ANALYSIS OF TRANSGENIC

using

PCR AND QPCR

 Polymerase chain reaction

is a method for

Amplifying DNA sequences

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.

Use highly conserved chloroplast

gene from Photosystem II – part of the
light reaction of photosynthesis.
 CaMV 35S – Sequence for the
Why use CaMV promoter of 35S transcript of the
35S and NOS? Cauliflower mosaic virus.
Used because it functions in every
plant cell

NOS- Sequence for nopaline

synthase terminator from soil
bacterium Agrobacterium tumefacians
Used because it evolved to be
recognized in most plants
 Analysis of
1 2 3 4 5 6 7

Results GMO
positive
1: non-GMO food with plant primers

2: non-GMO food with GMO primers

3. Test food with plant primers

1 2 3 4 5 6 7
4: Test food with GMO primers

5: GMO positive template with plant

primers
GMO
6: GMO positive template with GMO
negative
primers
7: PCR MW Ruler
 PCR and False Positives

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

PCR products (amplicons) can be:

 analyzed using gel electrophoresis
 identified using blotting techniques or
hybridization
 cloned into the expression vector
 cloned into a vector (vector-T)
 sequenced
PCR quantitative
What is Real-Time PCR?
The Polymerase Chain Reaction (PCR)
is a process for the amplification of
specific fragments of DNA.

Real-Time PCR a specialized technique

that allows a PCR reaction to be
visualized “in real time” as the
reaction progresses.

As we will see, Real-Time PCR allows

us to measure minute amounts of
DNA sequences in a sample!
What is Real-Time PCR used for?
Real-Time PCR has become a cornerstone of
molecular biology:

• Gene expression analysis

– Cancer research
– Drug research
• Disease diagnosis and management
– Viral quantification
• Food testing
– Percent GMO food
• Animal and plant breeding
– Gene copy number
 What is
Differences with normal
Real-Time PCR?
PCR? • 20ul PCR reactions
• SYBR Green or probes

 94°C 4 min
 94°C 15 sec
40x
 61°C 30 sec
 72°C 30 sec
 Real-time Principles

•based on the detection and

quantitation of
a fluorescent reporter

•In stead of measuring the endpoint

we focus on the first significant
increase in the amount of PCR product.

• The time of the increase correlates

inversely to the initial amount of DNA
template
Measuring DNA: Ethidium Bromide
Ethidium Bromide

http://www.web.virginia.edu/Heidi/chapter12/chp12.htm
Measuring DNA: SYBR Green I
SYBR Green I

Ames test results from Molecular Probes

Singer et al., Mutat. Res. 1999, 439: 37- 47
 RT-PCR
• Isolate RNA from tissues of interest
• Eliminate all DNA from a sample
• Make cDNA from mRNA
• Perform PCR on sample using
transgene-specific primers
 Real-time PCR or Quantitative PCR
• Real-time PCR uses fluorescence as an output
for DNA amplification in real-time
• The amount of starting template DNA (or
cDNA for RNA measurement (real-time RT-
PCR) is correlated with the Ct number
• More DNA = lower Ct; Ct is the cycle number
when a threshold amount of DNA is produced
during the PCR experiment
 http://www.rt-pcr.com/
Advantages of qRT-PCR
over RT-PCR?
http://www.youtube.com/watch?v=QVeVIM1yRMU
 Q R

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

 Size-based discrimination only

 Results are not expressed as numbers

 based on personal evaluation

 Ethidium bromide staining is not very
quantitative
 End point analysis
 So… if YOU started with FOUR times as much
Imagining DNA template as I did…

Real-Time Then you’d reach 1,000,000 copies exactly

TWO cycles earlier than I would!
PCR 5000000

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

Real-Time DNA, by way of the formula:

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

Real-Time copies (Ct value).

PCR DNA amount = 2 ^ Cycle Number

C o p y N u m b e r v s. C t - St a n d a r d C u r v e
40

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

Prepare a 2-fold serial dilution of a DNA sample:

Recomendation: add always a standard curve in every

run
 “normalizer” gene
Quantification and
Normalization • Knowing the amount of mRNA in one sample
from one specific gene does not tell us much..
• You need to know the total amount of mRNA in
5000000
your sample
• You also dont know how much the mRNA level
4500000

4000000

has changed compared to other mRNA levels

3500000

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

It is possable that all (1) genexpression in the cell

has increased (2) the induced samples contained
more total mRNA

We have to compare the expression of

our gene to another gene which
expression is normally constant, a
housekeeping gene (ex. TBP, 18S)
 ΔΔCt method
experiment control

-[(Cttg-Ctcg)-(Cttg-Ctcg)]
2 Ex!
 Ct = target gene– ref gene
 Ct = 9.70

 Ct = target gene– ref gene

 Ct = -1.70

Difference = Ct-Ct
= Ct
= 9.70-(-1.7)
= 11.40
Fold change = 211.40 = 2702

Always in duplicate or triplicate!