Download Detection of genetically modified cotton seeds using PCR

Indian Journal of Biotechnology
Vol 11, April 2012, pp 176-181
Detection of genetically modified cotton seeds using PCR and real-time PCR
S Shree Vidhya, P H Ramanjini Gowda*, K N Yogendra, T M Ningaraju and T Salome
Department of Biotechnology, University of Agricultural Sciences, Bangalore 560 065, India
Received 3 June 2010; revised 8 April 2011; accepted 10 August 2011
Detection of genetically modified (GM) crops and products are necessary to comply with international labeling
regulations and to avoid spurious and unapproved GM planting. DNA based analytical tools involving PCR and real-time
PCR were used to detect GM cotton seeds. Four pairs of primers specific for CaMV 35S, Nos, nptII and cry1Ac genes were
used for the DNA detection of GM crop. The limit of detection in real-time PCR was found to be 0.1%. Evidently, these two
PCR techniques were successful in detecting transgenes in the DNA of GM cotton seeds.
Keywords: Detection method, GM cotton seeds, labeling regulations, PCR, real-time PCR
Introduction
The development of new technologies and
inventions in genetic engineering has given rise to
genetically modified organisms (GMOs). These
organisms carry genetic material that has been altered
by the insertion or deletion of genes in order to confer
pest resistance, herbicide tolerance or to improve the
quality of their produce. A number of these GMO
products and seeds are available in the markets today
after going through rigorous testing and labeling
procedures. Yet, there is concern amongst the general
public regarding the safety of consumption of
products derived from these GMOs. And
environmentalists are worried about the cross
contaminations of these GMOs with the local or
indigenous varieties of crops when both are being
cultivated simultaneously.
The infiltration of unapproved and spurious
varieties of genetically modified (GM) crop seeds into
the market has been the reason for controversies
regarding their acceptance amongst farmers because
planting of these varieties leads to crop failures and
huge commercial losses. Hence, there is a need for
GM testing and detection, which will help to identify
GMOs and stop the infiltration of spurious GM
varieties into the markets. GMO analysis is usually
carried out using protein and nucleic acid based
techniques, such as, enzyme linked immunosorbant
_________
*Author for correspondence:
Tel: +91-80-2330153 (Ext 263)
E-mail: [email protected]
assay (ELISA), lateral flow strip ELISA, polymerase
chain reaction (PCR), microarray, etc1-5.
The analytical techniques are often affected by the
quality and the quantity of the target analyte and,
hence, conventional PCR and real-time PCR methods
are widely accepted for this purpose because of their
specificity, sensitivity and reliability. The foreign
DNA insert in GMOs usually consists of a promoter,
a coding sequence and a terminator. The target
sequence is the most important factor that controls the
specificity of the PCR method. Screening of GMOs
using PCR is based on sequences for control elements
used in a majority of GM plants, such as, the
cauliflower mosaic virus (CaMV) 35S promoter or
terminator (P-35S or T-35S), Agrobacterium
tumefaciens nopaline synthase terminator (T-Nos),
and cloning vector genes coding for resistance to
ampicillin and neomycin/kanamycin (bla or nptII),
which are the targets for amplification. Using PCR,
construct-specific detection targets the junctions
between the adjacent elements of the gene construct,
such as, the region between the promoter and the gene
of interest. Similarly, event-specific detection targets
the junction at the integration locus between the
recipient genome and the inserted DNA6. Real-time
PCR provides a very convenient technique for rapid
and sensitive detection of these transgenics with very
limited amount of starting material. In real-time PCR,
the detection is based on the fluorescence
measurement emitted by the probes or the dyes used
during the amplification of the target.
VIDHYA et al: DETECTION OF GM COTTON SEEDS
Bt-Cotton was among the first GM crops to be
commercialized during the 1900’s at the global level
and was officially approved for sale in India in 2002.
It consists of a gene from the soil bacterium Bacillus
thuringensis (Bt), which provides resistance to
different bollworm species, a major pest in cotton
crops, and helps cotton growers benefit through
efficient pest control. India is the second largest
producer and consumer of cotton accounting for a
little over 21% of the global cotton production in
2008-2009, and is cultivated in an area of about 9.4 m
ha7,8. In this study, we have used the P-35S, T-Nos,
nptII and cry1Ac specific primers for the screening of
transgenic cotton using conventional PCR and realtime PCR (using SYBR Green I chemistry).
Materials and Methods
Seed Material
Three samples of transgenic cotton seeds, RCH2,
JK99 and R3, and non-transgenic cotton seeds, MRC
5156, were obtained from Dow AgroSciences India
Pvt. Ltd.
