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BasicTheoryofPCR
1
Denature Anneal
3
Extend
Repeatstep1-3
25-30cycles
94
Temperature
72
DenatureDNA
Single-strandDNA
Amplifytarget
DNAfragmentto
1millioncopies
Target
DNAdoubled
55
Anneal
Primers
22
DoublehelixDNA
3
Time
min
PCR Process
1. Temperature Cycling
Denaturation
Annealing
Extension
94
55
72
PCR Amplification
Exponential Amplification
What is RTRT-PCR?
PCR?
RT-PCR: Reverse Transcription Polymerase
Chain Reaction
A method to amplify a piece of a ribonucleic acid (RNA)
molecule. The RNA strand is first reverse transcribed into
its DNA complement or complementary DNA followed by
amplification of the resulting DNA using PCR. This can
either be a 1 or 2 step process.
Real--time PCR
Real
Real time PCR: a technique used to
monitor the progress of a PCR
reaction in real time
SYBR Green
Hydrolysis probe
1. TaqMan probe
The reporter dye (R) represents the quenching dye that disrupts
the observable signal from the quencher dye (Q) when it is within
a short distance.
2. Molecular Beacons
Molecular beacons are hairpin
shaped molecules with an
internally quenched fluorophore
whose fluorescence is restored
when they bind to a target
nucleic acid
Molecular Beacon Example Sequence
Fluorophore at 5' end;
5GCGAGCTAGGAAACACCAAAGATGATATTTGCTCGC 3'-DABCYL
3. Scorpion probe
The oligonucleotide that consists of a probe
and primer linked together is called a Scorpion
probe or Scorpion primer.
The advantage of
this technique is
that the detection
of the amplicon is
much faster due
to the unimolecular
reaction.
Hybridization probe
Instrument compatibility
AMOUNT OF DNA
1.6E+09
1.4E+09
1.2E+09
1E+09
800000000
600000000
400000000
200000000
0
0
AMOU N T OF D N A
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
AMOUNT OF DNA
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
10
15
20
25
PCR CYCLE NUMBER
30
35
30
35
10000000000
1000000000
100000000
10000000
1000000
100000
10000
1000
100
10
1
0
10
15
20
25
23
Amplification curve
Amplification curve
Baseline: The initial cycles of PCR, in which there is little
change in fluorescence signal.
Threshold: The location on the graph where the values of the
samples are determined. The threshold should be set in
the region associated with an exponential growth of PCR
product.
CT: (threshold cycle) The cycle number at which the
fluorescence passes the threshold. Lower Ct indicates
higher starting amount; lower Ct for the same starting
amount indicates higher detection sensitivity
Threshold line
The Threshold line is the level of detection or the
point at which a reaction reaches a fluorescent
intensity above background.
threshold
Ct
Relative Quantification
overview
Example
http://cels.uri.edu/gsc/cndna.html
Relative Quantification
Methods for relative quantitation of gene expression allow
you to quantify differences in the expression level of a
specific target (gene) between different samples. The data
output is expressed as a fold-change or a fold-difference of
expression levels
- Comparative Ct method
- delta delta Ct
Relative Quantitation
Comparative Ct (
Ct
Method)
qRT-PCR
- SuperScript III Plantinum SYBR Green One-step qRT-PCR kit
- EXPRESS One-Step SYBR GreenER Universal
- InnuScript One Step RT-PCR Sygreen kit
qRT-PCR
- SuperScript III Platinum one-step qRT-PCR SuperMix