Applied 

Genetics

Digital PCR and 

Quantitation

Ross Haynes

Research Biologist, Applied Genetics Group

Forensics@NIST 2012 Meeting

Gaithersburg, MD

November 28, 2012
Applied 

Genetics Agenda

• Why quantitate with qPCR?

• How digital PCR Will Help Quantitation

• Quantitative PCR versus Digital PCR

• Digital PCR at NIST

• Instruments at NIST

• Benefits of digital PCR
Applied 

Genetics Why Quantitate with qPCR

• Forensic samples often have non-human DNA

– Forensic standards require human specific DNA 

quantitation 

– qPCR measures specific (e.g. human) DNA targets

Pet Hair
Applied 

Genetics Why Quantitate with qPCR

• STR kits have a narrow working range for 

amount of DNA (0.5 ng to 2 ng DNA)

Too little DNA

 incomplete profile

Too much DNA

 artifacts/noise
Applied 

Genetics How dPCR Will Help Quantitation

• Predicted Major Uses of Digital PCR:

– Quantify higher order reference materials

• Standard Reference Materials

– Quantify calibrant materials for qPCR

• Manufacturers of calibrant materials

– Quantify DNA solutions for critical and sensitive 

processes

• E.g. Next Generation Sequencing
Applied 

Genetics qPCR

• Calibrant concentration is independently 

determined (e.g. UV spectrophotometer)

• Prepare a dilution curve of calibrant

10,000 

pg/uL

1,000 

pg/uL

100 

pg/uL

10 

pg/uL
Applied 

Genetics qPCR

• Use calibration dilutions plus samples of 

unknown concentration as template for qPCR

• Thermal cycle and measure florescence signal 

after each cycle of PCR

10,000 

pg/uL

1,000 

pg/uL

100 

pg/uL

10 

pg/uL

? 

pg/uL
Applied 

Genetics qPCR

• Apply a threshold while 

florescence signal is in 

exponential phase

• Determine point where 

florescence signal 

crosses threshold (Ct)

10,000 

pg/uL

1,000 

pg/uL

100 

pg/uL

10 

pg/uL

? 

pg/uL

-1

0

1

2

3

4

5

0 10 20 30 40

Delta Rn

Cycle

qPCR with Calibration 

curve

Ct=21.59 Ct=24.90 Ct=28.22 Ct=31.54 Ct=26.90
Applied 

Genetics qPCR

• Log transform concentration

• Plot Log(conc.) vs Ct

Conc. (pg/μL) Log(conc.) Ct 

10,000 4 21.59

1,000 3 24.90

100 2 28.22

10 1 31.54

unknown unknown 26.90

20

22

24

26

28

30

32

34

1 2 3 4

CT

Log(concentration) (pg/uL)

Calibration Curve
Applied 

Genetics qPCR

• Log transform concentration

• Plot Log(conc.) vs Ct

Conc. (pg/μL) Log(conc.) Ct 

10,000 4 21.59

1,000 3 24.90

100 2 28.22

10 1 31.54

unknown 2.40 26.90

20

22

24

26

28

30

32

34

1 2 3 4

CT

Log(concentration) (pg/uL)

Calibration Curve
Applied 

Genetics qPCR

• Log transform concentration

• Plot Log(conc.) vs Ct

Conc. (pg/μL) Log(conc.) Ct 

10,000 4 21.59

1,000 3 24.90

100 2 28.22

10 1 31.54

251 2.40 26.90

20

22

24

26

28

30

32

34

1 2 3 4

CT

Log(concentration) (pg/uL)

Calibration Curve
Applied 

Genetics qPCR “Goldilocks Zone”

• Very high and very low concentrations do not fit 

on the line

• Best data obtained from the middle

http://www.sabiosciences.com/pathwaymagazine/pathways7/designing-validating-real-time-pcrprimers.php

