ORCID Profile
0000-0001-9995-1508
Current Organisation
National Measurement Institute
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Publisher: IOP Publishing
Date: 2020
DOI: 10.1088/0026-1394/57/1A/08004
Abstract: Key comparison CCQM-K86.c was performed to demonstrate the capacity of National Metrology Institutes (NMIs) and Designated Institutes (DIs) in the determination of the relative quantity of two specific genomic DNA fragments present in a canola powder. The study provides direct support for the following measurement claim: "Quantification of the ratio of the number of copies of specified intact sequence fragments of a length up to 150 nucleotides following extraction from an unprocessed, high fat/oil ground seed matrix, with a copy number ratio from 0.001 to 1". The study was carried out under the auspices of the Nucleic Acids Working Group (NAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) and was jointly coordinated by the National Research Council of Canada (NRC) and the EU Joint Research Centre, Geel (JRC). The following laboratories (in alphabetical order) submitted measurement results in this key comparison study: Centro Nacional de Metrología, Mexico ("CENAM") D.I. Mendeleyev Institute of Metrology, Russia ("VNIIM") EU Joint Research Centre, Geel (JRC) Hong Kong Government Laboratory ("GLHK") Instituto Nacional de Metrología de Colombia ("INM") LGC (United Kingdom) National Institute of Biology, Slovenia ("NIB") National Institute of Metrology, P.R. of China ("NIM China"/"NIMC" [figures]) National Institute of Metrology, Thailand (NIMT) National Measurement Institute, Australia ("NMIA") National Metrology Institute of Japan, AIST, Japan ("NMIJ") National Metrology Institute of Turkey ("TÜBITAK"). Main text To reach the main text of this paper, click on Final Report . Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/ . The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).
Publisher: American Chemical Society (ACS)
Date: 18-02-2019
DOI: 10.1021/ACS.ANALCHEM.8B05828
Abstract: Digital polymerase chain reaction (dPCR) is increasingly being adopted by reference material producers and metrology institutes for value assignment, and for homogeneity and stability studies of nucleic acid reference materials. A reference method procedure should fulfill several requirements, and the uncertainty and biases should be completely understood. A bias in target concentration when inaccurate droplet volume is used in the droplet dPCR measurement equation has previously been documented. In this study, we characterize both intrawell and interwell droplet volume variability using optical microscopy and determine the impact of these two sources of variability on target concentration estimates. A small optical distortion across the image was measured which, without correction, biased droplet volume measurements. Longitudinal monitoring of interwell droplet volume over 39 weeks using several lots of Mastermix demonstrated a mean droplet volume of 0.786 nL and intermediate precision of 1.7%. The frequency distribution of intrawell droplet volumes varied. Some wells displayed a skewed distribution which resulted in a small bias in estimated target concentration for a simulated dPCR with target concentrations of between 62 and 8000 copies μL
Publisher: MDPI AG
Date: 20-10-2023
Publisher: Royal Society of Chemistry (RSC)
Date: 2011
DOI: 10.1039/C0AN00484G
Abstract: Digital polymerase chain reaction (dPCR) has the potential to enable accurate quantification of target DNA copy number provided that all target DNA molecules are successfully lified. Following duplex dPCR analysis from a linear DNA target sequence that contains single copies of two independent template sequences, we have observed that lification of both templates in a single partition does not always occur. To investigate this finding, we heated the target DNA solution to 95 °C for increasing time intervals and then immediately chilled on ice prior to preparing the dPCR mix. We observed an exponential decline in estimated copy number (R(2)≥ 0.98) of the two template sequences when lified from either a linearized plasmid or a 388 base pair (bp) licon containing the same two template sequences. The distribution of lifiable templates and the final concentration (copies per µL) were both affected by heat treatment of the s les at 95 °C from 0 s to 30 min. The proportion of target sequences from which only one of the two templates was lified in a single partition (either 1507 or hmg only) increased over time, while the proportion of target sequences where both templates were lified (1507 and hmg) in each in idual partition declined rapidly from 94% to 52% (plasmid) and 88% to 31% (388 bp licon) suggesting an increase in number of targets from which both templates no longer lify. A 10 min incubation at 95 °C reduced the initial lifiable template concentration of the plasmid and the 388 bp licon by 59% and 91%, respectively. To determine if a similar decrease in lifiable target occurs during the default pre-activation step of typical PCR lification protocol, we used mastermixes with a 20 s or 10 min hot-start. The choice of mastermix and consequent pre-activation time did not affect the estimated plasmid concentration. Therefore, we conclude that prolonged exposure of this DNA template to elevated temperatures could lead to significant bias in dPCR measurements. However, care must be taken when designing PCR and non-PCR based experiments by reducing exposure of the DNA template to sustained elevated temperatures in order to improve accuracy in copy number estimation and concentration determination.
Publisher: Cold Spring Harbor Laboratory
Date: 06-09-2023
Publisher: IOP Publishing
Date: 2018
DOI: 10.1088/0026-1394/55/1A/08017
Abstract: A key comparison CCQM-K86.b was performed to demonstrate and document the capacity of National Metrology Institutes (NMIs) and Designated Institutes (DIs) in the determination of the relative quantity of two specific genomic DNA fragments present in a rice powder. The study provides the support for the following measurement claim: "Quantification of the ratio of the number of copies of specified intact sequence fragments of a length up to 150 nucleotides following extraction from an unprocessed, high starch ground seed matrix, with a copy number ratio from 0.005 to 1". The study was carried out under the auspices of the Nucleic Acids Working Group (NAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) and was jointly coordinated by the National Institute of Metrology, China (NIMC) and Government Laboratory Hong Kong (GLHK).The following laboratories (in alphabetical order) participated in this key comparison study: CENAM (Mexico) GLHK (Hong Kong) LGC (United Kingdom) NIB (Slovenia) NIMC (P.R. of China) NIMT (Thailand) NMIA (Australia) NMIJ (Japan) TÜBITAK-UME (Turkey). Good agreement was observed between the reported results. KEY WORDS FOR SEARCH Genetically modified (GM) crops, digital PCR, copy number ratio, GM rice Main text To reach the main text of this paper, click on Final Report . Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/ . The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).
Publisher: American Chemical Society (ACS)
Date: 30-11-2016
DOI: 10.1021/ACS.ANALCHEM.6B03076
Abstract: Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the in idual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.
No related grants have been discovered for Jacob McLaughlin.