Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT;
Department of Mathematics, Utah Valley University, Orem, UT;
Clin Chem. 2015 May;61(5):724-33. doi: 10.1373/clinchem.2014.236208. Epub 2015 Mar 10.
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 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%.
Relative quantification by restricting the dNTP concentration with melting curve display is a simple and precise way to assess targeted copy number variation.
DNA 拷贝数变异与遗传疾病和癌症有关。目前用于辨别拷贝数变异的方法通常成本高、速度慢、需要专门的设备，并且/或者精度不足。
使用不同引物对和有限的脱氧核苷酸三磷酸（dNTP）（3-12 μmol/L）的多重 PCR 用于相对定量和拷贝数评估。设计了 50-121bp 的小 PCR 产物，具有 1 个融解域、分离良好的 Tm 值、最小的内部序列变异和没有共同的同源物。PCR 产物在导数图上显示为融解曲线，并与参考峰进行归一化。每个靶标的不同拷贝数聚集在一起，并通过无偏层次聚类进行分组。
使用参考基因和靶基因的双 PCR 检测 X、Y、13、18、21 染色体、表皮生长因子受体（EGFR）、运动神经元生存 1、端粒（SMN1）和运动神经元生存 2、着丝粒（SMN2）的拷贝数变化。X 和 Y 以及 CFTR 外显子 2 和 3 使用三 PCR。对 50 个潜在三体样本（13、18、21 或正常）和 50 个潜在性染色体异常样本进行的盲法研究与核型分析一致，除了 2 个最初是嵌合体的样本，在生长后显示出单一核型。还证明了大囊性纤维化跨膜电导调节因子（ATP 结合盒亚家族 C，成员 7）（CFTR）缺失、EGFR 扩增以及 SMN1 和 SMN2 拷贝数评估。在理想条件下，通过限制 dNTP 浓度并使用融解曲线显示来进行相对定量，可以辨别 1.11 倍或更低的拷贝数变化，其 CV 约为 1%。
通过融解曲线显示限制 dNTP 浓度的相对定量是一种简单而精确的方法，可用于评估靶向拷贝数变异。
