IN BRIEF ABOUT POLYMERASE CHAIN REACTION.

1. PCR AND ITS VARIANTS
BY :
SHILPA C ( PALB9313)
SOWMYAPRIYA R (PALB9315)

2. Polymerase chain reaction (PCR) is a
technique that results in exponential
amplification of a desired region of a DNA
molecule in vitro using a set of the sequence-
specific complementary primers in the
enzymatic cyclic temperature dependent
reaction.
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3. The of PCR technique was invented by Kary Mullis,
a Research Scientist at a California Biotech
Company, in 1983.
For this work, Mullis received the 1993 Noble Prize
in Chemistry.
Working principle:
As the name implies , it is a chain reaction ,a
small fragment of the DNA of interest which serves
as the template for producing the primers that
initiate the reaction. One DNA molecule is used to
produce 2 copies, then 4 copies ,then 8 copies and so
forth..
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4. COMPONENTS OF PCR
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5. Steps in PCR :
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6. PCR Steps
Initial
Denaturation
Denaturation Annealing Extension
Final
extension
Temperature 90 ̊C-95 ̊C 90 ̊C-95 ̊C 55 ̊C-6o ̊C 72 ̊C 72 ̊C
Time 5min 1min 50sec 1min 7 min
——————– ——————- 25-28 cycles ————— ———————
The reaction conditions are,
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7. Why different types of PCR ?
• Reduce contamination caused due to non-specific binding (Primer dimer,
misprimed or false primed targets) leads to wrong amplification.
• If unknown flanking DNA sequence adjacent to gene of interest
• If RNA is there
• Whether amplification is taking place or not
• What is the amplified quantity.
• More than one template as to be amplified at the same time.
• One side sequence is known to design only one primer
• Detect mutation
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8. BASIC
MODIFICATION
• ANCHORED PCR
• NESTED PCR
• MULTIPLEX PCR
• HOT START PCR
• TOUCHDOWN PCR
• ASYMMETRIC PCR
• COLD PCR
• INVERSE PCR
PRE TREATMENT
AND EXTENSION
• RT PCR
• 2 TAILED PCR
• LIGATION
MEDIATED PCR
• METHYLATION
SPECIFIC PCR
OTHER
MODIFICATIONS
• SITE DIRECTED
MUTAGENESIS
USING PCR
• OVERLAP
EXTENSION PCR
• ARMS PCR
• REAL TIME PCR
VARIANTS OF PCR
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9. ANCHORED PCR
Where only enough information to
make a single primer is known , for
second primer a known sequence is
added to the end of the DNA by
enzymatic addition of a polynucleotide
stretch of homopolymer tailing of
cDNA catalyzed by the terminal
deoxynucleotidyl transferase or by
ligation of known sequence.
 This technique of amplification with
single sided specificity has been known
as one-sided PCR or anchored PCR.
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10. Applications
•The production of cDNA libraries from very small amounts of starting
material
Limitations
• It is relatively difficult protocol.
• Large amount of starting templates are required.
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11. NESTED PCR
 Designed to increase the sensitivity and
specificity.
 It involves the use of two primer sets directed
against the same target and two successive
PCR reactions.
 The first set of primers is designed to anneal
to sequences upstream from the second set of
primers, whereas the second set of primers is
situated internally or nested with respect to
the first set of primers.
 First set of primers also called “outer
primers” amplify a large fragment of the
gene which is used as a template in the second
round of PCR that targets a smaller region of
the amplicon using the second set of primers
also known as “inner primers or nested
primers.”
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12. Application
Nested PCRs have proven valuable for the detection of microorganisms
when they are present in very low quantities.
Limitations
• Susceptible to contamination: The extreme sensitivity of nested PCR
comes with its own set of problems. Contamination mostly occurs during
the transfer of the first-round product to the second tube for the second
round of amplification.
• Costly: This PCR assay is also more costly as it involves the use of two
separate reactions to arrive at one result.
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13. LIGATION MEDIATED PCR (LM-PCR)
Ligation-mediated PCR uses
small DNA oligonucleotide
linkers or adaptors that are
first ligated to fragments of the
target DNA.
PCR primers that anneal to the
linker sequences are then used
to amplify the target
fragments.
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14. USES OF LM PCR :
• Adapted to map DNA damage and reveal DNA–protein
interactions inside living cells.
