Simple and versatile molecular method of copy-number measurement using cloned competitors

PLoS One. 2013 Jul 30;8(7):e69414. doi: 10.1371/journal.pone.0069414. Print 2013.

Abstract

Variations and alterations of copy numbers (CNVs and CNAs) carry disease susceptibility and drug responsiveness implications. Although there are many molecular methods to measure copy numbers, sensitivity, reproducibility, cost, and time issues remain. In the present study, we were able to solve those problems utilizing our modified real competitive PCR method with cloned competitors (mrcPCR). First, the mrcPCR for ERBB2 copy number was established, and the results were comparable to current standard methods but with a shorter assay time and a lower cost. Second, the mrcPCR assays for 24 drug-target genes were established, and the results in a panel of NCI-60 cells were comparable to those from real-time PCR and microarray. Third, the mrcPCR results for FCGR3A and the FCGR3B CNVs were comparable to those by the paralog ratio test (PRT), but without PRT's limitations. These results suggest that mrcPCR is comparable to the currently available standard or the most sensitive methods. In addition, mrcPCR would be invaluable for measurement of CNVs in genes with variants of similar structures, because combination of the other methods is not necessary, along with its other advantages such as short assay time, small sample amount requirement, and applicability to all sequences and genes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Breast Neoplasms / genetics
  • Cell Line, Tumor
  • Cluster Analysis
  • DNA Copy Number Variations*
  • Female
  • GPI-Linked Proteins / genetics
  • Gene Amplification
  • Gene Dosage*
  • Gene Expression Profiling
  • Humans
  • Polymerase Chain Reaction / methods*
  • Polymerase Chain Reaction / standards
  • Receptor, ErbB-2 / genetics
  • Receptors, IgG / genetics
  • Reproducibility of Results

Substances

  • FCGR3A protein, human
  • FCGR3B protein, human
  • GPI-Linked Proteins
  • Receptors, IgG
  • Receptor, ErbB-2

Grants and funding

This work was supported by a research grant to KMH from the National Cancer Center (1110130). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.