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Table 14: Primer sequences and amplicon length in PCR-assays to detect GMOs
Target sequence |
Primer 1 (5' => 3') Primer 2 (5' => 3') |
Amplicon length (bp) |
Application (ID) |
Cycling parameter |
Ref. |
I.) Across interfaces |
Structural gene / regulatory sequence |
antisense PG P-35S |
AGGGGAAAGTGGAAAACCATC CCACTGACGTAAGGGATGACG |
427 |
Tomato (1) |
x |
12 |
antisense PG P-35S |
TTTGGAGCTAAGGGTGATGGA AGTTCATTTCATTTGGAGAGGACA |
472 |
Tomato (12) |
|
17 |
sense PG P-35S |
GAAGATCTGCATGGACCTGAAAA AGTTCATTTCATTTGGAGAGGACA |
478 |
Tomato (12) |
|
17 |
nptII P-nos |
GAACTCGTCAAGAAGGCGATA GTTCAAATGCGCCTAAGGTCA |
943 |
Tomato (12) |
x |
17 |
gene IIIA nos 3' |
CTACTGATTACGGTGCTGCTA TGAATCCTGTTGCCGGTCTTG |
658 |
Tomato (12) |
x |
17 |
P-35S PVX cp |
CCACTGACGTAAGGGATGACG CCAGTTCCATACCACTGGAGC |
502 |
Potato |
x |
5 |
P-35S CTP (of CP4 epsps) |
TGATGTGATATCTCCACTGACG TGTATCCCTTGAGCCATGTTGT |
172 |
Soybean (8) |
x |
24 |
P-35S dhfr |
ATCATTGCGATAAAGGAAAGGC CTGCCTCCGACTATCCAAACCA |
540 |
Corn |
x |
4 |
P-35S dhfr |
ATCATTGCGATAAAGGAAAGGC AAAGCCACAAAAGTCCCAT |
840 |
Corn |
x |
4 |
CP4 epsps nos 3' |
nda nda |
213/314 |
Soybean (8) |
x |
13 |
Regulatory sequence / regulatory sequence |
P-35S nos 3' |
CAATCCCACTATCCTTCGC
CATCGCAAGACCGGCAACAG |
890 |
Tomato (12) |
x |
17 |
Structural gene / structural gene |
cat lacZ |
TTTGTATTCTGAGCATAGTGA ATAGCGACGAGAGTTAG |
623 |
Microorganism |
x |
10 |
kat A cat |
CAGCGACTTGAGAAAAACGAGTG TGTCAGATAGGCCTAATGACTG |
1321 |
Microorganism |
x |
9, 20 |
nptII ocd |
TATCGCCTTCTTGACGAGTTC CTGTGGCGGGAACTCCACGA |
401 |
Tomato (12) |
x |
17 |
ocd gene IIIA |
CGATCCTGAGCGACAATATGA TAGCAGCACCGTAATCAGTAG |
660 |
Tomato (12) |
x |
17 |
PVY-cp nptII |
nda nda |
ca. 1800* |
Potato§ |
x * |
22 |
II.) Within single structural genes |
als |
CAGGTCAAGTGGCACGTAGGATG GGCTGCTTGTTCTTTCCAATCT |
642 |
Cotton |
|
14 |
aphIV (hygromycin-phosphotransferase) |
CGCCGATGGTTTCTACAA GGCGTCGGTTTCCACTAT |
839 |
Potato |
x |
8, 20 |
barnase |
CTGGGTGGCATCAAAAGGGAACC TCCGGTCTGAATTTCTGAAGCCTG |
160 |
Corn (21) |
x |
16 |
barstar |
TCAGAAGTATCAGCGACCTCCACC AAGTATGATGGTGATGTCGCAGCC |
235 |
Corn (21) |
x |
16 |
gus |
TCCGTAGAAACCCAACC GCTAGCCTTGTCCATTG |
674 |
Papaya (26?) |
x |
25 |
gus |
ACGTCCTGTAGAAACCCCAA CCCGCTTCGAAACCAATGCC |
1097 |
Alfalfa |
x |
1 |
nptII |
GAACAAGATGGATTGCACGC GAAGAACTCGTCAAGAAGGC |
785 |
Alfalfa |
x |
1 |
nptII |
nda nda |
506 |
Tom.paste (51, 54) |
x |
11 |
nptII |
GGATCTCCTGTCATCT GATCATCCTGATCGAC |
173 |
