ITALY TOUR 2008 April Amongst the Ruins. San Bernardo in Rome, old convent.
Coextra Rome 2008
Transcript of Coextra Rome 2008
•Gene of Interest
• Promoter (e.g.. NOS)
• Terminator (e.g. 35S)
• Selectable marker (e.g. GFP)
Plant Transformation Technology
Ecological demands
(Increased Biomass)
Agricultural
Sustainability
(Resistance)
“Pro et Contra”
Food quality
(Phyto-ceuticals)
Research
(Gene function)
DNA replication
BART Reaction Scheme
PPi + APS ATP + SO4-
ATP sulphurylase
AMP + PPiATP + Luciferin O2 CO2+ ++Luciferase
0
5
10
15
20
25
30
1 6 11 16 21 26 31 36
20
25
30
35
40
2000 (0.%5)
2000 (1%)
5000 (2%)
4000 (5%)
35S-p
ADH ref
qPCR Analysis of Bt11 maize GM reference series
0
5
10
15
1 6 11 16 21 26 31 36
0
5
10
15
20
25
30
1 6 11 16 21 26 31 36
300 (0.1%)
2000 (0.%5)
300 (0.1%)
2000 (0.%5)
2000 (1%)
5000 (2%)
4000 (5%)
NOS-t
Rn
Cycle
0
5
10
15
20
25
30
35
0 0.1 0.5 1 2 5
Ct
QPCR Ct values for GM titration series
ADH CAMV 35S-p NOS-t
0, 0.1, 0.5, 1, 2, 5 %, 0,0.1,0.5,1 2, 5 % 0,0.1,0.5,1 2, 5 %
0 0.1 0.5 1 2 5
ADH 35s NOSt
1200
1400
1600
1800
0
200
400
600
800
1000
1200
1400
1600
1 11 21 31 41 51 61 71 81
All 105
104 (5%)
5 x 103 (2%)
2 x 103 (1%)
900 (0.5%)
ADH1
35s
LAMP-BART Analysis of Bt11 maize GM reference seriesIntensity
0
200
400
600
800
1000
1 11 21 31 41 51 61 71 81
900 (0.5%)
200 (0.1%)
NOSt
0
200
400
600
800
1000
1200
1400
1 11 21 31 41 51 61 71 81
104 (5%)
5 x 103 (2%)
2 x 103 (1%)
900 (0.5%)
200 (0.1%)
Intensity
0
10
20
30
40
50
60
70
0 0.1 0.5 1 2 5
ADH 35s NOSt
Tm
ax
LAMP-BART Tmax values for GM titration series
ADH 35s NOSt
ADH CAMV 35S-p NOS-t
0, 0.1, 0.5, 1, 2, 5 %, 0,0.1,0.5,1 2, 5 % 0,0.1,0.5,1 2, 5 %
0
200
400
600
800
1000
1200
1400
1600
1800
1 11 21 31 41 51 61 71 81
Bt11 (5%)
ADH
35S
NOSt
1200
1400
1600
1800Roundup (5%)
ADH
35S
NOSt
Specificity of GM LAMP-BARTintensity
0
200
400
600
800
1000
1 11 21 31 41 51 61 71 81
0
200
400
600
800
1000
1200
1400
1600
1800
1 11 21 31 41 51 61 71 81
Mono810 (100%)
ADH35S
NOSt
ADH
intensity
λ DNA standard 5 to 50ng
Promega Wizard
Nucleon Phytopure
DNA integrity and quantification
NanoDrop
CTAB
0, 0.1, 0.5, 1 2, 5 %
Comparison of genomic DNA
quantifications (density vs. spectra)
200
250
0
50
100
150
Promega Phytopure CTAB
Gel NanoDrop
ng/ ul
DNA Quality vs. Amplification Linearity & Reproducibility
0
10
20
30
40
50
60
70
80
90
Phytopure0
10
20
30
40
50
60
70
80
90
Promega Wizard 35S-p NOSt
70
80
90
CTAB
LAMP-BART QT-PCR
copy No
1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06
0
10
20
30
40
50
60
70
1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06 1.E+01 1.E+02
10x10x
ADH1
35s
All 105
2 x 103 (2%)
5 x 103 (5%)
1 x 103(1%)
5 x 102(0.5%)
Portable solution for GM detection (PDQ)
NOSt
5 x 102(0.5%)
102(0.1%)
2 x 103 (2%)
5 x 103 (5%)
1 x 103(1%)
5 x 102(0.5%)
102(0.1%)
90
100
Linearity of CHELEX-Saline extracted DNA in LAMP (10min DNA prep)
qPCR
0
10
20
30
40
50
60
70
80
10 100 1000 10000
CHELEX - SALINE
35S
NOSt
Summary
GM LAMP-BART
A simple rapid technique
Quantifiable
Portable
Sensitive & reproducible
Specific
Good value