拷贝数变化
生物
多重连接依赖探针扩增
基因剂量
遗传学
多路复用
基因组DNA
基因
分子生物学
拷贝数分析
断点
实时聚合酶链反应
塔克曼
基因表达
外显子
基因组
染色体
作者
Márton Doleschall,Otto Darvasi,Zoltán Herold,Zoltán Doleschall,Gábor Nyírő,Anikó Somogyi,Peter Igaz,Attila Patócs
出处
期刊:PLOS ONE
[Public Library of Science]
日期:2022-12-01
卷期号:17 (12): e0277299-e0277299
标识
DOI:10.1371/journal.pone.0277299
摘要
Quantitative PCR (qPCR) is used for the determination of gene copy number (GCN). GCNs contribute to human disorders, and characterize copy number variation (CNV). The single laboratory method validations of duplex qPCR assays with hydrolysis probes on CYP21A1P and CYP21A2 genes, residing a CNV (RCCX CNV) and related to congenital adrenal hyperplasia, were performed using 46 human genomic DNA samples. We also performed the verifications on 5 qPCR assays for the genetic elements of RCCX CNV; C4A , C4B , CNV breakpoint, HERV-K(C4) CNV deletion and insertion alleles. Precision of each qPCR assay was under 1.01 CV%. Accuracy (relative error) ranged from 4.96±4.08% to 9.91±8.93%. Accuracy was not tightly linked to precision, but was significantly correlated with the efficiency of normalization using the RPPH1 internal reference gene (Spearman’s ρ: 0.793–0.940, p>0.0001), ambiguity (ρ = 0.671, p = 0.029) and misclassification (ρ = 0.769, p = 0.009). A strong genomic matrix effect was observed, and target-singleplex (one target gene in one assay) qPCR was able to appropriately differentiate 2 GCN from 3 GCN at best. The analysis of all GCNs from the 7 qPCR assays using a multiplex approach increased the resolution of differentiation, and produced 98% of GCNs unambiguously, and all of which were in 100% concordance with GCNs measured by Southern blot, MLPA and aCGH. We conclude that the use of an internal (in one assay with the target gene) reference gene, the use of allele-specific primers or probes, and the multiplex approach (in one assay or different assays) are crucial for GCN determination using qPCR or other methods.
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