Kannabiran Chitra
Kallam Anji Reddy Molecular Genetics Laboratory, Prof. Brien Holden Eye Research Centre, L.V. Prasad Eye Institute, Hyderabad, India.
Methods Mol Biol. 2018;1726:19-28. doi: 10.1007/978-1-4939-7565-5_3.
Copy number changes comprising deletions or insertions involving single or multiple exons of a gene are known to occur in a significant proportion of cases in retinoblastoma. The protocol described here involves a two-step quantitative multiplex PCR process which is suitable for the detection of such mutations in the RB1 as well as in other genes. This is achieved through the use of suitable gene-specific primers designed to amplify individual exons, with universal tags attached to the 5' end of each primer. These tagged primers are used in the first step of PCR of the RB1 gene in patients. The second step is carried out through the use of "universal" primers complementary to the tag sequences alone. This technique facilitates the detection of fluorescent PCR products from multiple exons through the use of a single fluorescent tagged primer.
已知在相当比例的视网膜母细胞瘤病例中会发生拷贝数变化，包括涉及一个基因的单个或多个外显子的缺失或插入。本文所述方案涉及两步定量多重PCR过程，适用于检测RB1基因以及其他基因中的此类突变。这是通过使用合适的基因特异性引物来实现的，这些引物设计用于扩增各个外显子，每个引物的5'端都连接有通用标签。这些带标签的引物用于患者RB1基因PCR的第一步。第二步仅通过使用与标签序列互补的“通用”引物来进行。该技术通过使用单个荧光标记引物促进了对来自多个外显子的荧光PCR产物的检测。
