Polymerase Chain Reaction (PCR)
• Polymerase chain reaction:
Starting with VERY SMALL AMOUNTS OF DNA
(sometimes a few molecules), one can amplify
the DNA enough to detect it by electrophoresis.
5’
3’
3’
5’
Denaturation
5’
3’
3’
5’
Annealing
5’
3’
3’
5’
3’
5’
5’
5’
5’
5’
5’
5’
5’
3’
3’
3’
3’
5’
3’
5’
3’
5’
3’
5’
3’
Cycle 1
5’
3’
5’
3’
3’
Extension
5’
3’
5’
3’
Cycle 2
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
3’
Cycle 3
3’
3’
5’
• Rate of PCR
2n
Initial
DNA
1
2
4
Number of DNA molecules
8
DNA copy number
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
Copies of DNA=2N
1.8E+09
1.6E+09
1.4E+09
DNA copy number
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
1.2E+09
1.0E+09
8.0E+08
6.0E+08
4.0E+08
2.0E+08
0.0E+00
0
5
10
15
20
PCR cycle
25
30
35
RT-PCR
• Polymerase chain reaction amplification of cDNA
can also be used to detect specific transcripts in
a RNA sample.
• In this procedure, known as RT-PCR, reverse
transcriptase is used to copy all of the mRNAs in
an RNA sample into cDNA.
• Usually, oligo dT molecules, that anneal to the
poly A tails of the mRNA, are used as primers.
• This single stranded cDNA can then be amplified
by PCR using primers that anneal to a specific
transcript sequence.
RT-PCR
5‘-Cap
mRNA
(dT)12~18
primer
AAA(A)n
anneal
3‘
5‘
AAA(A)n
dNTP
Reverse transcriptase
5‘
cDNA:mRNA hybrid
AAA(A)n
Regular
PCR
• The amplified DNA fragments that are
produced can by analyzed by agarose gel
electrophoresis.
• The amount of amplified fragment
produced is proportional to the amount of
target mRNA in the original RNA sample.
• Although less quantitative than Northern
blots, RT-PCR is extremely sensitive and
can be used to detect very rare mRNA
species.
How do we accurately quantify the amount of DNA?
Real-time PCR
Real Time PCR
3. intensifier
1. halogen
tungsten lamp
2b. emission
filters
2a. excitation
filters
4. sample plate
5. ccd
detector
350,000
pixels
Amplification Plot of real-time PCR
DNA copy number (log)
Log phase
Level off/ plateau
2
4
8
16
32
PCR cycle (Ct)
DNA sequencing
• Sequencing is used to determine the precise
order of nucleotides in a DNA molecule.
• The Sanger di-deoxy
method involves the
synthesis of DNA by a DNA
polymerase.
• DNA synthesis is terminated
at specific nucleotides by
the incorporation of di-deoxy
nucleotides that are missing
the 3’ OH.
Sequence analysis
• Four different reactions produce DNA
fragments that are terminated (randomly)
at each of the four nucleotides.
• These samples are resolved by
electrophoresis.
• The shortest fragments, those terminated
closest to the primer, run faster than the
longer fragments.
ACGT
• DNA sequencing
reactions can be
radioactively labeled.
• Bands detected by Xray film exposure.
• Sequence can be read
in the 5’ to 3’ direction
from the bottom of the
image towards the top.
A
A
T
C
T
A
A
C
G
This is great but…
Wouldn’t it be great to run everything
in one lane?
Saves space and time, more
efficient
Fluorescently label the ddNTPs so
that they each have a different
color…
07_03.jpg
• Automated DNA
sequencers use
dideoxy terminators
labeled with four
different fluorescent
dyes.
• All four reactions
can be carried out
simultaneously in a
single reaction.
• Fluorescently tagged fragments are
resolved by capillary electrophoresis and
detected by a flourometer.
• The DNA sequence is read automatically.
Beckman CEQ 2000, 8 capillary
ABI Prism 3730, 96 capillary
Affymetrix Gene Chip
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Affymetrix Expression Arrays
http://www.affymetrix.com/technology/ge_analysis/index.affx
Analysis of microarrays
• Microarrays allow for the simultaneous
analysis of the expression of thousands of
mRNAs.
• Useful for determining changes in gene
expression patterns from one sample
tissue to another.
• For example, microarrays have been used
to study differences in gene expression in
different tumor tissues.
Genes with similar expression patterns are clustered together.
Gene expression patterns can be associated with different
disease patterns.
Samples
Microarray example #1: Biomarker
identification - lung cancer
Garber, Troyanskaya et al. Diversity of gene expression in adenocarcinoma of the
lung. PNAS 2001, 98(24):13784-9.
Data partitioning clinically important:
Patient survival for lung cancer subgroups
1
Cum. Survival (Group 1)
Cum. Survival (Group 2)
Cum. Survival
.8
Cum. Survival (Group 3)
.6
p = 0.002
for Gr. 1 vs. Gr. 3
.4
.2
0
0
10
20
30
40
50
60
Time (months)
Garber, Troyanskaya et al. Diversity of gene expression in adenocarcinoma of the
lung. PNAS 2001, 98(24):13784-9.
Another microarray example: understanding malaria
IDC Transcriptomes for three
P.falciparum strains
Llinas, M. et al. Nucl. Acids Res. 2006 34:1166-1173; doi:10.1093/nar/gkj517
Yeast 2 Hybrid
Allows the genetic detection of
physical interactions between
proteins
Yeast Two Hybrid Assay
•
•
The two-hybrid system is a molecular genetic tool which
facilitates the study of protein-protein interactions.
If two proteins interact, then a reporter gene is
transcriptionally activated.
– e.g. gal1-lacZ - the beta-galactosidase gene
•
•
A colour reaction can be seen on specific media.
You can use this to
– Study the interaction between two proteins which you expect to
interact
– Find proteins (prey) which interact with a protein you have
already (bait).
GAL4
has two domains:
one binds DNA (UAS) ,
the other activates transcription
{
GAL1
UAS
{
{
GAL4
DNA-binding Activating
GAL1
GAL1
POL
POL
.
If protein X and Protein Y interact,
they lead to expression of the
reporter gene.
Two-hybrid assay
SNF4
SNF1
1.
B
A
2.
GAL4-DBD
3.
Transcription activation domain
UASG
Fields S. Song O.
Nature. 1989 Jul 20;340(6230):245-6.
PMID: 2547163
4.
GAL1
Allows growth on galactose
Two-Hybrid Selection
UAS-Reporter
GAL4-dbd/BAIT
GAL4-ad/X
Library
No expression
UAS-Reporter
GAL4-dbd/BAIT
GAL4-ad/X
Expression