Copy Number Assessment by Competitive PCR with Limiting Deoxynucleotide Triphosphates and High-Resolution Melting

Abstract

BACKGROUND DNA copy number variation is associated with genetic disorders and cancer. Available methods to discern variation in copy number are typically costly, slow, require specialized equipment, and/or lack precision. METHODS Multiplex PCR with different primer pairs and limiting deoxynucleotide triphosphates (dNTPs) (3–12 μmol/L) were used for relative quantification and copy number assessment. Small PCR products (50–121 bp) were designed with 1 melting domain, well-separated Tms, minimal internal sequence variation, and no common homologs. PCR products were displayed as melting curves on derivative plots and normalized to the reference peak. Different copy numbers of each target clustered together and were grouped by unbiased hierarchical clustering. RESULTS Duplex PCR of a reference gene and a target gene was used to detect copy number variation in chromosomes X, Y, 13, 18, 21, epidermal growth factor receptor (EGFR), survival of motor neuron 1, telomeric (SMN1), and survival of motor neuron 2, centromeric (SMN2). Triplex PCR was used for X and Y and CFTR exons 2 and 3. Blinded studies of 50 potential trisomic samples (13, 18, 21, or normal) and 50 samples with potential sex chromosome abnormalities were concordant to karyotyping, except for 2 samples that were originally mosaics that displayed a single karyotype after growth. Large cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7) (CFTR) deletions, EGFR amplifications, and SMN1 and SMN2 copy number assessments were also demonstrated. Under ideal conditions, copy number changes of 1.11-fold or lower could be discerned with CVs of about 1%. CONCLUSIONS Relative quantification by restricting the dNTP concentration with melting curve display is a simple and precise way to assess targeted copy number variation.

Spinal muscular atrophy within Amish and Mennonite populations: Ancestral haplotypes and natural history

“…For SMN1 carrier testing and SMN2 copy number analysis, competitive PCR with limiting deoxynucleotide triphosphates was used similar to the procedure outlined in Zhou et al [ 46 ]. This procedure was modified to use unique primer sets for SMN1 and SMN2 ( S3 Table ) and a different reference gene, ALB .…”

Accurate diagnosis of spinal muscular atrophy and 22q11.2 deletion syndrome using limited deoxynucleotide triphosphates and high-resolution melting

“…PCR products having only one melting domain were selected. The ΔTm between selected reference and one or more targets were in the necessary range of between 2 °C to 10 °C [ 4 ].…”

“…The only coding nucleotide difference is SMN1 c.840C > T in exon 7 [ 7 ]. In the duplex PCR to determine the copy number of SMN1 , an allele specific primer was used to amplify only SMN1 c.840C, but not the SMN2 homolog [ 4 ]. Another primer pair amplified a reference segment of CFTR exon 7.…”

“…Competitive PCR with restricted dNTPs and high resolution melting (HRM) is a simple, fast, and economical approach with high accuracy that can be used for targeted evaluation of relative copy number [ 4 ]. The necessary multiplex PCR assays can be easily designed using uMELT melting curve prediction software.…”

High-Resolution Assessment of Copy Number Variation

“…Zhou et al now address this lack in the current issue of Clinical Chemistry (9 ). The authors chose multiplex PCR for relative quantification.…”