High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

被引：2019

作者：

Hindson, Benjamin J. ^{[1
]}

Ness, Kevin D. ^{[1
]}

Masquelier, Donald A. ^{[1
]}

Belgrader, Phillip ^{[1
]}

Heredia, Nicholas J. ^{[1
]}

Makarewicz, Anthony J. ^{[1
]}

Bright, Isaac J. ^{[1
]}

Lucero, Michael Y. ^{[1
]}

Hiddessen, Amy L. ^{[1
]}

Legler, Tina C. ^{[1
]}

Kitano, Tyler K. ^{[1
]}

Hodel, Michael R. ^{[1
]}

Petersen, Jonathan F. ^{[1
]}

Wyatt, Paul W. ^{[1
]}

Steenblock, Erin R. ^{[1
]}

Shah, Pallavi H. ^{[1
]}

Bousse, Luc J. ^{[1
]}

Troup, Camille B. ^{[1
]}

Mellen, Jeffrey C. ^{[1
]}

Wittmann, Dean K. ^{[1
]}

Erndt, Nicholas G. ^{[1
]}

Cauley, Thomas H. ^{[1
]}

Koehler, Ryan T. ^{[1
]}

So, Austin P. ^{[1
]}

Dube, Simant ^{[1
]}

Rose, Klint A. ^{[1
]}

Montesclaros, Luz ^{[1
]}

Wang, Shenglong ^{[1
]}

Stumbo, David P. ^{[1
]}

Hodges, Shawn P. ^{[1
]}

Romine, Steven ^{[1
]}

Milanovich, Fred P. ^{[1
]}

White, Helen E. ^{[2
]}

Regan, John F. ^{[1
]}

Karlin-Neumann, George A. ^{[1
]}

Hindson, Christopher M. ^{[1
]}

Saxonov, Serge ^{[1
]}

Colston, Bill W. ^{[1
]}

机构：

[1] Biorad Labs Inc, Pleasanton, CA 94566 USA

[2] Salisbury Dist Hosp, Natl Genet Reference Lab, Salisbury SP2 8BJ, Wilts, England

来源：

ANALYTICAL CHEMISTRY | 2011年 / 83卷 / 22期

关键词：

POLYMERASE-CHAIN-REACTION; CELL-FREE DNA; REAL-TIME; ON-CHIP; QUANTIFICATION; CANCER; FETAL; AMPLIFICATION; ANEUPLOIDY;

D O I：

10.1021/ac202028g

中图分类号：

O65 [分析化学];

学科分类号：

070302 ; 081704 ;

摘要：

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of similar to 2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more p:recision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

引用

页码：8604 / 8610

页数：7

共 37 条

[1] Measurement methods and accuracy in copy number variation: failure to replicate associations of beta-defensin copy number with Crohn's disease
Aldhous, Marian C.
Abu Bakar, Suhaili
Prescott, Natalie J.
Palla, Raquel
Soo, Kimberley
Mansfield, John C.
Mathew, Christopher G.
Satsangi, Jack
Armour, John A. L.
[J]. HUMAN MOLECULAR GENETICS, 2010, 19 (24) : 4930 - 4938
[2] Personalized copy number and segmental duplication maps using next-generation sequencing
Alkan, Can
Kidd, Jeffrey M.
Marques-Bonet, Tomas
Aksay, Gozde
Antonacci, Francesca
Hormozdiari, Fereydoun
Kitzman, Jacob O.
Baker, Carl
Malig, Maika
Mutlu, Onur
Sahinalp, S. Cenk
Gibbs, Richard A.
Eichler, Evan E.
[J]. NATURE GENETICS, 2009, 41 (10) : 1061 - U29
[3] External Quality Assurance of HER2 FISH and ISH Testing Three Years of the UK National External Quality Assurance Scheme
Bartlett, John M. S.
Ibrahim, Merdol
Jasani, Bharat
Morgan, John M.
Ellis, Ian
Kay, Elaine
Connolly, Yvonne
Campbell, Fiona
O'Grady, Anthony
Barnett, Sarah
Miller, Keith
[J]. AMERICAN JOURNAL OF CLINICAL PATHOLOGY, 2009, 131 (01) : 106 - 111
[4] On-chip single-copy real-time reverse-transcription PCR in isolated picoliter droplets
Beer, N. Reginald
Wheeler, Elizabeth K.
Lee-Houghton, Lorenna
Watkins, Nicholas
Nasarabadi, Shanavaz
Hebert, Nicole
Leung, Patrick
Arnold, Don W.
Bailey, Christopher G.
Colston, Bill W.
[J]. ANALYTICAL CHEMISTRY, 2008, 80 (06) : 1854 - 1858
[5] On-chip, real-time, single-copy polymerase chain reaction in picoliter droplets
Beer, N. Reginald
Hindson, Benjamin J.
Wheeler, Elizabeth K.
Hall, Sara B.
Rose, Klint A.
Kennedy, Ian M.
Colston, Bill W.
[J]. ANALYTICAL CHEMISTRY, 2007, 79 (22) : 8471 - 8475
[6] Detection of BRAF V600E mutation in colorectal cancer:: Comparison of automatic sequencing and real-time chemistry methodology
Benlloch, Susana
Paya, Artemio
Alenda, Cristina
Bessa, Xavier
Andreu, Montserrat
Jover, Rodrigo
Castells, Antoni
Llor, Xavier
Aranda, F. Ignacio
Massuti, Bartomeu
[J]. JOURNAL OF MOLECULAR DIAGNOSTICS, 2006, 8 (05) : 540 - 543
[7] Methods and strategies for analyzing copy number variation using DNA microarrays
Carter, Nigel P.
[J]. NATURE GENETICS, 2007, 39 (Suppl 7) : S16 - S21
[8] Digital quantification of mutant DNA in cancer patients
Diehl, Frank
Diaz, Luis A., Jr.
[J]. CURRENT OPINION IN ONCOLOGY, 2007, 19 (01) : 36 - 42
[9] BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions
Diehl, Frank
Li, Meng
He, Yiping
Kinzler, Kenneth W.
Vogelstein, Bert
Dressman, Devin
[J]. NATURE METHODS, 2006, 3 (07) : 551 - 559
[10] Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations
Dressman, D
Yan, H
Traverso, G
Kinzler, KW
Vogelstein, B
[J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2003, 100 (15) : 8817 - 8822

← 1 2 3 4 →