Materials for Nucleic Acids Marcia Holden, Ross Haynes, Margaret - - PowerPoint PPT Presentation

NIST Standard Reference Materials for Nucleic Acids

Marcia Holden, Ross Haynes, Margaret Kline, John Butler National Institute of Standards and Technology Applied Genetics Group Gaithersburg, Maryland, USA

SoGAT Blood Virology meeting Vilnius, Lithuania April 16 – 17 2012

NIST is a non-regulatory federal agency within the U.S. Department

• f Commerce. NIST’s primary

mission is to promote economic

growth by working with industry to develop and apply technology, measurements, and standards.

Measurement and Standards Program

planned and conducted in cooperation with industry and focused on infrastructural technologies

NIST is the national measurement institute for the United States

Hollings Manufacturing Extension Partnership Program

a nationwide network of extension centers that provides hands-on technical assistance to smaller manufacturers

Baldridge National Quality Program

an outreach program recognizing organizational performance excellence

Applied Genetics Group

Biochemical Science Division Mission Statement

Advancing technology and traceability through

quality genetic measurements and standard reference materials (SRMs) to aid work in

• Forensic DNA testing
• DNA biometrics
• Clinical diagnostics

– Infectious disease – genetic biomarkers – cancer-linked biomarkers

NIST DNA Reference Materials (1) Forensic Applications

• STR PCR DNA Profiling (SRM 2391b)
• Human Y-Chromosome DNA Profiling (SRM 2395)
• Human DNA Quantitation (SRM 2372)
• Mitochondrial DNA Sequencing (SRM 2392-1

SRM 2392)

NIST DNA Reference Materials (2) Clinical Diagnostics

• Cytomegalovirus (SRM 2366)
• Fragile X Human DNA triplet repeat (SRM 2399)
• Huntington's Disease CAG Repeats (SRM 2393)

Platform Testing

• Heteroplasmic mtDNA Mutation Detection (SRM

2394)

• DNA Sequence Library for External RNA Controls

(SRM 2374)

Certified Reference Material (CRM)

• Reference material (RM), one or more of whose

property values are certified by a procedure which establishes its traceability to an accurate realization

• f the unit in which the property values are

expressed, and for which each certified value is accompanied by an uncertainty at a stated level of confidence and accompanied by a certificate

• Standard Reference Materials (SRMs) are CRMs

certified by NIST

Traceability to the International System of Units (SI)

• We want to develop characterized, stable,

homogeneous nucleic acid SRMs that are traceable to the SI unit, the mole (amount of substance)

• The approach to quantitative traceability is to

count the number of molecules of a dilute solution of DNA in a known volume.

• Our goal is to achieve accurate counting that is

assay independent

Why quantitative SRMs for clinical diagnostics

• To provide higher order standards for clinical

diagnostics that can be used in the validation

• f a measurement system or to assign values

to calibrants

Characterization - What and How

• DNA sequence of regions of the

genome is verified using Sanger sequencing

• The copy number per volume is

certified using digital PCR

Cytomegalovirus SRM 2366

• CMV material consists of the Towne Strain

cloned into a bacterial artificial chromosome

• The BAC (Towne 147) was propagated, the

DNA was purified and packaged in Teflon tubes

• Three different concentrations were prepared

and tubes were randomly selected for quantification

Sequenced regions of the CMV genome

• Regions that were chosen to be sequenced were selected

because they are targets for amplification assays, based on 65 published PCR assays and information from commercial assay manufacturers

• Sequencing was performed in both directions
• The sequencing primers were designed to have significant overlap
• The sequenced regions (total of 15,000 bp) was an exact match for

the Towne strain as deposited in GenBank with the exception of

• ne base (UL54, base 78651)

Digital PCR (dPCR)

for certification of SRM 2366

• dPCR is a process where individual molecules are

counted using the fluorescent signal generated by a PCR reaction when amplification of DNA occurs

