Analysis of transgenic

Why the analysis??

• The analysis of the transgenic plants gives a confirmation
that gene of interest is present in the transgenic plant.
• If present, analysis confirms the transgene integration
with the host genome.
• It determines the no. of sites at which the transgene is
integrated.

• It determines no. of copies of the transgene.

• Detection of transgene transcription and the transcription
level.
• Production and accumulation of the transgene encoded
protein.
 Techniques to analyse
transgenic plants:

1. PCR
DNA level
2. Southern blotting
3. Northern blotting RNA level

4. Western blotting
Protein level
5. ELISA
1. Polymerase chain reaction(PCR):

v PCR can be used to analyse transgenic plants by using:

v The primers specific to the transgene of interest.
v The plasmid as a positive control. This is because, positive
control reveals that the PCR condition is working.
v The DNA of a non-transformed plant as a negative control.
v But the PCR do not give info. about the copy number.
 A. Double
50º strand DNA

96º B. Denature

50º C. Anneal
primers

Taq
72º D. Polymerase
binds
Taq
 Taq Taq
72º E. Copy
strands
Taq Taq

96º
2 F.
Denature
First round 3
of cDNA
synthesis (4
strands)
4
 Basic Polymerase Chain Reaction (I)

A thermostable DNA polymerase to catalyze template-dependent synthesis

A pair of synthetic oligonucleotides to prime DNA synthesis
Deoxynucleoside triphosphates (dNTPs)
Divalent cations
Buffer to maintain pH
Monovalent cations
Template DNA

Template DNA (105 to 106 molecules)

20 pmol of each primer
20 mM Tris-HCl (pH 8.3 at 20˚C)
1.5 mM MgCl2
25 mM KCl
0.05% Tween 20
100 µg/ml autoclaved gelatin or nuclease free BSA
50 µM dNTP each
2 units of Taq DNA polymerase
Total volume 100 µl
 Basic Polymerase Chain Reaction (II)
ü (92-98 ˚C) Denaturation 96 ˚C for 15 sec
ü (37-70 ˚C) Primer annealing 55 ˚C for 30 sec
ü (70-74 ˚C) Primer extension 72 ˚C for 90 sec
Repeat for 20 to 30 cycles
ü (70-74 ˚C) Final extension 72 ˚C for 5 min
Stop the reaction: Chill at 4 ˚C or adding EDTA to 10 mM
PCR products gel purified for further work

Primer Design
• Length: 18 to 28 nt
• GC content: 50 to 60%
• Tm: 55 to 80 ˚C
• Others: avoid 3’-end complementarity

Anneal Temperature = 2 x ( A + T ) + 4 x ( G + C )
 PCR analysis by gel electrophoresis

-
Ladder Sample
1500
bp
1000
bp

750 bp

500 bp

+
 PCR and False Positives

Genomic DNA
Transgenic plant produced from
Agrobacterium-mediated
transformation
• In T0 plants, Agrobacterium left over from the initial
transformation is still present in all tissues.
• Contamination of the genomic DNA with the initial transformation
vector that is still present in the agrobacterium can produce a PCR
band.
 2. Southern blotting:
• It provides the confirmation of transgene integration and reveals
the no. of sites at which the transgene is integrated.
• PCR positive controls are used for the analysis.
Steps
i. Genomic DNA isolation.
ii. Restriction enzyme digestion of genomic DNA
iii. Then it is treated with NaOH to make ss DNA.
iv. Electrophoresis.
v. Blotting.
vi. Hybridization with DNA probe.
vii.Autoradiography.
Southern analysis:
 How SB is used to know copy
number??
• Here Agrobacterium mediated transformation is used for the
illustration.
• In order to determine the no. of copies integrated, the genomic
DNA is digested with two restriction enzymes, one cleaving
outside the T-DNA borders and another within the gene
construct.
In case of single locus single copy insertion, two
bands will be detected. One fragment contains the
right border and the other containing the left border.
In case of single locus two copy insertion, three
fragments will be generated. The additional
fragment represent the gene construct inclusive of
T-DNA.
In case of two loci with single copy, each locus
would yield two distinct fragments and a total of four
fragments will be seen. In case of single copy
insertions, 2n bands can be obtained, where “n” is
no. of insertion loci.
Southern blotting results showing the copy number:

