Copy number assessment by competitive PCR with limiting deoxynucleotide triphosphates and high-resolution melting

Clinical Chemistry
Luming ZhouCarl T Wittwer

Abstract

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. 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. 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 sam...Continue Reading

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Citations

Oct 24, 2019·Laboratory Investigation; a Journal of Technical Methods and Pathology·Mei LiXu Sun
Nov 5, 2019·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Jan M RuijterMaurice J B van den Hoff
Apr 5, 2015·Clinical Chemistry·Alexander Dobrovic
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Feb 16, 2019·Journal of Human Genetics·Zhongmin XiaQiwei Guo
Jun 23, 2021·Journal of Infection and Chemotherapy : Official Journal of the Japan Society of Chemotherapy·Akira AokiHideto Jinno

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