• This concept has been around since the 1999 but

was only practical very recently with the development of microfluidic and droplet platforms where thousands of reactions are conducted simultaneously in chambers with nanoliter volumes/chamber

• No calibration curve is needed

X 1 2 3 4 5 6 7 8 9101112 X H H

Pressurized valve Samples: 1-12 Water: H Pressurized valve

dPCR microfluidics array

765 individual chambers / panel 12 panels/chip = 9180 chambers Each chamber – 6 nL

Concentration

• Saturated

≥ 1 copy in each well

• Binary detection

Calculate concentration

• No amplification

No target

Recommendations for the optimal range/765 chamber panel to minimize measurement uncertainty

Institution Positive wells Targets/panel Average Copies/well Platform 200 to 700 232 to 1902 0.3 to 2.5 manufacturer National Cancer 300 to 600 382 to 1178 0.5 to 1.5 Institute USA Published* 409 to 734 585 to 2453 0.8 to 3.2 * Bhat S. et al. 2009

• Anal. Bioanal. Chem.

394:457-467

dPCR

Determines absolute concentration of DNA through the use of counting and statistics

550 wells with target (+) 215 wells without target (-) 550 positive wells statistics  212 copies/ L 765 wells / sample

Poisson Distribution

• Probability statistics of counting rare

events

• Rare  not every partition (time or space)

has event (in this case, DNA)

• dPCR  If you know how many wells

have target you can estimate the total number of targets

0% 10% 20% 30% 40% 50% 60% 70% 1 2 3 4 5 6 7 8 9 10 Wells (%) molecules/well

5,000 molecules 10,000 molecules 30,000 molecules

Poisson Distribution

Thousands of molecules over 10,000 wells

Positive wells Negative wells More conc. means more likely to have well with >1 molecule/well Change in % negative wells with concentration 10,000 molecules 25%

• f wells with >1

copy 30,000 molecules 80%

• f wells with >1

copy

Quantification of SRM 2366 components

• Each component was

separately quantified using multiple digital arrays

• Utilized separate assays

targeting different regions

• f the genome, and found

that the results were not significantly different – no bias

• One assay was used for the

rest of the measurements

Commutability

• Component B of SRM 2366 was included in the Quality

Control for Molecular Diagnostics (QCMD) 2010 CMV EQA program

• Participants were asked to add the DNA directly to the assay

rather than extracting the DNA along with the QCMD test samples

• 178 participants submitted data. Laboratory developed

assays were used to generate 78 datasets and commercial assays were used for 100 datasets

• The median consensus value was 5.9 log10 (Component B =

6.2 log10 based on dPCR)

Commutability

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 1 2 3 4 5 6 7 8 9 Log10(Copies per mL)

Centered Histogram Kernal Density Gaussian Model FWHM

Relative Area, #Bin / (#Total Δx)

QCMD CMV EQA - Participants & assays # Data sets Mean SD Median MADe Total Datasets 178 5.845 0.674 5.900 0.486 Conventional Commercial 5 5.670 0.672 5.854 0.872 Real-Time Laboratory developed - Total 78 5.909 0.756 6.002 0.650 Real-Time Commercial - Total 95 5.795 0.600 5.826 0.451 Commercial kit A (1) 6 5.859 0.386 5.864 0.150 Commercial kit A (2) 15 6.224 0.345 6.205 0.332 Commercial kit B 21 5.632 0.880 5.733 0.794 Commercial kit C 28 5.767 0.321 5.821 0.326 Commercial kit D 12 5.789 0.233 5.776 0.298

dPCR

• Digital PCR has the possibility to be a realized

reference method for quantification of nucleic acids

• NIST is working with other national measurement

institutions such as IRMM, LGC, NMI Australia and KRISS in collaboration with each other and through BIPM (CCQM) to validate digital PCR

• NIST is working on other candidate SRMs and will

be using dPCR for quantification

Thank you for the opportunity to make a presentation at SoGAT

Clinical Diagnostics Team

Ross Haynes Marcia Holden Margaret Kline John Butler, Group Leader