Competitive PCR for Copy Number Assessment by Restricting dNTPs

  • Luming Zhou
  • Robert A. Palais
  • Yotam Ardon
  • Carl T. WittwerEmail author
Part of the Neuromethods book series (NM, volume 131)


Copy number variation (CNV) reflects a gain or loss in the number of copies of DNA fragments in a genome. CNV is common in genetic diseases and is known to cause particular neurodegenerative diseases. We developed a dNTP-limited, competitive PCR technique to identify relative copy number differences between a reference and one or more target genes. Suitable fragments with single melting domains, well-separated melting temperatures, and no common homologs were identified by uMelt melting curve prediction software. Relative product amounts were maintained during multiplex PCR into the plateau phase by limiting dNTPs. After PCR, fluorescent melting curve analysis was automatically performed with the saturating DNA dye, LCGreen® Plus. Exponential background was removed, melting curves were plotted as negative derivative melting peaks, and the reference peak was normalized by both position (temperature) and height of each peak. With the reference peak normalized, the height of the target peaks established the copy number order that can be quantified against standards. Using chromosome X variation, the best dNTP concentration to distinguish copy numbers was about 6 μM each and CVs of about 1% were obtained with high-resolution melting analysis. The method was applied to spinal muscular atrophy, trisomies 13, 18, and 21, and cystic fibrosis gene deletions. The method is rapid, economical, and closed tube, and can be used for diagnosis or confirmation of copy number differences identified by high-throughput screening methods.

Key words

Copy number variation CNV High-resolution melting analysis HRMA Melting temperature Tm Spinal muscular atrophy 


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Luming Zhou
    • 1
  • Robert A. Palais
    • 1
    • 2
  • Yotam Ardon
    • 1
  • Carl T. Wittwer
    • 1
    Email author
  1. 1.Department of PathologyUniversity of Utah Medical SchoolSalt Lake CityUSA
  2. 2.Department of MathematicsUtah Valley UniversityOremUSA

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