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首页> 外文期刊>Genetic testing and molecular biomarkers >Quantitative Real-Time Polymerase Chain Reaction for the Verification of Genomic Imbalances Detected by Microarray-Based Comparative Genomic Hybridization
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Quantitative Real-Time Polymerase Chain Reaction for the Verification of Genomic Imbalances Detected by Microarray-Based Comparative Genomic Hybridization

机译：定量实时聚合酶链反应验证基因的失衡检测到芯片比较基因组杂交

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摘要

The American College of Medical Genetics guidelines for microarray analysis for constitutional cytogenetic abnormalities require abnormal or ambiguous results from microarray-based comparative genomic hybridization ( aCGH) analysis be confirmed by an alternative method. We employed quantitative real-time polymerase chain reaction (qPCR) technology using SYBR Green I reagents for confirmation of 93 abnormal aCGH results (50 deletions and 43 duplications) and 54 parental samples. A novel qPCR protocol using DNA sequences coding for X-linked lethal diseases in males for designing reference primers was established. Of the 81 sets of test primers used for confirmation of 93 abnormal copy number variants (CNVs) in 80 patients, 71 sets worked after the initial primer design (88%), 9 sets were redesigned once, and 1 set twice because of poor amplification. Fifty-four parental samples were tested using 33 sets of test primers to follow up 34 CNVs in 30 patients. Nineteen CNVs were confirmed as inherited, 13 were negative in both parents, and 2 were inconclusive due to a negative result in a single parent. The qPCR assessment clarified aCGH results in two cases and corrected a fluorescence in situ hybridization result in one case. Our data illustrate that qPCR methodology using SYBR Green I reagents is accurate, highly sensitive, specific, rapid, and cost-effective for verification of chromosomal imbalances detected by aCGH in the clinical setting.
机译：美国医学遗传学微阵列分析指南宪法的细胞遗传学异常需要异常或模棱两可的结果芯片比较基因组杂交(aCGH)分析证实了一个替代方法。实时聚合酶链反应(qPCR)技术使用SYBR绿色我试剂确认93 (50 aCGH异常结果删除和43个重复)和54父母样本。序列编码x连锁致命的疾病引物是雄性的设计参考建立。93年确认异常的拷贝数变异基因拷贝数异变在80名患者,71套在最初的引物设计(88%)、9套被重新设计一次,和1套两次因为可怜的放大。测试使用测试33套引物跟进34基因拷贝数异变在30个病人。确认为继承,13 -父母,2是不确定的原因单亲的阴性结果。评估澄清aCGH导致两种情况并纠正荧光原位杂交结果在一个案例中。说明qPCR方法使用SYBR绿色我高度敏感的试剂是准确的,特定的、快速的和具有成本效益的验证发现的染色体不平衡aCGH在临床设置。

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