Lane 1- pattern of single locus single copy insertion

Lane 2- single locus two copy insertion
Lane 3-Two locus two copy insertion
 Northern blotting:
• It Gives relative amount of gene expression-at the transcript
level.
• Steps:
i. Isolate mRNA of good quality (not degraded)
ii. Separate transcripts on an agarose gel
iii. Transfer to nylon filter
iv. The radiolabeled probe is complementary to the mRNA
from transgene is used as a probe(cDNA), which
hybridizes the RNA.
v. The hybridized RNA is detected using autoradiography.
• The intensity of the bands on X-ray film helps to know
how much of the transgene is transcribed to mRNA.
 Northern Blot
(an old technique for measuring mRNA expression)
1. mRNA extracted and
purified.
4. mRNA are
transferred from the
gel to a membrane.
2

2.mRNA loaded for

electrophoresi s.
Lane 1: size standards .
5. A labeled probe
Lane 2: RNA to be tested. •
specific for the RNA
fragment is incubated
w ith the b lo t. So the
3.The gel is charged RNA of interest can be
and RNA " swim " detected . Hybridization
through gel according
to weight.
Need relatively large amount of mRNA
'----------' +
 Northern Blot
No digestion with
RE is necessary…
why is this?

RNA loading controls

are necessary to
ensure an equal
amount of RNA is
loaded in each well.
 Western Blotting:
It is used to measure gene expression at the
protein level. Steps:
• Extract proteins
• Separate proteins on a SDS- PAGE
• Electroblotting: gel to nitrocellulose sheet
• Primary antibody specific to the protein is added
to nitro cellulose sheet and bands containing the
protein binds with Ab
• Radio labelled secondary Ab containing enzyme
is added, which binds to primary Ab and helps to
visualize the Ab-protein complex through
colorimetry.
Western blot example

What is missing in this experiment?

 Real-Time PCR
Real-time PCR monitors the fluorescence emitted during
the reaction as an indicator of amplicon production at
each PCR cycle (in real time) as opposed to the endpoint
detection
(www)
 Log-view augments
this part

Nigel Walker, NIEHS (www)

 Real-time PCR advantages
* not influenced by non-specific amplification
* amplification can be monitored real-time
* no post-PCR processing of products
(high throughput, low contamination risk)

* ultra-rapid cycling (as fast as 25 minutes)

* wider dynamic range of up to 1010-fold
* requirement of 1000-fold less RNA than conventional assays
(minimum 6 picogram = one diploid genome equivalent)

* detection is capable down to a two-fold change

* confirmation of specific amplification by melting curve analysis
* most specific, sensitive and reproducible
* not much more expensive than conventional PCR
(except equipment cost)
 Real-time PCR disadvantages
* not ideal for multiplexing (it is possible to multiplex though)
* setting up requires high technical skill and support
* high equipment cost

***
* intra- and inter-assay variation
* RNA lability
* DNA contamination (in mRNA analysis)
 Real-time PCR Principles
* based on the detection and quantitation of a fluorescent
reporter

* the first significant increase in the amount of PCR product

(CT - threshold cycle) correlates with the initial amount of
template
(www
log view
 Linear vs Log View

linear view log view

 Real-Time PCR Principles
Three general methods for the quantitative assays:
1. Hydrolysis probes
(TaqMan, Beacons)
2. Hybridization or FRET probes
(Light Cycler)
3. DNA-binding (intercalating) agents
(SYBR Green, Eva Green, LC Green)
(www
(www)
DNA Polymerase 5' Exonuclease Activity

Mocellin et al. Trends Mol Med 2003 (www)

 SYBR Green
(double-stranded DNA binding dye)

* emits a strong fluorescent signal upon binding to

double-stranded DNA
* nonspecific binding is a disadvantage
* requires extensive optimization
* requires melting curve analysis to ensure specificity
* longer amplicons create a stronger signal
* may be multiplexed when coupled with melting
curve analysis
 SYBR Green
(1) At the beginning of amplification, the reaction mixture contains the denatured
DNA, the primers and the SYBR Green. The unbound dye molecules weakly fluoresce,
producing a minimal background fluorescence signal which is subtracted during
computer analysis. (2) After annealing of the primers, a few dye molecules can bind
to the double strand. DNA binding results in a dramatic increase of the SYBR Green
molecules to emit light upon excitation. (3) During elongation, more and more dye
molecules bind to the newly synthesized DNA. If the reaction is monitored
continuously, an increase in fluorescence is viewed in real-time. Upon denaturation
of the DNA for the next heating cycle, the dye molecules are released and the
fluorescence signal falls.