DNA Extraction and Quantification
Genomic DNA from the transgenic and nontransgenic cotton seeds were extracted using a
modified hexadecyltrimethylammonium bromide
(CTAB)-based method along with Qiagen’s Genomictip 20/G kit (Qiagen, Hilden, Germany) as per the
protocol provided. The extracted DNA was dissolved
in 1× TE buffer.
For sensitivity tests, different per cent transgenic
contamination standards (5, 1 & 0.1%) were prepared
by mixing appropriate amounts of transgenic seed
powder with non-transgenic cotton seed powder. A
mixture of 9.5 g non-GM cotton seed powder and
0.5 g GM cotton seed powder, resulting in a 5%
(w/w) sample was used as the starting material. 2 g of
this 5% (w/w) mixture was added to 8 g of the nonGM cotton seed powder giving a 1% (w/w) sample. A
0.5% (w/w) mixture was prepared using 5 g of the 1%
(w/w) sample and 5 g of the non-GM cotton seed
powder, while 8 g of non-GM cotton seed powder
was mixed with 2 g of 0.5% (w/w) sample giving
0.1% (w/w) sample9. DNA was extracted from these
mixtures using the same CTAB-Genomic-tip 20/G
method. The real-time PCR was carried out with the
DNA extracted from 5, 1 and 0.1% mass fraction
mixtures. The concentration of extracted DNA was
quantified by measuring the UV absorption at 260 nm
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using an Eppendorf Biophotometer. DNA was
checked for fragmentation by gel electrophoresis.
Working stocks of 100 ng/µL were prepared and
stored at –20°C for use.
PCR Analysis
PCR analysis of transgenic and non-transgenic
cotton was carried out using the published primers,
P-35S, nptII, T-Nos and cry1Ac. The primer
sequences and their amplicon size are shown in
Table 1. All the primers were synthesized by Sigma
Aldrich Chemicals Pvt. Ltd.
PCR was carried out in 25 µL final volume reaction
mixtures using 10× Taq DNA polymerase buffer with
15 mM MgCl2, Taq DNA polymerase (3 U/µL), dNTP
mix (2.5 mM solution) (Bangalore Genei),
0.25 mM of each primer and 100 ng of sample DNA.
The thermocycler (TECHNE, Touchgene Gradient,
UK) was programmed with initial denaturation at 94oC
for 3 min. The cycling conditions for 35 cycles were:
denaturing at 94oC for 30 sec, annealing at 55oC (nptII,
T-NOS and Cry1Ac) and 54oC (P-35S) for
40 sec, and extension at 72°C for 30 sec. The final
extension was at 72°C for 10 min. The PCR amplicons
were analyzed on 2% agarose/ethidium bromide gel
(Sigma Aldrich Chemicals Pvt. Ltd.) along with 100 bp
DNA ladder (New England BioLabs Inc.), visualized
by direct observation on a UV transilluminator and the
images were recorded using a gel documentation
system AlphaImager (Alpha Innotech).
Real-time PCR Analysis
To find out sensitivity in detection of GMO’s, realtime PCR was carried out using SYBR Green I
chemistry [iQ SYBR Green Supermix containing
100 mM KCl, 40 mM Tris-HCl (pH 8.4), 0.4 mM of
Table 1—Details of primers sequences and the respective
amplicon size used for qualitative PCR analysis of GM cotton
Primer
Sequence
(5'—3')
P-35S
F: GCTCCTACAAATGCCATCA
R: GATAGTGGGATTGTGCGTCA
nptII
F: GAGGCTATTCGGCTATGACTG
R: ATCGGGAGCGGCGATACCGTA
T-Nos
F: GAATCCTGTTGCCGGTCTTG
R: TTATCCTAGTTTGCGCGCTA
cryIAc
F: GCCAATGCCTCGTGATTGTTCTCTGC
R: GATTTGCGAGGCTGGCCAGCTCCACG
F: Forward; R: Reverse
[
Amplicon
size (bp)
19510,11
70012
18010,11
28013
178
INDIAN J BIOTECHNOL, APRIL 2012
each dNTP, 50 U/mL DNA polymerase (iTaq), and
6 mM MgCl2, SYBR Green I, 20 nM fluorescein, and
stabilizers; Bio-Rad, Hercules, CA) and analyzed by
an iCycler iQ Real Time Detection System (Bio-Rad).
