This document discusses various techniques used to analyze transgenic plants, including PCR, Southern blotting, Northern blotting, Western blotting, ELISA, and strip tests. PCR can detect the presence of a transgene but not copy number. Southern blotting determines transgene integration sites and copy number by detecting DNA fragments after restriction enzyme digestion. Northern blotting analyzes relative transgene expression at the mRNA level. Western blotting and ELISA detect and quantify transgene protein production. Strip tests provide a rapid, qualitative method to detect transgene proteins. Together, these techniques allow analysis of transgenic plants at the DNA, RNA, and protein levels.

1. Analysis of transgenic plants
Likhith R K
TNAU
Coimbatore

2. Transgenic plants:
• The transgenic plants refer to the those plants which carry the
gene from the other organism in its genome and can carry to
their successive generations.
• The transgenic plants are developed for various reasons, such
as
i. To improve nutritional quality(Golden rice)
ii. For the resistance against the insect pest(Bt cotton)
iii. For Herbicide tolerance(transgenic soyabean-against
glyphosate)
iv. For Virus resistance(transgenic tobacco-against TMV)
v. For the resistance against diseases(transgenic potato
resistant to late blight) and so on.

3. How the gene from one organism to
other is transferred??
1. Direct methods: it includes the physical
methods(biolistic, electroporation, laser
mediated, liposome mediated etc..) and
chemical methods(calcium phosphate
mediated and PEG mediated).
2. Indirect methods: it includes the viral
mediated and Agrobacterium mediated.

4. 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.

5. •It determines no. of copies of the transgene.
•Detection of transgene transcription and the
transcription level.
•Production and accumulation of the transgene
encoded protein.

6. Techniques to analyse transgenic plants:
1. PCR
2. Southern blotting
3. Northern blotting
4. Western blotting
5. ELISA
6. Strip test
DNA level
RNA level
Protein level

7. 1.Polymerase chain reaction(PCR):
PCR can be used to analyse transgenic plants by using:
The primers specific to the transgene of interest.
The plasmid as a positive control. This is because, positive
control reveals that the PCR condition is working.
The DNA of a non-transformed plant as a negative control.
But the PCR do not give info. about the copy number.

9. 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.

10. Southern analysis:

11. 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.

12. 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.

13. In case of single locus two copy insertion, three
fragments will be generated. The additional
fragment represent the gene construct inclusive of
T-DNA.

14. 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.

15. 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

16. 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.

18. 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.

19. Real time-PCR
• Isolate RNA from tissues of interest
• Eliminate all DNA from a sample
• Make cDNA from mRNA
• PCR is done using transgene-specific primers

20. 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.

22. 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.
www.gmotesting.com

23. • The enzyme linked detection antibody will bind any
protein already immobilized to the well by the capture
antibody.
• 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.
www.gmotesting.com

24. www.gmotesting.com
Image showing the contents in the wells

25. Advantages:
More sensitive, can detect even a small
amount of protein.
Disadvantage:
Need of intact protein and a good facilitated
laboratory.
www.gmotesting.com

26. Strip test:
The most commonly used immuno or antibody-
based test for transgenic protein detection is the strip
test (also called a lateral flow device or
dipstick). Strip tests are thin strips comprised of a
nitrocellulose membrane covered by a sample pad on
one end and a wicking pad on the other end.
www.gmotesting.com

27. Dipstick:
www.gmotesting.com

28. • The sample pad is submersed in a solution of
homogenized test sample. The solution wicks up
the nitrocellulose membrane on the strip, causing
the fluid to pass over an area containing an excess
of gold-labelled antibody specific to the protein
being tested.
• If that particular protein is present in the sample
then it will specifically bind to the gold-labelled
antibody, and the antibody-protein complex will
continue moving up the strip with the flow of fluid.
www.gmotesting.com

29. • The sample fluid then passes over two additional
areas on the strip, a test line and a control line.
• The test line contains a second antibody which is
specific for the protein being tested.
• When the gold-labelled antibodies with bound
proteins pass over the test line, the antibody-protein
complex forms a sandwich with the immobilized
second capture antibody.
• This results in formation of a visible line on the
strip indicating that that particular protein is present
in the sample
www.gmotesting.com

30. • Excess unbound gold-labelled antibody continues to
flow up the strip and passes over the control line
which contains a third immobilized antibody specific
for the gold-labelled antibody.
• When the excess gold-labelled antibodies bind to this
immobilized antibody in the control line, it produces
a second visible line that serves as an internal control
to ensure that gold-labelled antibodies have passed
through all three check points.
www.gmotesting.com

31. www.gmotesting.com
Image showing the binding of protein bound gold Ab with the
Ab present at the test line

32. The sample is considered
positive for the transgene
expressed interest if the test
and control lines are visible
whereas the sample is
considered negative for the
GMO protein if only the
control line is visible. Absence
of both lines indicates that the
test is invalid and should be
repeated
www.gmotesting.com

33. Advantage:
The assay is rapid, is appropriate for qualitative or
semi-quantitative detection of proteins expressed by
transgene.
Disadvantage:
The sensitivity is not as great as that of PCR test or
ELISA.
www.gmotesting.com

34. Summary:
Is my plant transgenic?
• PCR
• Southern blot analysis
Is my plant expressing
the transgene?
• Northern blot analysis
• Western blot analysis
• ELISA
• RT-PCR
• Strip test

35. Thankyou