The importance of transgenic plants, gene identification and methods of gene identification.

1. Transgenic Plants,
Gene Identification
Methods &
Localization and
Sequencing of Genes
By: Annu
M. Pharmacy [DRA 2nd Sem]
2239807

2. Transgenic Plants

3. Definition
"A transgenic plant is a modified organism where
genes are transferred from one organism to
another through genetic engineering techniques".
Purpose: The purpose of producing a transgenic
plant is to obtain a species that has ideal traits,
high yield and quality.
The first transgenic plant was created in 1994.

4. History
1st genetically modified crop approved for
sale in U.S. was FlavrSavr tomato.
1994
1st pesticide producing crop, Bt Potato was
approved by U.S. Environmental Protection
agency.
1995
Golden rice with B-
carotene developed with
increased nutrient value.
2000
1st genetically engineered crop developed by
Robert Fraley, Marc Van Montagu & Marry
Dell Chilton were awarded World Food Prize.
2013

5. Production
of
Transgenic
Plants
It can be carried out by several methods
such as:
• Particle Gun or Gene Gun or Biolistic or
microprojectile bombardment.
• PEG (polyethylene glycol mediated
transformation)
• Electroporation
• Agrobacterium –mediated gene transfer.

6. Particle gun/
Particle
bombardment
• Foreign DNA coated with high velocity gold or
tungsten particles to deliver DNA into cells.
• This method is widely being used because of its
ability to transfer foreign DNA into the
mammalian cells and microorganisms.

7. Polyethylene glycol-
mediated transformation
• PEG in the presence of divalent cations,
destabilizes the plasma membrane of
protoplasts and renders it permeable to
naked DNA.
• A large number of protoplasts can be
simultaneously transformed.
• This technique can be successfully used for
a wide variety of plant species.

8. Electroporation
• It involve the creation of pores in the
cells membrane using electric pulse
of high field strength. If DNA is
present in the buffer solution at
sufficient concentration, it will be
taken up through these pores.

9. Agrobacterium
mediated gene
transfer
• It is treated as nature's most
effective plant genetic engineer.
• A tumefaciens infects wounded
or damaged plant tissues results
in the formation of plant tumor
called crown gall.
• The bacterium releases Ti
plasmid into the plant cell
cytoplasm which induce crown
gall.
• Several dicots are affected by
this diseases e.g. grapes,
roses etc.

10. Importance of
transgenic
plants
Improving production yield.
Cutting down transportation costs.
Enhancing the nutritional values.
Preventing crop damage by producing
herbicide, insect, virus, and pest-resistant crops.
Development of drought-resistant crops.

11. Methods used in gene
identification

12. Gene identification
• Identification of important
components in genomic DNA.
• Identification of genes in a
genomic DNA Sequence.
• Prediction of protein-coding
genes:
• Prokaryotes
• Unicellular eukaryotes
• Multicellular eukaryotes

13. Gene identification methods
Genes are identified broadly via two methods:
 Similarity-based searches
 Ab-initio prediction

14. Similarity
-based
Searches

15. Ab-initio
Prediction

16. Localization & Sequencing
of genes

17. Gene sequencing
Sequencing: it means to determine the
primary structure of an unbranched
biopolymer.
Gene sequencing: it is the technique that
allows researchers to read the genetic
information found in DNA.
Sequencing involves determining the
order of bases.

18. Gene
sequencing
methods:
1. First generation sequencing:
• Sanger sequencing
• Maxam Gilbert sequencing
2. Second generation sequencing
3. Next generation sequencing:
• Template preparation
• Library preparation
• Library amplification

19. First generation
sequencing:
• Sanger Sequencing:
This method is used in
in-vitro DNA
replication. This
technique is developed
by British Biochemist
Frederick and his team
in 1977.

20. Maxam Gilbert
Sequencing:
• This technique developed by Allan Maxam
and Walter Gilbert in 1976.
• Principle: Chemical degradation
of Pyrimidines.

21. Second Generation Sequencing:
These SGSTs generate hundreds of thousands to billions of 25-800
nucleotide-long reads within days in a low-cost manner compared
to Sanger sequencing.
Following a shotgun approaches several of these technologies
today can re-sequence a human genome in 10-30 coverage for
under $5,000 in reagent costs in 8-10 days.
SGSTs have enjoyed a warm welcome from the academics and
industrial scientists.

22. Next
generation
sequencing:
• Basic Principle: Its works is similar to traditional
Sanger Sequencing methods involving capillary
electrophoresis.
1. Template preparation: The double-stranded DNA is
considered as the starting material. However, the
source from which this material is derived may vary.
Sources can be either genomic DNA, immuno-
precipitated DNA, reverse-transcribed RNA or
cDNA.
2. Library preparation: Sequence library preparation
involves some common steps of fragmentation, size
selection and adapter ligation. Adapter ligation is also
involved in this process, which adds platform specific,
synthetic DNAs at the end of the DNA fragments
present in this library to facilitate the sequencing
reactions.

23. 3. Library amplification :
to produce significant signal
for nucleotide addition. This
step involves either by the
attachment of DNA
fragment to micro bead or
attachment of the same to
glass slide, when some PCR
techniques are followed.

24. Thank you