Professional Documents
Culture Documents
using
is a method for
using an
which requires
Automated
thermal cycler
DNA which is Thermostable
Primers Which bind to amplified DNA
template which runs a cycle of
by polymerase
may be prepared from Denaturation
such as at > 90 °C
Taq
Random Gene-specific Genomic mRNA polymerase Primer binding
oligonucleotides primers DNA
requires such as
derived from Copying of DNA strands
by a polymerase
cDNA synthesis
followed by
Amino acid Known DNA
sequence data sequence
amplification
of cDNA
Polymerase chain reaction (PCR)
Amplifying spesifc DNA sequence
Polymerase chain reaction (PCR) was invented by
Kary Mullis.
Kary Mullis won Nobel prize in chemistry in 1993.
THE PRINCIPLE OF PCR
PCR components
1. Template DNA
DNA containing sequence for
amplification
2. Primer
• Oligonucleotide
• Providing free 3’OH to start
amplification
• bordering the fragment to be amplified.
DNA
3. DNA polymerase
• DNA polymerase
• Amplifying DNA fragment
4. dNTP
• Providing DNA bases
• dATP, dTTP, dGTP, dCTP
SIKLUS DAN TAHAPAN PCR
DENATURATION (95◦C)
Duplex DNA is heat
denatured to give single
strands
ANNEALING (55◦C)
two oligonucleotide primers
are annealed to their
complementary sequences
on the target DNA.
EXTENSION (72◦C)
Taq polymerase
(thermostable) is used to
synthesise complementary
strands from the template
strands by primer extension.
LO 64: menjelaskan prinsip dasar PCR
How to test for Test for GMOs by PCR:
GMOs
1. Grind food
2. Extract DNA from sample
3. Test sample DNA for viable
plant DNA
4. Test sample DNA for genetic
modifications
• Bio-Rad certified non-GMO food
– Verify PCR is not contaminated
• GMO positive control DNA
Kit – Verify GMO-negative result is not due
Controls to PCR reaction not working properly
• Primers to universal plant gene
(Photosystem II)
– Verify viable DNA was extracted
To confirm that viable DNA was
Why amplify a extracted and that negative GM result
plant gene? isn’t due to a non-viable template.
Results GMO
positive
1: non-GMO food with plant primers
Genomic DNA
Transgenic plant produced from
Agrobacterium-mediated
transformation
• In T0 plants, Agrobacterium left over from the initial
transformation may still be present in the plant tissue.
• Contamination of the genomic DNA with the initial transformation
vector that is still present in the agrobacterium can produce a PCR
band.
Varian PCR
1. Reverse transcriptase PCR (RT-PCR)
PCR using mRNA as a template
Used to determine the level of gene expression
requires reverse transcriptase
Oligo (dT) Primer was used to synthesize first
strand cDNA.
PCR primer is used after the first strand cDNA
synthesis
Fig. 7.4 RT-PCR. (a) Reverse transcriptase is used to synthesise a cDNA copy of the mRNA. In
this example oligo(dT)-primed synthesis is shown. (b) The cDNA product is amplified using
genespecific primers. The initial PCR synthesis will copy the cDNA to give a duplex molecule,
which is then amplified in the usual way. In many kits available for RT-PCR the entire
procedure can be carried out in a single tube.
Processing PCR product
94°C 4 min
94°C 15 sec
40x
61°C 30 sec
72°C 30 sec
Real-time Principles
http://www.web.virginia.edu/Heidi/chapter12/chp12.htm
Measuring DNA: SYBR Green I
SYBR Green I
5’ 3’
Extension
R
Q
Taq
Fluorescent 5’
R
3’
Hydrolysis
Dyes in PCR 5’
Q
Taq
5’
3’
R
Taq
Probes
5’
5’ 3’
l
R
Signal
Taq
5’
5’ 3’
What’s Wrong With
Agarose Gels?
Low sensitivity
Low resolution
Non-automated
4500000
4000000
3500000
Measuring 3000000
2500000
Quantities 2000000
1500000
1000000
500000
0
0 5 10 15 20 25 30 35 40
The “ct value”
• The value that represents the cycle number where the
Imagining •
amplification curve crosses an arbitrary threshold.
Ct values are directly related to the starting quantity of
Quantity = 2^Ct
PCR
5000000
4500000
Ct Values:
4000000
Measuring 3500000
3000000
25
23 28
Quantities Threshold
2500000
2000000
1500000
1000000
500000
0
0 5 10 15 20 25 30 35 40
real-time PCR looking for the exact
amount of a target sequence or gene in
the sample.
During the PCR reaction, you measure its
progress by accumulation of a fluorescent
signal during amplification.
the Ct, or “threshold cycle.” This spot
shows the number of cycles it took to
detect a real signal from your samples.
Any real-time PCR run will have many of
these curves from several samples, and
therefore many Ct values.
threshold
Ct
• Ct values are inverse to the amount of nucleic
acid that is in the sample, and correlate to the
number of copies in your sample.
• Lower Ct values indicate high amounts of
targeted nucleic acid,
• while higher Ct values mean lower (and even
too little) amounts of your target nucleic acid.
• Typically, Ct values below 29 cycles show
abundant nucleic acids, and Ct values above
38 cycles indicate minimal amounts, and
possibly an infection or environmental
contamination.
There’s a DIRECT relationship between the
Imagining starting amount of DNA, and the cycle number
that you’ll reach an arbitrary number of DNA
35
Measuring
30
y = -3 . 3 1 9 2 x + 3 9 .77 2
2
R = 0 .9 9 6 7
25
Quantities
Ct
20
15
10
0
0 1 2 3 4 5 6 7 8 9 10 11
n
L o g o f c o p y n u m b e r (1 0 )
Real-time PCR instruments consist
of TWO main components:
What Type • Thermal Cycler (PCR machine)
of • Optical Module (to detect
fluorescence in the tubes during
Instruments the run)
are used
with Real-
Time PCR?
Quantification
and
Normalization
• First basic underlying principle: every
cycle there is a doubling of product.
Quantification and
Normalization
• Second basic principle: we do not
5000000
need to know exact quantities of DNA,
4500000
4000000
instead we will only deal with relative
3500000
3000000
quantities.
2500000
2000000
1500000
1000000
500000
• Third basic principle: we have to have
not only a “target” gene but also a
0
0 5 10 15 20 25 30 35 40
“normalizer” gene.
• Key formula:
Quantity = 2 ^ (Cta – Ctb)
Standard Curve
Quantification and
Normalization
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0 5 10 15 20 25 30 35 40
4000000
3000000
2500000
2000000
1500000
• Example:
mRNA levels of a gene increase 2x after induction
1000000
500000
0
0 5 10 15 20 25 30 35 40
-[(Cttg-Ctcg)-(Cttg-Ctcg)]
2 Ex!
Ct = target gene– ref gene
Ct = 9.70
Difference = Ct-Ct
= Ct
= 9.70-(-1.7)
= 11.40
Fold change = 211.40 = 2702