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GMO Biosafety Research 2012, Vol.3, No.1, 1-7
http://gmo.sophiapublisher.com
6
Table 1 Specific primer sequences for the hiTAIL-PCR
Primer name
Primer sequences (5
'
-3
'
)
3-1SP1
ACCTCGTTAGACTCAACAGCAGTGG
3-1SP2
ACGATGGACTCCAGTCCGGCCTTCGTGTGAGGTATGCTTCTGTGACC
3-1SP3
TCCAATCCAGCGATTTCGGTTACTTTG
3-2SP1
TCCCGTCCTTTGTCTTCAATTTTGAG
3-2SP2
ACGATGGACTCCAGTCCGGCCCATAGTGAGCGAGGAAAGTATCGGGC
Specific primer
of 3
'
-end
3-2SP3
CTAATTTGATATGGTTGGCACTTGGCTCG
5-1SP1
CTAACGTCTCGAAGCACGCTGAGG
5-1SP2
ACGATGGACTCCAGTCCGGCCATGGGATAGCTGTGGTCAAGGCGCT
5-1SP3
GCTTCCCACTCTCTGAAGCTCTCTGCAT
5-2SP1
CTTCCCCTGGAGAGAGCGAGATTCT
5-2SP2
ACGATGGACTCCAGTCCGGCCCGTTATCCAGCTAAGCGCGAACTGC
5-2SP3
CCACGATCGACATTGATCTGGCTATCTTG
5-3SP1
ACCATGATATTTGGCAAGCAGGCAT
5-3SP2
ACGATGGACTCCAGTCCGGCCCTTGAGCCTGGCGAACAGTTTGG
5-3SP3
CAGGTAGCCGGATCAAGCGTATGCAG
c-1sp1
CCCCAGGTGTGATTCCTCCAAC
c-1sp2
ACGATGGACTCCAGTCCGGCCGTCGGTATTGGACCAAAGGAGGAGCT
Specific primer
of 5
'
-end
c-1sp3
GGTGTTTCAACATCTCTGGGGAAATCAAC
Table 2 The primer sequences for LD-PCR
Primer name
Primer sequences (5
'
-3
'
)
5
'
-F1
5
'
-F2
ATCGTGGCTGGCTCGAAGATACCTG
CACAACAATGGTGACTTCTACAGCGCG
5
'
-F3
GCTGCTCTGATGCCGCCGTGTT
5
'
-F4
ACCTGTCCGGTGCCCTGAATGAACT
Forward primer
5
'
-F5
CCTTTCAACTGCGCCGAATCAAGTAG
3
'
-R1
GAAACGACAATCTGATCCCAGCTTGC
3
'
-R2
AGAAACGTTCCTATTGGTGGTGGGTGT
3
'
-R3
CTGCGTGCAATCCATCTTGTTCAATC
Reverse primer
3
'
-R4
GCTTCCCACTCTCTGAAGCTCTCTGCAT
designed to detect the transformation events. The
primers MF-1 (5'-TGTGTACTTCAACTGTCTGC
TTAGC-3') and MR-2 (5'-CCCATCTTCTATCCAA
TCTAACCTC-3') were screened to detect the
specificity,
while
the
primers
MF-3
(5'-GAGGTTAGATTGGATAG AAGATGGG-3') and
MR-5 (5'-ATTCAAGAACTCCTT GGAGGTTGT-3')
were screened to verify the cotton genome. PCR
Reaction system was set as: 5 μL 5 × Go
Taq
buffer, 2 μL
dNTP (2.5 mmol/L) each primer 0.5 μL (10 μmol/L),
0.13 μL Go
Taq
(5 U/μL), 1μL Templates (50 ng/μL),
adding ultra-purified water up to total 25 μL. PCR
amplification
procedures
were
as
follows,
Pre-denaturation at 94 for 5min then 35 amplifying
℃
cycles with 94 for 30s, 60
℃
for 30 s, 72
℃
for 30
℃
s),
finally extension at 72 for 7 min.
℃
3.6.2 Sensitivity test
The GM sample DNA solutions with the concentrations
in weight percentage as 10.00% 1.00% 0.50% 0.10%
0.05% and 0% were prepared by using 100 ng/μL
genetically modified cotton samples DNA and 100 ng/μL
non-transgenic cotton DNA to conduct PCR
amplification with the transformation event-specific
primers; the sensitivity of the test-specific primers were
determined by 2.0% agarose gel electrophoresis. PCR
reaction system was as follows, 5 μL 5 × Go
Taq
buffer, 2 μL
dNTP (2.5 mmol/L), each primer 0.5 μL (10 μmol/L),
0.13 μL GoTaq (5U/μL), 1 μL Templates (100 ng/μL),
adding ultra-purified water up to total 25 μL. PCR
amplification
procedures
were
as
follows:
Pre-denaturation at 94 for 5
℃
min then 35 amplifying
cycles with 94 for 30
℃
s, 60 for 30 s, 72
℃
for 30
℃
s),
finally extension at 72 for 7 min.
℃