Easy One-Step Amplification and Labeling Procedure for Copy Number Variation Detection

Clin Chem. 2020 Mar 1;66(3):463-473. doi: 10.1093/clinchem/hvaa002.


Background: The specific characteristics of copy number variations (CNVs) require specific methods of detection and characterization. We developed the Easy One-Step Amplification and Labeling procedure for CNV detection (EOSAL-CNV), a new method based on proportional amplification and labeling of amplicons in 1 PCR.

Methods: We used tailed primers for specific amplification and a pair of labeling probes (only 1 labeled) for amplification and labeling of all amplicons in just 1 reaction. Products were loaded directly onto a capillary DNA sequencer for fragment sizing and quantification. Data obtained could be analyzed by Microsoft Excel spreadsheet or EOSAL-CNV analysis software. We developed the protocol using the LDLR (low density lipoprotein receptor) gene including 23 samples with 8 different CNVs. After optimizing the protocol, it was used for genes in the following multiplexes: BRCA1 (BRCA1 DNA repair associated), BRCA2 (BRCA2 DNA repair associated), CHEK2 (checkpoint kinase 2), MLH1 (mutL homolog 1) plus MSH6 (mutS homolog 6), MSH2 (mutS homolog 2) plus EPCAM (epithelial cell adhesion molecule) and chromosome 17 (especially the TP53 [tumor protein 53] gene). We compared our procedure with multiplex ligation-dependent probe amplification (MLPA).

Results: The simple procedure for CNV detection required 150 min, with <10 min of handwork. After analyzing >240 samples, EOSAL-CNV excluded the presence of CNVs in all controls, and in all cases, results were identical using MLPA and EOSAL-CNV. Analysis of the 17p region in tumor samples showed 100% similarity between fluorescent in situ hybridization and EOSAL-CNV.

Conclusions: EOSAL-CNV allowed reliable, fast, easy detection and characterization of CNVs. It provides an alternative to targeted analysis methods such as MLPA.

Keywords: Lynch syndrome; Mutation detection system; copy number variations; familial hypercholesterolemia; genetic diagnosis; hereditary breast cancer.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Copy Number Variations*
  • DNA Probes / chemistry
  • DNA Probes / metabolism
  • Fluorescent Dyes / chemistry
  • Humans
  • In Situ Hybridization, Fluorescence
  • Multiplex Polymerase Chain Reaction
  • Polymerase Chain Reaction / methods*
  • Receptors, LDL / genetics*
  • Sequence Analysis, DNA


  • DNA Probes
  • Fluorescent Dyes
  • LDLR protein, human
  • Receptors, LDL