Use of SNPDNA analysis in authenticating Basmati rice

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Use of SNP-DNA analysis in authenticating
Basmati rice
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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

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Authentication of basmati rice by DNA genotyping

• Micro-satellite markers
• Single nucleotide polymorphisms

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Single nucleotide polymorphisms
Single nucleotide differences exist
betweenindividuals, plant and animal varieties
and bacterial strains
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Authentication using SNPs
Single nucleotide polymorphisms

• PCR based SNP genotyping
• MALDI-ToF MS based SNP genotyping

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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
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Assaying SNPs by MALDI-ToF MS
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MALDI-TOF Mass Spectrometry Samples mixed with a
matrix in great excess and spotted onto a MALDI
plate and loaded into the Mass Spectrometer. Samp
le 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.
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Assaying SNPs by MALDI-ToF MS
C 273 Da T 288 Da A 297 Da G 313 Da
Difference in peak size reveals the SNP allele
for that rice variety
Unextended SNP primer
Extended SNP primer
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Multiplexing SNP assays
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Quantitative SNP assays

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MALDI-TOF-based DNA authentication of basmati
rice

• Sponsored by the FSA

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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

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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
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Assembling a SNP database for Basmati varieties
and adulterants
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Assessing reproducibility
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Examples of survey work
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SNP Genotyping using primer I
Primer
Primer T
Primer C
Tilda basmati Rice ASDA basmati rice
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SNP Genotyping using 11 primers
Tilda basmati rice behaves like traditional
variety Kernal Asda basmati rice is most like the
basmati hybrid Pusa
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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.

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SNP analysis as a QC tool
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SNP analysis as a QC tool
SNP markers revealed that results with the 2003
crop could NOT have arisen from mixing Super
Basmati (S) with Thai (T) rice
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Providing a commercial genotyping service
Applying cutting-edge DNA technology to provide
analytical services for product authentication
to the food industry. http//Authentigene.com ht
tp//Authentigene.info
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The Authentigene team

• Prof. David Archer
• Prof. Malcolm Bennett
• Prof. Ian Connerton
• Dr. Sean May
• Prof. Gregory Tucker