• Determination of primary nucleotide sequences.
• Determination of cytosine methylation pattern.
• DNA sequencing, genome walking, and DNA footprinting.
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15. MULTIPLEX PCR
Amplifying different DNA sequences or different DNA templets by using
the different set of primers in a single PCR reaction is refers to as a
multiplex PCR.
• Based on the type of the template the multiplex PCR can be divided into
two categories:
1. Single template PCR reaction:
This technique uses a single template which can be a genomic DNA along
with several pairs of forward and reverse primers to amplify specific regions
within a template
2. Multiple template PCR reaction:
This technique uses multiple templates and several primer sets in the same
reaction tube. Presence of multiple primer may lead to cross hybridization
with each other and the possibility of mis-priming with other templates.
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16. Uni-template multiplex PCR is used in the
detection of inherited genetic disorders.
Multi-template multiplex PCR is not a valid
technique for the detection of inherited genetic
disorders. It is applicable in the detection of
different strains or species of pathogens.
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17. How to Avoid Primer Dimer Formation ?
• Annealing temperatures for each of the primer sets must be optimized.
• Base pair length, should be different enough to form distinct bands
when visualized by gel electrophoresis.
Limitations of multiplex PCR:
• Although the technique is advantageous, the multiplexing is not
applicable to all types of reaction.
(For the larger amplicon such as 800bp or 1000bp, multiplex PCR might
not work efficiently always)
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18. Applications :
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Multiplex PCR in
combination with the real-
time PCR is even more
valuable and useful in the
quantitative studies.

19. HOT START PCR
 Which reduces the non-specific
bindings by limiting one of the
reagents until the heating step
of the PCR.
Hot start PCR = One of the
components starts its activity
under the hot condition of PCR.
 The aim of the Hot start PCR is
to limit the reaction at an early
stage, by limiting Taq DNA
polymerase in the reaction.
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20. Advantages:
• The hot start PCR technique decreases the nonspecific bindings.
• Also, it prevents mis-priming and primer dimer formation.
• By using the hot start Taq DNA polymerase, the reaction can even be
prepared at room temperature.
• It increases the yield and accuracy of the results.
Disadvantages:
• The overall cost of the reaction is increased, due to the use of the
antibody.
• The heating step is predominant in the hot start PCR, hence due to the
higher temperature for a longer time the template DNA can damage or
break down badly.
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21. TOUCHDOWN PCR
• “By sequentially decreasing the
annealing temperature during each
PCR cycle, the chance of the non-
specific binding can be reduced.”
1. By using higher annealing
temperatures at the earlier cycles,
only very specific base pairing
between the primer and the
template will occur
2. Increases the efficiency by lowering
the annealing temperatures
gradually toward the end of cycles.
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22. • The annealing temperature of
the primers is the temperature
at which the primers bind to its
specific complementary
sequence on DNA.
• The melting temperature is a
temperature at which the
primer dissociate from the
complementary DNA
sequence.
• The annealing temperature is
5°C lower than the melting
temperature
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24. Advantages
• The touchdown PCR reduces the primer-dimer formation capacity of
primers.
• It will also provide higher specificity by reducing the non-specific and
unwanted bindings of the primer to the template DNA.
• The technique is extremely useful in the templates having higher GC
contents.
Disadvantages
• Although it is highly specific, if not performed well the touchdown PCR
also gives non-specific results.
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25. COLD(Co-amplification at lower denaturation
temperature) PCR
• Common PCR will amplify
both the major (wildtype) and
minor (mutant) alleles with
the same efficiency, the ability
to easily detect the presence of
low-level mutations.
• COLD –PCR has the ability to
preferentially amplify and
identify minority alleles and
low-level DNA mutations in
the presence of excess
wildtype alleles is useful for
the detection of mutations.
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26. 26

27. Full COLD-PCR
Denaturation stage. DNA is denatured at a high
temperature – usually 94 °C .
Intermediate annealing stage. Allows hybridization
of mutant and wild type allele DNA to one another.
Because the mutant allele DNA forms the minority
of DNA in the mixture they will be more likely to
form mismatch heteroduplex DNA with the wild
type DNA.
Melting stage. These heteroduplexes will more
readily melt at lower temperatures. Hence they are
selectively denatured at the Tc.
