Digital Droplet PCR: CNV Analysis and Other Applications

Erica Mazaika1, Jason Homsy2

1 Department of Genetics, Harvard Medical School, Boston, Massachusetts, 2 Cardiovascular Research Center, Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.24
DOI:  10.1002/0471142905.hg0724s82
Online Posting Date:  July, 2014
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Abstract

ABSTRACT

Digital droplet PCR (ddPCR) is an assay that combines state‐of‐the‐art microfluidics technology with TaqMan‐based PCR to achieve precise target DNA quantification at high levels of sensitivity and specificity. Because quantification is achieved without the need for standard assays in an easy to interpret, unambiguous digital readout, ddPCR is far simpler, faster, and less error prone than real‐time qPCR. The basic protocol can be modified with minor adjustments to suit a wide range of applications, such as CNV analysis, rare variant detection, SNP genotyping, and transcript quantification. This unit describes the ddPCR workflow in detail for the Bio‐Rad QX100 system, but the theory and data interpretation are generalizable to any ddPCR system. Curr. Protoc. Hum. Genet. 82:7.24.1‐7.24.13. © 2014 by John Wiley & Sons, Inc.

Keywords: ddPCR; TaqMan; CNV; genotyping; rare variant detection; transcript quantification; copy number variant

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: CNV Detection Using BIO‐RAD's QX100 Digital Droplet PCR Platform
  • Alternate Protocol 1: Rare Variant Detection
  • Alternate Protocol 2: SNP Genotyping
  • Alternate Protocol 3: Transcript Quantification
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: CNV Detection Using BIO‐RAD's QX100 Digital Droplet PCR Platform

  Materials
  • DNA
  • Nuclease‐free water
  • 10× restriction enzyme buffer
  • AluI or alternate restriction enzyme
  • 2× ddPCR master mix (includes hot‐start DNA Polymerase, dNTPs including dUTP; Bio‐Rad)
  • 20× ROI target primer/TaqMan probe mix (see recipe)
  • 20× REF target (RPP30) primer/TaqMan probe mix (see recipe)
  • 2× control buffer (Bio‐Rad, cat. no. 186‐3052)
  • Heat block or water bath
  • 96‐well plates
  • Centrifuge
  • DG8 droplet generator cartridges (single‐use; Bio‐Rad)
  • DG8 droplet generator cartridge holder (Bio‐Rad)
  • ddPCR droplet generation (DG) oil (Bio‐Rad)
  • DG8 gaskets (single‐use; Bio‐Rad)
  • QX100 droplet generator (Bio‐Rad)
  • Eppendorf twin.tec semi‐skirted 96‐well plate
  • Heat sealer
  • Heat sealing PCR foil
  • Thermal cycler
  • Bio‐Rad QX100 droplet reader
  • QuantaSoft software
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Figures

Videos

Literature Cited

Literature Cited
  Bio‐Rad. 2012. QX100 Droplet Digital PCR System Guide. Bio‐Rad, Pleasanton, Calif.
  Fraga, D., Meulia, T., and Fenster, S. 2014. Real‐time PCR. Curr. Protoc. Essential Lab. Techn. 8:10.3.1‐10.3.40.
  Hindson, B.J., Ness, K.D., Masquelier, D.A., Belgrader, P., Heredia, N.J., Makarewicz, A.J., Bright, I.J., Lucero, M.Y., Hiddessen, A.L., Legler, T.C., Kitano, T.K., Hodel, M.R., Petersen, J.F., Wyatt, P.W., Steenblock, E.R., Shah, P.H., Bousse, L.J., Troup, C.B., Mellen, J.C., Wittmann, D.K., Erndt, N.G., Cauley, T.H., Koehler, R.T., So, A.P., Dube, S., Rose, K.A., Montesclaros, L., Wang, S., Stumbo, D.P., Hodges, S.P., Romine, S., Milanovich, F.P., White, H.E., Regan, J.F., Karlin‐Neumann, G.A., Hindson, C.M., Saxonov, S., and Colston, B.W. 2011. High‐throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal. Chem. 83:8604‐8610.
  Kutyavin, I.V., Afonina, I.A., Mills, A., Gorn, V.V., Lukhtanov, E.A., Belousov, E.S., Singer, M.J., Walburger, D.K., Lokhov, S.G., Gall, A.A., Dempcy, R., Reed, M.W., Meyer, R.B., and Hedgpeth, J. 2000. 3′‐Minor groove binder‐DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res. 28:655‐661.
  Tarailo‐Graovac, M. and Chen, N. 2009. Using RepeatMasker to identify repetitive elements in genomic sequences. Curr. Protoc. Bioinform. 25:4.10.1‐4.10.14.
Internet Resources
  http://genome.ucsc.edu/
  Use In‐Silico PCR Tool to ensure that PCR primers will only amplify one region.
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