High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.
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 ~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 precision 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 100,000-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.
Bio-Rad Laboratories, Inc., Pleasanton, California 94566, United States. ben_hindson@bio-rad.com
Benjamin J
BJ
Hindson
Kevin D
KD
Ness
Donald A
DA
Masquelier
Phillip
P
Belgrader
Nicholas J
NJ
Heredia
Anthony J
AJ
Makarewicz
Isaac J
IJ
Bright
Michael Y
MY
Lucero
Amy L
AL
Hiddessen
Tina C
TC
Legler
Tyler K
TK
Kitano
Michael R
MR
Hodel
Jonathan F
JF
Petersen
Paul W
PW
Wyatt
Erin R
ER
Steenblock
Pallavi H
PH
Shah
Luc J
LJ
Bousse
Camille B
CB
Troup
Jeffrey C
JC
Mellen
Dean K
DK
Wittmann
Nicholas G
NG
Erndt
Thomas H
TH
Cauley
Ryan T
RT
Koehler
Austin P
AP
So
Simant
S
Dube
Klint A
KA
Rose
Luz
L
Montesclaros
Shenglong
S
Wang
David P
DP
Stumbo
Shawn P
SP
Hodges
Steven
S
Romine
Fred P
FP
Milanovich
Helen E
HE
White
John F
JF
Regan
George A
GA
Karlin-Neumann
Christopher M
CM
Hindson
Serge
S
Saxonov
Bill W
BW
Colston
D004247:DNA / Q000235:genetics*; D018628:Gene Dosage / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016133:Polymerase Chain Reaction
D004247:DNA