July 27, 2001

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CCQM 27 July 2001NIST/LGC Pilot study
proposalDNA Quantification
CCQM/02-08
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DNA Quantitation Pilot Study

• Purpose - to verify the ability of a laboratory
  to determine the relative ratios of specific DNA
  sequences
• How - provide plasmid based DNA testing materials
  and calibration materials

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The idea!
John Butler Gary Gilliland Marcia Holden David
McDowell Helen Parkes Allison Woolford
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Nucleic Acid measurements

• Sequence - analysis of target base sequence
• Length (bases) - size important for
  identification of correct target
• Amount - quantification of specific nucleic acids

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DNA quantification

• Real time PCR
• LightCycler
• ABI7700
• iCycler
• PCR
• Gel analysis (visual/densitometry)
• CE

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Quantitative DNA Applications

• GM detection - relative ratios of GM insert vs
  housekeeping gene to quantify amount of GM
  material present in particular food ingredient
• Viral load - HIV detection
• Pharmaceutical discovery - relative gene
  expression in response to drug exposure

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DNA quantification

• Real time PCR
• LightCycler
• ABI7700
• iCycler
• PCR
• Gel analysis (visual/densitometry)
• Capillary Electrophoresis

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Generic ratio system

• Q-PCR testing system will not specific for any
  particular type of detection
• The amplifiable inserts will not resemble any
  known gene
• Useful across platforms and methods

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PCR (Polymerase Chain Reaction)

• PCR copies specific sections of DNA - primers to
  target
• genetic photocopying
• million fold amplification to easily detectable
  levels

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GM soya and maize detection multiplex PCR /
LabChip CE
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Real time analysis - LightCycler
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Real time amplification of GM maize - Scorpion
detection
Key A Maize taxon B Maize bt C negative
controls
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Proposed CCQM Pilot Study
Set of samples prepared at various ratios and
sent out for testing by labs
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Probe Possibilities
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Technologies Covered by Proposed Study
Generic primer pr 1 Same size product
Generic primer pr 2 Different size product
Different specific primers Different size product
Quantitative PCR
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TaqMan probes
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?
?
?
Scorpion probes
?
?
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Molecular Beacons
?
X
?
SYBR Green
PCR with Separation
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X
?
Gel/eyeballing
X
?
?
Gel/densitometry
X
?
?
Capillary electrophoresis
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Pilot Study Materials

• Calibration curve
• Materials consisting of several defined insert
  ratio which can be run in parallel with unknowns
• Unknowns
• Several materials to be analyzed by the
  measurement laboratories
• Recommended primer and probe sequences

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Timescale of activity