DNA Quantitation
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Transcript of DNA Quantitation
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
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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