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GMO Biosafety Research 2012, Vol.3, No.1, 1-7
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5
be the cotton genome sequence by blasting the region
of the negative control sequence. These insertion sites
might map on the chromosome once the cotton whole
genome sequencing is completed.
According to the study of Pan et al (2003), the
sensitivity of the primer detection reaching 0.1% can
meet the requirements for detecting genetically
modified crop. On the basis of the 3' end flanking
sequence, 3' end of the transformation event-specific
detection methods of the transgenic cotton 06N-119
was established. PCR sensitivity analysis showed that the
sensitivity of primers MF-1/MR-2 was 0.05%, which the
detection limit was equivalent to 11 exogenous DNA
insertion copies. Therefore, the 3' end of the qualitative
PCR primer screened in this study had very good
stability, strong specificity, and high sensitivity, which
would be suitable for specific detection of genetically
modified cotton varieties (lines), and might improve and
enrich the methods for transgenic
Bt
cotton transformants
specific detection as well as provide a reference for
safety testing of transgene.
3 Materials and Methods
3.1The experimentalmaterials and enzymes and reagents
Transgenic cotton material 06N-119 and other tested
varieties such as Zhongmiansuo 41, GK12, and
non-genetically modified cotton Ji 668 are cultivated
or possessed in this laboratory.
Ex
Taq
DNA polymerase, LA
Taq
DNA polymerase
were purchased from TaKaRa Company; Go
Taq
DNA
polymerase purchased from Promega; DNA molecular
weight marker, dNTP were purchased Beijing
Quanshijin Biotechnology Co., Ltd.; primers were
synthesized by Sangon (Shanghai) limited company;
other biochemical reagents are imported from abroad
for repackaging or domestic analytical grade.
3.2 DNA extraction method
The genomic DNA of young cotton leaves was
extracted by using the CTAB method. DNA purity and
concentration was determined by UV spectrophotometer,
DNA solution was diluted to 50 ng/μL and 100 ng/μL
ready for use.
3.3 Obtaining the flanking sequence of the trans-
genic cotton
The 5' and 3' end of the flanking sequence of
transgenic cotton were determined following the
method hiTAIL-PCR of Liu et al (2007) combined
with long distance PCR. PCR products were
sequenced by Beijing Saino Genome Research Center
Co., Ltd., and the sequence alignment were completed
with CExpress software and then blasted in BLASTn.
3.4 HiTAIL-PCR reaction and primer designing
HiTAIL-PCR reaction system, procedure and random
primer designing followed the literature (Liu and Chen,
2007). 5' and 3' end specific primers were designed
based on Cry1Ac sequence; the 5' end specific primers
were designed based on the cotton genome sequence.
The specific primers of 5' and 3' flanking sequence for
amplification were shown in Table 1.
3.5 Long distance PCR (LD-PCR)
To verify the specificity of hiTAIL-PCR products,
forward and reverse primers were designed according
to the spliced sequence. Long-distance PCR
amplification was carried out by using different primer
combinations. The reaction system was as follows, F/R
primers 1.0 μL (primer concentration 10 μM) LA
Taq
of
DNA polymerase 0.25 μL (5 U/μL), 10× LA PCR
buffer 2.5 μL, dNTPs 4.0 μL (2.5 mM), 50 ng/μL cotton
genomic DNA 1 μL and double distilled water to make
up the volume to 25 μL. Reaction procedures was as
follows, Pre-denaturation at 94 for 1 min in advance,
℃
and then 30 amplifying cycles with 98 10
℃
s and 66
℃
15 min; finally extension at 72 for 10
℃
min. 5' and 3'
end of primer sequences of
cry1Ac
gene for LD-PCR are
shown in Table 2.
3.6 The specific PCR detection method for 3' end of
the transformation event
3.6.1 The specific verification
The specificity verification of PCR amplification was
carried out by employing the genomic DNAs extracted
from 12 GM rice samples, 4 GE cotton samples, 4 GE
soybean samples, 4 GE oilseed rapes, 2 GE wheat
samples and 1 GE beet PCR products were examined by
2% agarose gel electrophoresis.
The specific PCR primers based on between
exogenous insertion sequences close to the 3' flanking