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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Abstract

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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Materials

Basic Protocol 1:

  Materials
  • 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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Figures

Videos

Literature Cited

Literature Cited
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