Very high 

concentration

Very low 

concentration

Best data
Applied 

Genetics qPCR

• Relative quantitation between 

calibrant of known concentration 

(aka standard) and samples of 

unknown concentration

– Just as using a tape measure is a 

relative measurement if the calibrant 

is inaccurate the measurement will 

be inaccurate

• Spectrophotometer measures 

everything that absorbs at 

260 nm (i.e. DNA, RNA, protein, 

monomers)

22 inches

http://vickiwelsh.typepad.com/field_trips_in_fiber/tips/
Applied 

Genetics

1) Create a dPCR

PCR mastermix

as if for qPCR

2) Aliquot 

across 100s or 

1000s of wells

3) Thermal cycle as if for 

qPCR & count wells with 

detectible amplification at 

any cycle

4) Use Poisson statistics to 

determine concentration of 

starting material

? 

pg/uL
Applied 

Genetics

1) Create a dPCR

PCR mastermix

as if for qPCR

2) Aliquot 

across 100s or 

1000s of wells

3) Thermal cycle as if for 

qPCR & count wells with 

detectible amplification at 

any cycle

4) Use Poisson statistics to 

determine concentration of 

starting material

? 

pg/uL

Number 

of copies =

Number 

of wells ln( )

Number 

of wells

Number of 

negative wells

Volume of all PCR reactions

Concentration 

(c/μL)
Applied 

Genetics Range of Concentrations

• Saturated

Every well has at least 

one copy

• Binary detection

Calculate concentration

• No amplification

< 1 copy/total volume

Concentration increases 
Applied 

Genetics dPCR

• Absolute quantitation of target sequence

• Relies on PCR amplification 

– Only detects specific target DNA or RNA

– Will not detect proteins or monomers 

– Will not detect fragmented or degraded DNA 

molecules

– Affected by PCR inhibitors 
Applied 

Genetics Comparison

Quantitative PCR Digital PCR

Quant of unknown is based on amplifiable DNA

Quant is based on a 

previously characterized 

material

(a.k.a “calibrant”)

Quant is based on Poisson 

sampling statistics

(i.e. calibrant free)

Samples must be bracketed 

by calibrant dilution curve

Samples must be within 

“digital” range 

Older technology 

Widely accepted

New technology 

Gaining acceptance

Currently less expensive Currently more expensive
Applied 

Genetics dPCR at NIST

• Digital PCR has been used at NIST:

– To certify the concentration of a Standard Reference 

Material for Cytomegalovirus SRM 2366

• Evaluated using dPCR to measure the 

concentration of the SRM 2372: Human DNA 

Quantitation Standard

– Further validation of targets required
Applied 

Genetics Instruments at NIST

• Fluidigm BioMark

– Array based PCR reactions

– Gathers real-time data 

• Better for validation of PCR 

conditions

• Bio-Rad QX100

– Emulsion based PCR reactions

– End-point data

• Less expensive

• Better statistics
Applied 

Genetics Benefits of dPCR

• Digital PCR: 

– Is a PCR based quantitation method

• Specificity of DNA target 

– Uses Poisson counting statistics to determine number 

of molecules of DNA

• Does not require a calibrant

• Primarily a counting method with statistics to compensate for 

PCR reactions that had more than one template molecule

– Used to characterize higher order reference materials 

and qPCR calibrants

• Standard Reference Materials

• Commercial qPCR calibrants
Applied 

Genetics Acknowledgments

Contact Info: 

ross.haynes@nist.gov

301-975-4469

Marcia Holden

Applied Genetics Group: Clinical Team

Margaret Kline

NIST Disclaimer: Certain commercial equipment, instruments and materials are identified in order to specify experimental procedures as 

completely as possible. In no case does such identification imply a recommendation or it imply that any of the materials, instruments or 

equipment identified are necessarily the best available for the purpose.

Points of view are those of the presenters and do not necessarily represent the official position of the National Institute of Standards and 

Technology or the U.S. Department of Justice.

Ross Haynes
Applied 

Genetics Thank you for your attention!