National Center for Environmental HealthCenters for Disease Control and Prevention

DNA Quantitation

NBS Molecular Training ClassJune 28 – 30, 2011

Suzanne Cordovado, PhDMolecular Quality Improvement Program, CDC

DBS DNA Quantitation: When and How?

• Typically unnecessary for routine PCR based assays

• Important for validating new assay limits and sensitivity– Too little DNA may lead to allele drop-

out (not always obvious)– Some assays require a minimum DNA

quantity

• Absorbance– Measure not specific to DNA – DBS DNA contains contaminants resulting in

inaccurate measures– Not recommended for DBS DNA

• Pico-green – Measure specific to double stranded DNA– Recommended for DBS DNA

• Quantitative PCR– Measure specific to amplifiable DNA – PCR inhibitors may underestimate DNA

concentration– Different genomic targets may give different

concentrations– Recommended for DBS DNA

DBS DNA Quantitation: When and How cont.

DNA Quantitation: Absorbance• DNA absorbs UV light at 260nm

• Spectrophotometer reads the amount of light that passes through the sample to determine the amount of DNA present

• Disadvantage: cannot distinguish between dsDNA, ssDNA, RNA or aromatic organic compounds

• Proteins absorb UV light near 280nm• A sample with little protein contamination will

have A260/280 ratio of 1.8.

• Fluorescent dye binds to dsDNA• Absorbs light at 480nm (blue) and emits light

at 520nm (green)• Using a known standard curve, the amount of

light emitted can be used to calculate DNA quantity

• Unincorporated dye does not absorb light at 480nm

• Contaminants typically do not impact this measure

DNA Quantitation: Picogreen

DNA florescence is measured during each cycle of amplification which is used to calculate quantity.

DNA Quantitation: quantitative PCR

quencher

The fluorescent labeled probe anneals to the genomic DNA. The label is not visible prior to amplification.Taq polymerase begins to synthesize the new DNA strand. Once the enzyme encounters the probe, the exonuclease activity of the Taq will degrade the probe allowing visualization of the label.

reporter

Taq

Standard curve amplification – 8 points, each run in duplicate (pink curves) Unknown sample amplification – 4 samples, each run in duplicate (blue curves)

Unknown (~10.8 ng/µL)

Unknown (~0.27 ng/µL)

Unknown (~4 ng/µL)

Unknown (~3 ng/µL)

qPCR: RNaseP Amplification Plot

DBS DNA Extraction Evaluation Decision Tree• Evaluate DNA quantitation methods

– Spectrophotometer (Nanodrop)– Picogreen– qPCR RNase P

• Evaluate qPCR standard curve sources– Pooled genomic DNA– Lymphocyte DNA– Plasmid DNA

Public Health Collaborators: California, Massachusetts, New York, Texas,

Washington, Wisconsin and CDC

DBS DNA Extraction Evaluation Decision Tree

Partners

Conc

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ng/u

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DBS DNA Extraction by Method, and Age of Spot /Storage Conditions

Picogreen and qPCR using RNAseP (genomic DNA standard curve) were most similar

Average DNA Concentration from 20 Newborn DBS

Qiagen 5Prime Omega Manual Omega KF6 mo/opt 6 mo/opt 6 mo/opt 6 mo/opt

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00PicoGreen avg_conc ng/ulRNAseP_pooled gDNA avg_conc ng/ulRNAseP_Lym DNA avg_conc ng/ulRNAseP_plasmid DNA avg_conc ng/ulNanodrop avg_conc ng/ul

Average DNA Concentration from 20 Newborn DBS

Methods: PicoGreen, qPCR, and Spectrophotometry

PicoGreen RNAseP_pooled gDNA RNAseP_Lym DNA RNAseP_plasmid DNA Nanodropavg_conc ng/ul avg_conc ng/ul avg_conc ng/ul avg_conc ng/ul avg_conc ng/ul

Qiagen 3.06 3.13 1.85 2.71 13.715Prime 2.81 2.99 1.68 2.60 22.13Omega Manual 3.68 3.90 2.23 3.45 12.09Omega KF 3.13 3.08 1.83 2.68 28.84

Extraction method

DNA Quantitation Conclusions• Spectrophotometry overestimates

the quantity of DNA extracted from a DBS

• qPCR and PicoGreen DNA quantitation methods gave similar results and were consistent with the expected outcome