Quantitative Analysis of Copy Number Variants Based on Real‐Time LightCycler PCR

Lijiang Ma1, Wendy K. Chung2

1 Department of Pediatrics, Columbia University, New York, New York, 2 Department of Medicine, Columbia University, New York, New York
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.21
DOI:  10.1002/0471142905.hg0721s80
Online Posting Date:  January, 2014
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Quantitative real‐time PCR is PCR visualized in real time by the use of fluorescent or intercalating dyes, which are employed to measure gene expression or gene quantification including contiguous gene deletions or duplications. A simple method is described here to quantify DNA copy number from human samples. Curr. Protoc. Hum. Genet. 80:7.21.1‐7.21.8. © 2014 by John Wiley & Sons, Inc.

Keywords: qPCR; CNV; copy number analysis

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • Samples of interest (e.g., genomic DNA isolated from normal control and unknown)
  • DNA isolation kit (genomic DNA from blood, e.g., Puregene reagents, Gentra Systems)
  • TE buffer (10 mM Tris/0.1 mM EDTA, pH 8)
  • Primers (for gene of interest)
  • 2× SYBR Green master mix containing enzyme, buffer, dNTPs, and SYBR Green dye (e.g., Roche Molecular Biochemicals)
  • Hybridization probe that detects gene of interest consisting of a primer pair with a 3′‐fluorescein and a 5′‐LightCycler Red 640
  • Nanodrop ND‐1000 (e.g., Nanodrop Technologies)
  • qPCR‐compatible 96‐well plates (e.g., LightCycler 480 Multiwell plate 96)
  • Robotic liquid handling equipment (e.g., Thermo Scientific)
  • Optical plate seals (e.g., LightCycler 480 Multiwell Sealing Foil)
  • LightCycler instrument (e.g., LightCycler 480, Roche Diagnostics)
  • Additional reagents and equipment for spectrophotometry ( )
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Literature Cited

Literature Cited
   Aarskog, N.K. and Vedeler, C.A. 2000. Real‐time quantitative polymerase chain reaction. A new method that detects both the peripheral myelin protein 22 duplication in Charcot‐Marie‐Tooth type 1A disease and the peripheral myelin protein 22 deletion in hereditary neuropathy with liability to pressure palsies. Hum. Genet. 107:494‐498.
   Anhuf, D. , Eggermann, T. , Rudnik‐Schöneborn, S. , and Zerres, K. 2003. Determination of SMN1 and SMN2 copy number using TaqMan technology. Hum. Mutat. 22:74‐78.
   Feldkötter, M. , Schwarzer, V. , Wirth, R. , Wienker, T.F. , and Wirth, B. 2002. Quantitative analyses of SMN1 and SMN2 based on real‐time LightCycler PCR: Fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy. Am. J. Hum. Genet. 70:358‐368.
   Heid, C.A. , Stevens, J. , Livak, K.J. , and Williams, P.M. 1996. Real time quantitative PCR. Genome Res. 6:986‐994.
   Higuchi, R. , Fockler, C. , Dollinger, G. , and Watson, G. 1991. Kinetic PCR: Real‐time monitoring of DNA amplification reactions. Biotechnology 11:1026‐1030.
   Kulka, J. , Tôkés, A.M. , Kaposi‐Novák, P. , Udvarhelyi, N. , Keller, A. , and Schaff, Z. 2006. Detection of HER‐2/neu gene amplification in breast carcinomas using quantitative real‐time PCR—A comparison with immunohistochemical and FISH results. Pathol. Oncol. Res. 12:197‐204.
   Laccone, F. , Junemann, I. , Whatley, S. , Morgan, R. , Butler, R. , Huppke, P. , and Ravine, D. 2004. Large deletions of the MECP2 gene detected by gene dosage analysis in patients with Rett syndrome. Hum. Mutat. 23:234‐244.
   Linzmeier, R.M. and Ganz, T. 2005. Human defensin gene copy number polymorphisms: Comprehensive analysis of independent variation in alpha‐ and beta‐defensin regions at 8p22‐p23. Genomics 86:423‐430.
   Livak, K.J. and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real‐time quantitative PCR and the 2(‐delta delta C(T)) method. Methods 25:402‐408.
   Logan, C. , Habington, A. , Lennon, G. , Cronin, F. , and O'Sullivan, N. 2010. Evaluation of the efficacy of real‐time polymerase chain reaction for the routine early detection of Pseudomonas aeruginosa in cystic fibrosis sputum and throat swab specimens. Diagn. Microbiol. Infect. Dis. 68:358‐365.
   Longo, A.V. , Rodriguez, D. , da Silva Leite, D. , Toledo, L.F. , Mendoza Almeralla, C. , Burrowes, P.A. , and Zamudio, K.R. 2013. ITS1 copy number varies among Batrachochytrium dendrobatidis strains: Implications for qPCR estimates of infection intensity from field‐collected amphibian skin swabs. PLoS One 8:e59499.
   Ramalho, A.S. , Clarke, L.A. , and Amaral, M.D. 2011. Quantification of CFTR transcripts. Methods Mol. Biol. 741:115‐135.
   Schaeffeler, E. , Schwab, M. , Eichelbaum, M. , and Zanger, U.M. 2003. CYP2D6 genotyping strategy based on gene copy number determination by TaqMan real‐time PCR. Hum. Mutat. 22:476‐485.
   Schmittgen, T.D. and Livak, K.J. 2008. Analyzing real‐time PCR data by the comparative C(T) method. Nat. Protoc. 3:1101‐1108.
   Schmittgen, T.D. and Zakrajsek, B.A. 2000. Effect of experimental treatment on housekeeping gene expression: Validation by real‐time, quantitative RT‐PCR. J. Biochem. Biophys. Methods 46:69‐81.
   Schmittgen, T.D. , Zakrajsek, B.A. , Mills, A.G. , Gorn, V. , Singer, M.J. , and Reed, M.W. 2000. Quantitative reverse transcription‐polymerase chain reaction to study mRNA decay: Comparison of endpoint and real‐time methods. Anal. Biochem. 285:194‐204.
   Terribas, E. , Garcia‐Linares, C. , Lázaro, C. , and Serra, E. 2013. Probe‐based quantitative PCR assay for detecting constitutional and somatic deletions in the NF1 gene: Application to genetic testing and tumor analysis. Clin. Chem. 59:928‐937.
   Thiel, C.T. , Kraus, C. , Rauch, A. , Ekici, A.B. , Rautenstrauss, B. , and Reis, A. 2003. A new quantitative PCR multiplex assay for rapid analysis of chromosome 17p11.2‐12 duplications and deletions leading to HMSN/HNPP. Eur. J. Hum. Genet. 11:170‐178.
   Wilke, K. , Duman, B. , and Horst, J. 2000. Diagnosis of haploidy and triploidy based on measurement of gene copy number by real‐time PCR. Hum. Mutat. 16:431‐436.
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