Transcript MALDI-TOF-based DNA authentication of rice

Use of SNP-DNA analysis in
authenticating Basmati rice
Summary
• DNA based authentication of basmati rice
• Background to Single nucleotide
polymorphisms (SNPs)
• Introduction to SNP genotyping using
MALDI-ToF MS
• Authentication of basmati rice using
MALDI-ToF MS-based SNP genotyping
• Launching a commercial genotyping service
Authentication of basmati rice
by DNA genotyping
• Micro-satellite markers
• Single nucleotide polymorphisms
Single nucleotide polymorphisms
Single nucleotide differences exist between
individuals, plant and animal varieties
and bacterial strains
Variety 1 TCGAAATGCATGCGCATGATT
Variety 2 TCGAAATGCATGCGCGTGATT
Single nucleotide polymorphisms
Authentication using SNPs
• PCR based SNP genotyping
• MALDI-ToF MS
based SNP genotyping
Assaying SNPs by MALDI-ToF MS
Once a SNP is identified, 3 primers are required to enable genotyping including:-
- Two PCR primers to amplify the region around the SNP
5’
3’
- One Extension primer which anneals directly adjacent to the SNP.
5’
3’
Assaying SNPs by MALDI-ToF MS
TCGAAATGCATGCGCATGATT
TCGAAATGCATGCGCGTGATT
Anneal primer
TTACGTACGCG
TCGAAATGCATGCGCATGATT
TTACGTACGCG
TCGAAATGCATGCGCGTGATT
+polymerase
+ddNTP’s
TTACGTACGCGT
TCGAAATGCATGCGCATGATT
TTACGTACGCGC
TCGAAATGCATGCGCGTGATT
Analyse extension products by MALDI-TOF MS
MALDI-TOF Mass Spectrometry
Samples mixed with a matrix in great excess and spotted onto a MALDI plate
and loaded into the Mass Spectrometer.
Sample spot is subjected to repeated pulses of a nitrogen laser at 337nm in a
vacuum which vaporises and ionises sample. Matrix absorbs the majority of
incident laser energy, preventing degradation or fragmentation of the sample,
and allows the vaporisation and ionisation of some of the substrate.
Delayed application of an electric
field causes ions to enter flight tube
and accelerate towards detector.
Delayed Extraction applies high
voltage electric pulse after
predetermined time delay to minimise
energy spread of ions. All ions gain
same kinetic energy, so larger ions
take longer to reach detector. This
variation in Time of Flight allows the
separation of ions on the basis of size.
Assaying SNPs by MALDI-ToF MS
120
C = 273 Da
100
Difference in peak
size reveals the SNP
allele for that rice
variety
T = 288 Da
A = 297 Da
80
G = 313 Da
60
40
Unextended
SNP primer
Extended SNP
primer
20
0
4300
4400
4500
4600
4700
4800
4900
5000
Multiplexing SNP assays
Quantitative SNP assays
MALDI-TOF-based
DNA authentication of basmati rice
Sponsored by the FSA
FSA sponsored work includes:• Completing a SNP database for the main
commercial varieties and their adulterants.
• Assessing the reproducibility of the
protocols to determine the uncertainty of
the method.
• Performing a limited survey of Basmati
Products being sold in UK supermarkets.
• Developing a Standard Operating Procedure
Basmati rice varieties
Traditional breeds
Basmati 386
Basmati 370
Dehradun
Ranbir Basmati
Taraori (HBC-19)
Hybrid
Basmati 385
Super Basmati
Pusa Basmati
Kasturi
Basmati 198
Common Adulterants
Pusa 169
Sabarmati
PR 106
Kali-much
Lakra
Saket 4
Parimal
Sherbati
Terricot
Assembling a SNP database
for Basmati varieties and adulterants
RICE VARIETY
LINEAGE
ory-122-1
ory-58-2
ory-262
ory-S0186
ory-S0157
ory-S0153
ory-462
ory-592
ory-599
ory-624-1
ory-668
Basmati 370 Derhadun Kernal PK370 l Pak 385
Pure Bred Pure Bred Pure BredPure Bred Hybrid
GA
AA
GG
TT
GG
GG
GA
GG
TC
GG
AG
GG
AA
GG
TT
GG
GG
GA
GG
TC
GG
AA
AA
AA
GG
TT
GG
GG
GA
AA
TC
GG
AA
GG
AA
GG
TT
GG
GG
Ga
GG
TC
GG
GA
GG
TT
GG
TT
GG
GG
GA
GG
CC
CC
GG
Pusa 1
Hybrid
Sherbati
Non-Bas
Super
Hybrid
Taraori
Pure Bred
AA
AA
GG
CC
CG
GG
GA
AA
CC
GG
GG
GG
TT
CC
CC
CC
CC
GA
GG
CC
GC
GG
GG
AA
GG
TT
GG
GG
GA
GG
TC
GG
AA
GA
AA
GG
TT
GG
GG
GA
AA
TC
GG
AA
Assessing reproducibility
Examples of survey work
SNP Genotyping using primer I
Primer
Primer + C Primer + T
120
100
80
Tilda basmati
Rice
60
40
ASDA basmati
rice
20
0
4300
4400
4500
4600
4700
4800
4900
5000
SNP Genotyping using 11 primers
A
B
C
D
E
F
G
H
I
J
K
Tilda D6 Asda D5 Kernal (a1)
AA
AA
AA
AA
AA
AA
GG
GG
GG
TT
CC
TT
GG
CC
GG
GG
GG
GG
GA
GA
GA
AA
AA
AA
TC
CC
TC
GG
GG
GG
AA
GG
AA
Tilda basmati rice behaves like traditional variety Kernal
Asda basmati rice is most like the basmati hybrid Pusa
Summary
MALDI-ToF MS based genotyping represents
an effective analytical approach to assess:• Product authentication using a diagnostic
panel of multiplexed SNP markers based
on an expanded database.
• Product adulteration, exploiting the
techniques ability to quantitate SNPs in
the 5-100% range.
SNP analysis as a QC tool
Marker
ory-122-1
ory-58-2
ory-262
ory-S0186
ory-S0157
ory-S0153
ory-462
ory-592
ory-599
ory-624-1
ory-668
Super
GG
AA
GG
TT
GG
GG
AG
GG
CT
GG
AA
Thai
GG
AA
GG
TT
GG
CC
AG
AA
CT
GG
AA
2003
GG
AT
CG
CT
CG
GG
AG
GG
CC
CG
AG
SNP analysis as a QC tool
S+T
GG
AA
GG
TT
GG
GC
AG
GA
CT
GG
AA
2003
GG
AT
CG
CT
CG
GG
AG
GG
CC
CG
AG
SNP markers revealed that
results with the 2003 crop
could NOT have arisen
from mixing
Super Basmati (S)
with Thai (T) rice
Providing a commercial genotyping service
THE gold standard
Applying cutting-edge DNA technology
to provide analytical services
for product authentication
to the food industry.
http://Authentigene.com
http://Authentigene.info
The Authentigene team
Prof. David Archer
Prof. Malcolm Bennett
Prof. Ian Connerton
Dr. Sean May
Prof. Gregory Tucker