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Quantification of Plasmid Standards

Digital PCR Applications Grant Program

Shirley Chu & Patricia HegerichApplications ScientistsLife Technologies

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Agenda

Introduction to Digital PCR Objectives of Study Results

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How Digital PCR Works

Use the number of negative PCR reactions to count the number of target molecules.

Digital PCR is an analytical technique for quantification of nucleic acid samples based on PCR amplification of single template molecules, without reference to a

standard curve.

Positive reactionsNegative reactions

Preparation Distribution PCR ReactionPoisson fit

and readout

gDNA or cDNA

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QuantStudio™ 3D Digital PCR 20K Chip

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QuantStudio™ 3D Digital PCR 20K Chip

1mm

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Simple Workflow With Minimal Sample Handling

Sealed System

AmplifyLoad ReadMix Analyze

SEALED SYSTEM

LIMITED HANDS-ON

MINIMAL SAMPLE LOSS

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Objectives of Study

Quantify absolute copy numbers of plasmids

Provide concentration for each plasmid

Provide a ratio between GMO and reference target genes for the maize GMO sample

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Sample #1 (NK – maize GM0)

TargetCopies per µL

ng/µL (calculated)

ng/µL (NIMC)

Target 1*Target 2

Target 2*Target 1

1 - AIGJQPQ 1.82E+08 0.632 - AIHSOVY 1.87E+08 0.58

1.081 0.9250.89

* Ratio calculations generated outside of the QS3D software

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Sample #2 (BAZS)

TargetCopies per µL

ng/µL (calculated)

ng/µL (NIMC)

3 - AIIM16 1.87E+09 10.29 29

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Single Target Precision of dPCR

0.01.02.03.0

1 : 80,000 1 : 800,000Co

pies

per

reat

ion

Dilution

Sample 1 / Target 1

Chip Dil. Group Target1 : 80,000 Singleplex 2.39 1.89E+08 1.79% 1.79%

0.28 2.25E+08 2.81%0.31 2.44E+08 2.76%

0.79 1.57E+08 1.92%0.86 1.71E+08 1.85%

1.33%

1.96%0.94%

Dilution

1 : 200,000

1 : 800,000 Singleplex

Multiplex

Assay Format

PrecisionCopies/ Reaction

Copies/ul

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Summary

dPCR on QuantStudio™ 3D is a highly precise method for the quantification of gene targets in a sample

dPCR can be used to quantify plasmid-GMO samples

Multiplex assay provides the same results as a Singleplex assay

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APPENDIX

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Digital PCR Workflow

Calculate sample dilution needed to achieve an estimated 1.0 -1.6 copies of the target per reaction

Perform dPCR Assays − Assay Mix

> 1x dPCR Master Mix> 1x Assay> Nuclease Free Water> Test Sample

Calculate concentrations for each assay and plasmid

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Example of dPCR Results Output

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Example of dPCR Data Export Output

[Results]Target Sample Copies/microliter CI Copies/microliter Precision Chips CommentTarget 1 NTC 3.075 2.397 -- 3.944 28.26% 2Target 1 Sample_1 1.82E+08 1.80E8 -- 1.84E8 0.94% 5Target 2 Sample_1 1.87E+08 1.86E8 -- 1.89E8 0.95% 5Target 3 NTC 0 NA NA 1Target 3 Sample_2 1.87E+09 1.84E9 -- 1.89E9 1.30% 2

[Replicates]Target Sample Dilution Chip Copies/Rxn CI Copies/Rxn Copies/microliter CI Copies/microliterTarget 1 NTC 1 China Metrology NK NT 0.003106 0.002421 -- 0.003984 3.075 2.397 -- 3.944Target 1 NTC 1 China Metrology 1 NTC_ 0 NA 0 NATarget 1 Sample_1 1 to 8E4 China Metrology 1B1_13 2.387 2.345 -- 2.429 2363 2321.0 -- 2405.0Target 1 Sample_1 1 to 2E5 China Metrology NK_17 0.7977 0.7827 -- 0.813 789.8 775.0 -- 804.9Target 1 Sample_1 1 to 2E5 China Metrology NK_17 0.8326 0.8173 -- 0.8481 824.3 809.2 -- 839.7Target 1 Sample_1 1 to 8E5 China Metrology 1C1a_1 0.2858 0.278 -- 0.2938 283 275.2 -- 290.9Target 1 Sample_1 1 to 8E5 China Metrology 1C1b 1 0.3069 0.2987 -- 0.3154 303.9 295.7 -- 312.3Target 2 Sample_1 1 to 8E4 China Metrology 1B2_13 2.297 2.255 -- 2.34 2274 2233.0 -- 2317.0Target 2 Sample_1 1 to 2E5 China Metrology NK_17 0.8013 0.7863 -- 0.8166 793.3 778.5 -- 808.5Target 2 Sample_1 1 to 2E5 China Metrology NK_17 0.9043 0.8881 -- 0.9208 895.4 879.3 -- 911.7Target 2 Sample_1 1 to 8E5 China Metrology 1C2b_ 0.359 0.3497 -- 0.3685 355.4 346.3 -- 364.9Target 2 Sample_1 1 to 8E5 China Metrology 1C2a_1 0.2889 0.2809 -- 0.2971 286 278.1 -- 294.2Target 3 NTC 1 China Metrology 2 NTC_ 0 NA 0 NATarget 3 Sample_2 1 to 2E6 China Metrology 2Ba_16 0.8874 0.8708 -- 0.9043 878.6 862.2 -- 895.4Target 3 Sample_2 1 to 2E6 China Metrology 2Bb_16 0.9969 0.9794 -- 1.015 987 969.7 -- 1005.0

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The QuantStudio™ 3D Digital PCR System is For Research Use Only. Not for use in diagnostic procedures.

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