Primer annealing stage. The homo-duplex DNA
will preferentially remain double stranded and not
be available for primer annealing.
Extension stage. The DNA polymerase will extend
complementary to the template DNA. Since the
heteroduplex DNA is used as template, a larger
proportion of minor variant DNA will be amplified
and be available for subsequent rounds of PCR.
Fast COLD-PCR
 Denaturation stage and Intermediate annealing
stages are skipped. This is because, in some
cases, the preferential amplification of the
mutant DNA is so great that ensuring the
formation of the mutant/wildtype heteroduplex
DNA is not needed. Thus the denaturation can
occur at the Tc, proceed to primer annealing,
and then polymerase-mediated extension.
 Each round of amplification will include these
three stages in that order. By utilizing the
lower denaturation temperature, the reaction
will discriminate towards the products with
the lower Tm – i.e. the variant alleles.
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28. Applications :
 Useful for the detection of mutations
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29. Advantages:
•Single-step method capable of enriching both known and unknown minority alleles
irrespective of mutation type and position.
•Does not require any extra reagents or specialized machinery. Therefore, the cost is not
increased.
•Better than conventional PCR for the detection of mutations in a mixed sample.
Disadvantages :
•A suitable critical temperature may not be available that differentiates between mutant and
wildtype DNA sequences.
•Restricted to analyzing sequences smaller than approximately 200bp.
•No guarantee that all low-level mutations will be preferentially enriched. 29

30. INVERSE PCR
• It is a variant of PCR that is used to amplify
DNA with only one known sequence .
• limitation of conventional PCR is that it
requires primers complementary to both
termini of the target DNA,
• It is commonly used to identify the flanking
sequences around genomic inserts.
• The inverse PCR method includes a series of
digestions and self-ligations with the DNA
being cut by a restriction endonuclease. This
cut results in a known sequence at either end
of unknown sequences.
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31. • Primers oriented in the reverse direction of the usual
orientation.
• The template for the reverse primers is a restriction
fragment that has been ligated upon itself to form a
circle.
• Target DNA - restriction endonuclease digestion.
• Self-ligation- gives a circular DNA ligation product.
• Target DNA is then restriction digested with a known
endonuclease.
• This generates a cut within the known internal
sequence generating a linear product with known
terminal sequences.
• Standard PCR is conducted with primers
complementary to the now known internal sequences.
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32. SIGNIFICANCE :
• Identification of unknown flanking regions. For example, the identification
and investigation of promoter and enhancer regions of DNA upstream or
downstream to the exon region can be possible by using the inverse PCR.
• In the site-directed mutagenesis.
• Construction of end specific probes for chromosome walking.
• It is further useful in the identification of unknown mutations such as gene
rearrangements, gene fusion, oncogenic gene arrangement on a
chromosome.
• The inverse PCR is the first choice for transposable element studies,
identification and characterization.
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33. RT (Reverse Transcriptase) PCR
• It is used for amplifying DNA from RNA.
• Reverse transcriptase reverse transcribes RNA into
cDNA, which is then amplified by PCR.
• The RT-PCR is broadly divided into two steps,
1. reverse transcription
2. amplification.
Depending upon that the RT- PCR can be performed by
two methods:
• One-step RT-PCR
• Two-step RT-PCR
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34. • Both the reverse transcription and the
amplification can be performed in the single
reaction.
• Contamination is very less in comparison with
two-step RT-PCR.
• The major limitation of the one-step RT-PCR :
1.the stock of cDNA
2.non-specific bindings and primer dimers
formation are also higher in this method because
one-step RT PCR utilizes only sequence-specific
primers.
• First reaction, the cDNA is synthesised from the RNA
for that reverse transcriptase
• In the second reaction, instead of reverse transcriptase,
the normal DNA polymerase along with other PCR
essentials are added into the tube.
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35. Applications :
• RT-PCR is widely used in expression profiling, to determine the
expression of a gene or to identify the sequence of an RNA transcript,
including transcription start and termination sites.
• If the genomic DNA sequence of a gene is known, RT-PCR can be
used to map the location of exons and introns in the gene.
• Scientists are working on ways to use RT-PCR in cancer detection to
help improve and monitor response to theraphy.
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36. REAL TIME PCR / Quantitative PCR
• A real-time polymerase chain reaction (real-time PCR), also known as quantitative polymerase chain
reaction (qPCR)
• key feature - amplification of DNA is detected in real time as PCR is in progress by the use of fluorescent
reporter.
