chemically