P-35S, nptII and cry1Ac primers were used for this
analysis. The reactions were carried out at 95°C for
3 min, followed by 35 cycles of amplification:
denaturing at 94°C for 40 sec, annealing at 54°C
(P-35S and nptII) and 58oC (cry1Ac) for 30 sec and
extension at 72°C for 40 sec. The reactions were
further subjected to 91 cycles of 0.5°C increments
(30 sec each) beginning at 50°C for melting curve
analysis to confirm the specificity of the amplification
products. Thermocycling was performed in a final
volume of 25 µL (10.5 µL of water, 0.2 µM of each
primer, 1 µL of genomic DNA and 12.5 µL of 2X iQ
SYBR Green Supermix; Bio-Rad). The real-time PCR
analysis was performed with the 5, 1 and 0.1% GMO
dilution DNA standards along with the genomic DNA
of transgenic and non-transgenic cotton seed samples.
Results and Discussion
The transgenic construct usually contains the genes
of 35S promoter, nptII and Nos terminator. In the
present study, these genes were targeted in the DNA
samples of transgenic cotton seeds (RCH2, JK99 &
R3) and non-transgenic cotton seeds (MRC 5156) for
their presence using their specific primers. The
presence of cry1Ac gene was also checked with its
specific primers.
The primers specific to 35S promoter recorded a
PCR product of 195 bp (Fig. 1a) in all the three
transgenic samples. A study in the semi-quantitative
detection of GM grains conducted by Tozzini et al13
also showed similar amplification band size with 35S
promoter specific primers. An additional product of
about 490 bp was seen as a faint band on the gel with
35S primers. Since the cotton seeds used were from
hybrid plants, the parents may have different 35S
promoter sequences and, hence, two bands were
observed.
The PCR with Nos primers recorded 180 bp
products (Fig. 1b). Similar results with Nos primers
were obtained by Oraby et al10 in their work on
screening of food products for CaMV 35S promoter
and Nos terminator and also by Hardegger et al14 in
their study on quantitative detection of 35S and Nos
terminator. The amplification with nptII primers
recorded an amplicon size of 700 bp (Fig. 1c); similar
to the results observed by Surekha et al12 in their
Fig. 1—PCR amplifications using different primers: (a) PCR
amplicons of size 195 bp using P-35S primers, (b) amplicons of
size 180 bp using T-Nos primers, (c) amplicons of size 700 bp
using nptII primers & (d) amplicons of size 280 bp using cryIAc
primers. (M, 100 bp ladder; RCH2, JK99 and R3, transgenic
cotton; & MRC 5156, Non-transgenic cotton)
VIDHYA et al: DETECTION OF GM COTTON SEEDS
study on the development of transgenic pigeon pea
plants.
The primer pair cry1Ac, specific for the Bt gene in
cotton, yielded a PCR product of size 280 bp as shown
in Fig. 1d. The product was detected only in the
transgenic cotton seeds and not in the non-transgenics.
Further, the cry1Ac primer was less sensitive and
amplified a poor resolution fragment of 280 bp in
agarose gel on PCR run of R3 sample DNA. The light
amplification bands with cry1Ac specific primers may
be because the transgenic cotton had a Bt-event, such
as, a truncated cry1Ac, cry1Ab, cry1Ac+cry1Ab or
cry1C, which are approved for cultivation in India8.
Only the transgenic cotton showed amplifications
in all the PCR runs with the primers used and no
amplifications were recorded in the non-transgenic
seeds as shown in Fig. 1. Therefore, the primers for
35S promoter, Nos terminator, nptII and cry1Ac genes
can be used for routine screening and detection of
specific traits in GM cotton.
179
Real-time PCR allows highly sensitive detection of
the target with very little DNA. In this study, SYBR
Green I, a non-specific intercalating fluorescent dye,
was used to identify the amplified products. The
different percentage of transgenic contamination
standards (5, 1 , 0.1 & 100%) were used in each of the
real-time PCR runs along with the three transgenic
samples (RCH2, JK99 & R3) and one non-transgenic
sample (MRC 5156). Real-time PCR read-out is given
as the cycle threshold or threshold value (Ct), which
is the increasing fluorescence measured above a
predetermined set of cycles where the amplification
efficiency is constant15,16. The amplification curves
for each of the real-time PCR runs with specific
primers are indicated in Fig. 2.
The real-time PCR with P-35S specific primer
showed strong amplification with average Ct value of
21.6 for the 100% transgenic contaminated sample.
Similarly, for the 100% transgenic cotton DNA
Fig. 2—Real-time PCR amplification curves and melt curve charts for reactions performed using P-35S, nptII and cryIAc specific
primers.