• The fluorescent reporter signal strength is directly proportional to the number of amplified DNA.
• Based on the molecule used for the detection
1. Non-specific Detection using DNA Binding Dyes - that intercalate with any double-stranded DNA
2. Specific Detection Target Specific Probes - consisting of oligonucleotides that are labelled with
a fluorescent reporter
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37. RT-PCR can be divided into four stages:
• Linear ground phase - PCR is just starting,
fluorescent signal has not risen above background.
• Early exponential phase - fluorescent signal just rise
significantly above background, the cycle at which
occurs is called cycle threshold (Ct).
• Linear exponential phase (log phase) - PCR is in its
optimal amplification stage with doubling PCR
products in every cycle.
• Plateau phase - substrates are exhausted and Taq
DNA polymerase is in its end of life, fluorescent
signal will no long increase
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38. Non-specific Detection using DNA Binding Dyes -
that intercalate with any double-stranded DNA
PRINCIPLE :
• Nonspecific fluorescent dye SYBR Green bind to
double-stranded DNA, their fluorescence increases by
20–100-fold.
• As the amount of double-stranded DNA increases
during PCR process, the SYBR fluorescent signal
increases correspondingly.
LIMITATION
• SYBR can bind any double-stranded DNA,
even primer dimers . So it is critical to optimize PCR
reactions to amplify the target amplicon only.
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39. Specific Detection Target Specific Probes -
oligonucleotides that are labelled with a fluorescent reporter,
which permits detection only after hybridization of the probe
with its complementary sequence.
• TaqMan with a reporter fluorescent dye at 5′ end and a
quencher dye at 3′ end.
• When the probe is not hydrolyzed by Taq DNA polymerase,
reporter dye emitted fluorescent light is absorbed by
quencher dye .
• When probe is hydrolyzed by Taq DNA polymerase, the 5′
reporter dye is separated from quencher dye and thus 5′
reporter dye fluorescent light will be detected by RT-PCR
instrument.
• The released 5′ reporter dye signal is proportional to the
amount of PCR products.
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41. Real Time PCR VS Traditional PCR
• Real time PCR allows for the detection of PCR product during the early phases of
the reaction.
• Traditional methods use gel electrophoresis for the detection of PCR amplification in
the final phase or at end-point of the PCR reaction.
APPLICATIONS :
• Determination /monitoring of viral load
• Quantification of cancer genes
• Microarray verification
• Transgenic copy
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42. COLONY PCR
• Colony PCR is a rapid, high throughput
PCR method to determine the presence or
absence of the inserted DNA into plasmid
directly from the bacterial colonies.
• Colony PCR is a novel method in which by
designing the inserted DNA specific primers,
we can identify whether our DNA of interest is
inserted into the plasmid or not.
• Colony PCR is the modification of the
conventional PCR in which the bacterial
colonies are directly used as a PCR template.
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43. Primers used in Colony PCR:
The bacterial colony containing the plasmid can directly be amplified using two or three sets of primers.
• Insert specific primers bind to the specific location on both the side of the inserted DNA of our interest which
amplify the insertion sequence.
• vector-specific flanking primers, which amplifies the plasmid DNA other than the inserted DNA (flanking regions
on both the side of insert).It helps to determine the size of the insert. It expands regions other than the insert DNA.
• Orientation-specific primers are unique primers in which one primer binds inside the insert and another primer
binds to the plasmid DNA sequence (sequence other than the insert DNA).This types of primer set provide
information about the orientation of inserted DNA of our interest.
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44. Advantages of colony PCR:
 Identification of correct ligation and insertion of insert DNA into bacteria as well as yeast
plasmid.
 Colony PCR is a fast and reliable method for the screening of recombinants.
This method can easily be used for cDNA library screening
Disadvantages of colony PCR:
 Any mutation in the insert cannot be detected.
The sequence information can not be obtained by the colony PCR. we need to do sequencing for
the confirmation of the DNA transformation
The chance of false-positive results is high. Use only a few colonies, as many colonies increase the
chance of the non-specific bindings.
As an insert use short DNA sequences, longer DNA sequences increase the chance of non-specific
bindings and PCR reaction failure.
