Ahn Joo Wook, Coldwell Michael, Bint Susan, Mackie Ogilvie Caroline
Cytogenetics Department, Guy's & St Thomas' NHS Foundation Trust;
Cytogenetics Department, Viapath Analytics.
J Vis Exp. 2015 Feb 21(96):e51718. doi: 10.3791/51718.
Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient's sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.
用于检测基因组拷贝数变异的阵列比较基因组杂交（Array CGH）已取代了G带核型分析。本文描述了该技术及其在临床诊断服务实验室中的应用。从患者样本（血液、唾液或其他组织类型）中提取的DNA用荧光染料（花青素5或花青素3）标记。参考DNA样本用相反的荧光染料标记。在标记的DNA混合并重悬于杂交缓冲液中并应用于包含来自全基因组位点的约60,000个寡核苷酸探针的阵列之前，有一个清理步骤以去除未掺入的核苷酸，在临床重要区域具有高探针密度。杂交后，对阵列进行洗涤，然后扫描，并分析所得图像以测量每个探针的红色和绿色荧光。软件用于评估每个探针测量的质量，计算红色与绿色荧光的比率并检测潜在的拷贝数变异。