Mapping Protein/DNA Interactions by Cross-Linking (NCBI Books) (www)

 Molecular Beacons

Mocellin et al. Trends Mol Med 2003 (www)

See also Didenko et al, Biotechniques 2001 (www)

Principles of Real-Time Quantitative PCR Techniques
(a)SYBR Green I technique: SYBR Green I fluorescence is enormously
increased upon binding to double-stranded DNA. During the extension
phase, more and more SYBR Green I will bind to the PCR product, resulting
in an increased fluorescence. Consequently, during each subsequent PCR
cycle more fluorescence signal will be detected.
(b)Hydrolysis probe technique: The hydrolysis probe is conjugated with a
quencher fluorochrome, which absorbs the fluorescence of the reporter
fluorochrome as long as the probe is intact. However, upon amplification
of the target sequence, the hydrolysis probe is displaced and subsequently
hydrolyzed by the Taq polymerase. This results in the separation of the
reporter and quencher fluorochrome and consequently the fluorescence of
the reporter fluorochrome becomes detectable. During each consecutive
PCR cycle this fluorescence will further increase because of the
progressive and exponential accumulation of free reporter fluorochromes.
(c)Hybridization probes technique: In this technique one probe is labelled
with a donor fluorochrome at the 3’ end and a second –adjacent- probe is
labelled with an acceptor fluorochrome. When the two fluorochromes are
in close vicinity (1–5 nucleotides apart), the emitted light of the donor
fluorochrome will excite the acceptor fluorochrome (FRET). This results in
the emission of fluorescence, which subsequently can be detected during
the annealing phase and first part of the extension phase of the PCR
reaction. After each subsequent PCR cycle more hybridization probes can
anneal, resulting in higher fluorescence signals.
 Threshold Cycle
* threshold cycle or the CT value is the cycle at which
a significant increase in DRn is first detected
* it is the parameter used for quantitation
* CT value of 40 or more means no amplification and
cannot be included in the calculations
* theoretically a single copy of the target should
create a CT value of 40 (if efficiency is 100%), which
is the y-intercept in a standard curve experiment

See: ABI Understanding CT (www)

 What is CT?

log view

The Amplification Plot contains valuable information for the quantitative measurement of DNA or
RNA. The Threshold line is the level of detection or the point at which a reaction reaches a
fluorescent intensity above background. The threshold line is set in the exponential phase of the
amplification for the most accurate reading. The cycle at which the sample reaches this level is
called the Cycle Threshold, CT. These two values are very important for data analysis using the 5’
nuclease assay.
(www)
 Good efficiency,
good sensitivity
and good
predictive power.

Albumin (ALB) gene dosage by real-time PCR

Laurendeau et al. Clin Chem 1999 (www)
What is DRn?

(www)
 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.
• The fluorescence obtained allows us to know the presence of cDNA
of transgene.
 ELISA:
• More sensitive antibody-based protein detection method is the ELISA
(Enzyme-linked immunosorbent assay)
• In this assay, a sample solution predicted to contain a particular
protein is added to a multi-well solid plate on which protein
specific antibody has been immobilized. If the transgenic protein
is present in the sample it will bind to the immobilized capture
antibody.
• After washing, a different antibody, also specific for the protein of
interest and tagged with an enzyme, is added to the well.
• After another round of washing to remove any unbound antibody,
the substrate for the enzyme is added which induces a colour change
in the solution.
• The degree of colour change is directly proportional to the amount of
protein present in the well.
 Image showing the contents in the wells

Advantages:
More sensitive, can detect even a small amount of protein.

Disadvantage:
Need of intact protein and a good facilitated laboratory.
 Summary
• Is my plant transgenic? • Is my plant expressing
– Survives selection the transgene?
– Reporter gene – Northern blot analysis
expression – Western blot analysis
– Progeny analysis – ELISA
– PCR – RT-PCR
– Southern blot analysis – Real-time RT PCR