Tom (1), Pot, SuB |
x |
12,18 |
nptII |
nda nda |
512 |
Tomato (1) |
x |
19 |
nptII |
GGTGCCCTGAATGAACTG TAGCCAACGCTATGTCCT |
ca. 500 |
Microorganism§ |
x |
21 |
nptII |
CTCGACGTTGTCACTGAAGCGGGAAG AAAGCACGAGGAAGCGGTCAGCCCAT |
489b |
Microorganism§ |
x |
23 |
nptII |
CCGACCTGTCCGGTGCCC CCGCCACACCCAGCCGGCC |
475 |
Soybean (8) |
x |
15 |
pat (synthetic?) |
nda nda |
nda |
Rapeseed§ (66?) |
|
3 |
pat (synthetic?) |
nda nda |
552 |
Corn (15?) |
|
19 |
pat (synthetic) |
nda nda |
nda |
Rapeseed§ (66?) |
|
2 |
pat (synthetic) |
nda nda |
532 |
Corn/Rapeseed§ |
|
6 |
PG (polygalacturonase) |
CGTTGGTGCATCCCTGCATGG GGATCCTTAGAAGCATCTAGT |
180 (380)a |
Tomato (12) |
|
17 |
PG (polygalacturonase) |
nda nda |
427 |
Tomato (12) |
|
19 |
II.) Within single regulatory sequences |
P-35S |
GCTCCTACAAATGCCATCA
|
195 or 390 195 |
Tom (1), Soy (8), Corn, SuB, Potato |
x |
18,19 |
P-35S |
nda nda |
nda |
Rapeseed§ (66?) |
|
3 |
P-TA29 |
CTTTTTGGTTAGCGAATGC CTACCATGGTAGCTAATTTC |
880 |
Tobacco |
x |
7 |
T-nos ( = nos 3') |
GAATCCTGTTGCCGGTCTTG TTATCCTAGTTTGCGCGCTA |
180 |
Soybean (8), Pot. |
x |
18 |
Tom, Tomato; Pot, Potato; SuB, sugarbeet.
* cycling parameters had to be adjusted to the unusual amplicon size.
a) use of these primers resulted in a 380 bp amplicon in control lines, and
an additional 180 bp product in transgenic lines (no intron).
b) This amplicon was the result of the second set of primers in a nested
PCR; the size of the first product was 753 bp.
§ DNA was extracted from the soil (degraded plant material, or
microorganisms).
x Cycling parameter described
References:
1, Blake et al. (1991); 2, Ernst et al. (1996); 3, Feldmann et al. (1996);
4, Golovkin et al. (1993); 5, Jongedijk et al. (1992); 6, Kirchhof et al.
(1996); 7, Kriete et al. (1996); 8, LMBG-Methodensammlung (1996); 9,
LMBG-Methodensammlung (in press); 10, LMBG-Methodensammlung (in preparation);
11, Meyer, G.; Hanse Analytik, Bremen (personal communication); 12, Meyer
(1995a); 13, Padgette et al. (1995); 14, Petition from DuPont for
genetically modified cotton (1995); 15, Petition from Monsanto for
herbicide-tolerant soybean (1993); 16, Petition from PGS for SeedLink®
corn (1995); 17, Petition from Zeneca for genetically modified tomatoes
(1995); 18, Pietsch et al. (1997); 19, Pietsch and Waiblinger, (1996);
20, Schulze et al. (1996); 21, Smalla et al. (1993); 22, Stax et al. (1994);
23, Tsushima et al. (1995); 24, Wurz and Willmund, (1997); 25, Yang et al.
(1996)
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