Hosono Naoya, Kubo Michiaki, Tsuchiya Yumiko, Sato Hiroko, Kitamoto Takuya, Saito Susumu, Ohnishi Yozo, Nakamura Yusuke
Laboratory for Genotyping, SNP Research Center, The Institute of Physical and Chemical Research (RIKEN), Kanagawa, Japan.
Hum Mutat. 2008 Jan;29(1):182-9. doi: 10.1002/humu.20609.
We report the development of a real-time Invader assay combined with multiplex PCR (mPCR-RETINA), an SNP-based approach that can measure the allelic ratio in copy number variation (CNV) regions of a genome. RETINA monitors the real-time fluorescence intensity of each allele during the Invader assay and detects allelic asymmetries caused by genomic duplication/multiplication in heterozygous individuals. By combining mPCR-RETINA and real-time quantitative PCR that detects total copy number, we can estimate the copy number of each allele in CNV regions, which should be useful for investigating the functional significance of allele copy number with disease susceptibilities and drug responses. Also, mPCR-RETINA can efficiently refine the detailed structures of CNV regions. Due to the combination of RETINA with multiplex PCR, mPCR-RETINA requires a very small amount of genomic DNA for analysis (0.1-0.38 ng/locus). Additionally, mPCR-RETINA has clear advantages in its simple protocol and target-specific reaction, even in nonunique regions. We believe mPCR-RETINA will provide a significant contribution to identifying functional alleles in CNV regions.
我们报告了一种结合多重PCR的实时入侵检测法（mPCR-RETINA）的开发情况，这是一种基于单核苷酸多态性的方法，能够测量基因组拷贝数变异（CNV）区域中的等位基因比例。RETINA在入侵检测过程中监测每个等位基因的实时荧光强度，并检测杂合个体中由基因组复制/倍增引起的等位基因不对称性。通过将mPCR-RETINA与检测总拷贝数的实时定量PCR相结合，我们可以估算CNV区域中每个等位基因的拷贝数，这对于研究等位基因拷贝数与疾病易感性和药物反应之间的功能意义应该是有用的。此外，mPCR-RETINA能够有效地细化CNV区域的详细结构。由于RETINA与多重PCR相结合，mPCR-RETINA分析所需的基因组DNA量非常少（每个位点0.1 - 0.38 ng）。此外，即使在非独特区域，mPCR-RETINA在其简单的实验方案和靶向特异性反应方面也具有明显优势。我们相信mPCR-RETINA将为识别CNV区域中的功能等位基因做出重大贡献。