INDIAN J BIOTECHNOL, APRIL 2012
180
standard sample, average Ct values of 21.58 and
25.62 were obtained with nptII and cry1Ac specific
primers, respectively. As evident from Table 2, the
Ct value was found inversely proportional to the log
of the initial amount of the target molecule. The
Ct values of the 5, 1 and 0.1% transgenic
contamination standards are higher compared to the
average Ct value of the 100% transgenic
contaminated sample, indicating that the GM cotton
seeds have higher copy number of the transgenes. The
non-transgenic cotton seeds (0% transgenic sample),
MRC 5156, did not record any Ct values in any of
real-time PCR runs, indicating the absence of
transgenes in their DNA. Three independent runs
were performed with two replicates in each case. The
Ct mean values and the respective standard deviation
(SD) and the relative standard deviation (RSDr)
measured for each run using the three primers are
indicated in Table 2.
Since SYBR Green I binds non-specifically to
double stranded DNA, the measured fluorescence
may have been contributed by the non-specific PCR
products or by primer-dimers. In order to differentiate
such artifacts from the specific PCR products, a melt
curve cycle was incorporated into the real-time PCR
program. A melt-curve is the relative decrease in
fluorescence plotted against the temperature as shown
in Fig. 2. The amplified products with each of the
specific primers are of the same length and, hence,
show melt peaks at about the same temperature. The
melting temperature is usually determined by the
product’s length, GC content, concentration of the
Table 2—The threshold value (Ct) of the transgenic contamination standard samples using the target specific primers
a. Real time-PCR with 35S primers
Samples
Replicates
Run 1
Ct
Run 2
Run 3
5%
1
2
1
2
1
2
1
2
1
2
23.66
23.83
25.33
25.85
26.6
26.56
21.36
21.77
0
0
23.53
23.76
25.49
25.67
26.78
26.44
21.45
21.85
0
0
23.79
23.34
25.22
25.72
26.86
26.35
21.67
21.55
0
0
28.28
28.43
29.41
29.5
30.41
30.62
21.82
21.37
0
0
28.36
28.67
29.87
29.63
30.39
30.55
21.65
21.58
0
0
28.59
28.34
29.35
29.86
30.75
30.68
21.77
21.34
0
0
c. Real time-PCR with cry1Ac primers
5%
1
27.35
2
27.49
1%
1
28.55
2
28.57
0.10%
1
29.76
2
29.87
100%
1
25.71
2
25.87
0%
1
0
2
0
27.81
27.43
28.65
28.67
29.64
29.65
25.47
25.68
0
0
27.66
27.56
28.32
28.75
29.43
29.83
25.59
25.38
0
0
1%
0.10%
100%
0%
b. Real time-PCR with nptII primers
5%
1
2
1%
1
2
0.10%
1
2
100%
1
2
0%
1
2
Mean
SD
RSDr
23.65
0.18
0.79
25.54
0.24
0.95
26.59
0.19
0.73
21.6
0
0
0.18
0
0
0.87
28.44
0.15
0.54
29.6
0.22
0.75
30.56
0.14
0.47
21.58
0
0
0.19
0
0
0.92
27.55
0.16
0.6
28.59
0.14
0.52
29.7
0.16
0.54
25.62
0
0
0.18
0
0
0.68
0
0
0
The relative standard deviation (RSDr) was calculated from the Ct values obtained from three independent real-time PCR runs under
repeatability conditions
VIDHYA et al: DETECTION OF GM COTTON SEEDS
dye, salt and the specific template in the reaction
tubes. Hence, the nonspecific products normally melt
at a much lower temperature than the specific
products which are longer in size1,17.
Conclusion
In the present study, GM cotton has been detected
for the presence of transgene using PCR and real-time
PCR. The protocol developed can be used to detect
the transgenes in most of the GM crops since they
contain either an antibiotic gene like nptII with Nos
terminator or 35S promoter in most of the gene
constructs.
The real-time PCR could detect the presence of the
transgene to an extent of 0.1% as also reported by
Hubner et al18. Hence, it is very useful in detecting the
transgene contaminations and also to follow the
biosafety regulations. Such screening methods will
also help to control and identify the illegal/spurious
GM seeds in the market and avoid losses to the
farmers cultivating them.
Acknowledgement
The authors are grateful to the Government of
Karnataka for its financial support to carry out the
experiments and Dow AgroSciences India Pvt. Ltd.
for providing the GM cotton seeds for the
experiments.
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