title: 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.
authors.affiliation: Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT; carl.wittwer@path.utah.edu luming.zhou@path.utah.edu.
authors.forename: Luming
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Department of Mathematics, Utah Valley University, Orem, UT;
authors.forename: Robert A
authors.initials: RA
authors.lastname: Palais
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT.
authors.forename: Christian N
authors.initials: CN
authors.lastname: Paxton
authors.affiliation: Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT; ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT.
authors.forename: Katherine B
authors.initials: KB
authors.lastname: Geiersbach
authors.affiliation: Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT; ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT. carl.wittwer@path.utah.edu luming.zhou@path.utah.edu.
authors.forename: Carl T
authors.initials: CT
authors.lastname: Wittwer
mesh_terms: D018628:Gene Dosage; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010710:Phosphates / Q000737:chemistry*
chemical_list: D010710:Phosphates