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45. ASYMMETRIC PCR
The reaction amplifies one DNA
strand in a double-stranded DNA
template.
Thus it is useful when amplification
of only one of the two
complementary strands is needed
such as in sequencing and
hybridization probing.
 The amount of primer for the
targeted strand is much more than
that of the non-targeted strand.
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46. Limitation :
Asymmetric PCR is not widely used because it has low reaction
efficiency and it is hard to optimize the proper primer ratios, the amounts
of starting material, and the number of amplification cycles.
Limiting the concentration of one primer lowers its melting temperature
below the reaction annealing temperature .
Recently, this process has been changed to be known as Linear-After-The –
Exponential-PCR (LATE-PCR) where the limiting lower concentration
primer has a higher melting temperature than the higher concentration
primer to maintain reaction efficiency.
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47. SITE DIRECTED MUTAGENESIS using PCR
• Introduce specific and intentional mutations to the DNA
sequence of a gene or any genetic products
• Short DNA primer contains the expected mutation and
is complementary to the template DNA so it can
hybridize with the DNA sequence of interest.
• Thus the amplified gene then contains the mutated site,
which are then incorporated into a host cell as a vector
and cloned.
APPLICATION : Study the function of a gene or protein,
or for creating variants of an enzyme with new and
improved functions
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48. METHYLATION SPECIFIC PCR
• It is one of the most commonly used methods for
gene/sequence-specific detection of DNA methylation.
• The DNA undergoes bisulfite conversion of cytosine to uracil
and then the methylated sequences are selectively amplified
with primers specific for methylation.
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49. Treat sample DNA with sodium bisulfite
Sodium bisulfite treatment converts
unmethylated cytosine to uracil
PCR primers are designed for methylated and
unmethylated DNA
Final products of unmethylated DNA will
contain Ts in place of methylated
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50. Reasons Variant
Reduce contamination caused due to non-specific binding (Primer
dimer , misprimed or false primed targets) leads to wrong
amplification.
If unknown flanking DNA sequence adjacent to gene of interest
If RNA is there
Amplifies one DNA strand in a double-stranded DNA
What is the amplified quantity with change in time
Whether amplification is taking place or not
More than one template as to be amplified at the same time.
One side sequence is known to design only one primer
Detect mutation
Introduce mutation
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NESTED PCR
HOT START PCR
TOUCHDOWN PCR
LIGATION MEDIATED PCR
INVERSE PCR
REVERSE TRANSCRIPTASE PCR
ASYMMETRIC PCR
REAL TIME PCR
MULTIPLEX PCR
ANCHORED PCR
COLD PCR
SITE DIRECTED MUTAGENESIS USING
PCR

51. PROBLEMS AND SOME SIMPLE SOLUTIONS
No PCR product
•Check the template DNA concentrations and dilute them if necessary.
•Try to lower the annealing temperature in the PCR cycle and run 5-10 cycles at very low
annealing and extension temperatures (42°C and 68°C for instance), then run another 30-
40 cycles at higher temperatures.
•Check the primer concentrations; if there are faint bands.
Bright bands in the well of the gel
•Caused by over amplification of the PCR product. For this, first dilute the template DNA
and then raise the annealing temperature (>55°C).
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52. Smearing of PCR products or multiple bands
•Use a low concentration of template DNA and increase the annealing
temperature by 2-5°C.
•Standardize the optimum magnesium concentrations by amplifying at varying
concentrations. Then, decrease the number of cycles.
Bands in the blank
•Check all the solutions carefully and change the solutions if there is any
uncertainty. Wash the pipettes thoroughly and check for any contamination.
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PCR in agriculture
 Product development include gene discovery , transformant identification, screening and characterization
 Seed quality control .
 Detection of pathogen
1. species-specific identification
2. multiplexing, allowing for identification of more than one species in a single reaction.
 Grain handling and grain processing industry
1. Unapproved events- To verify the presence or absence of genetically modified(GM) material
during importing /exporting.
2. GM content -to quantify the amount of GM present in product(grain)
3. Non-GM crop labeling
4. For presence of high value commodity –GM crop that conveys the characteristic like low phytate
maize ,soybean with altered oil profile.
 Nutrigenomics – linking genetic information to information about food that might be better /worse for a
particular condition , in detection of food allergens
 For protecting against adulteration and ensuring standardization of processed foods and herbal medicine.

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