Wang D, Gao H, Zhang R, Ma X, Zhou Y, Cheng J
Tsinghua University, National Engineering Research Center for Beijing Biochip Technology, Beijing, P.R. China.
Biotechniques. 2003 Aug;35(2):300-2, 304, 306 passim. doi: 10.2144/03352st02.
Efficiencies of mismatch discrimination using size-varied capture probes were examined at various hybridization temperatures. The probes were 17, 15, 13, 11, 9, and 7 nucleotides long and contained single-base mismatches at their 3' ends. The optimal signal intensity and efficiency of base stacking hybridization on mismatch discrimination were observed for capture probes with a melting temperature (Tm) value of 36 degrees C, in the detection of DNA sequence variations at 40 degrees C. We employed asymmetric PCR to prepare single-stranded target DNA labeled with a fluorescent dye, and the PCR product was hybridized on the DNA microarray with no further purification. Our efforts have enhanced the sensitivity and simplified the procedures of base stacking hybridization on mismatch discrimination. As a model experiment, this improved technology was used to identify plasmid templates of human leukocyte antigen (HLA)-A alleles 2601, 2902, and 0206 on oligonucleotide microarrays. It is now possible to apply this simple, rapid, sensitive, and reliable base stacking hybridization technology to detect DNA sequence variations on microarrays in clinical diagnosis and other applications.
在不同的杂交温度下，研究了使用不同长度捕获探针进行错配识别的效率。这些探针长度分别为17、15、13、11、9和7个核苷酸，且在其3'端含有单碱基错配。在40℃检测DNA序列变异时，对于熔解温度（Tm）值为36℃的捕获探针，观察到了碱基堆积杂交对错配识别的最佳信号强度和效率。我们采用不对称PCR制备用荧光染料标记的单链靶DNA，且PCR产物无需进一步纯化即可与DNA微阵列杂交。我们的工作提高了碱基堆积杂交对错配识别的灵敏度并简化了操作程序。作为一个模型实验，这种改进技术用于在寡核苷酸微阵列上鉴定人类白细胞抗原（HLA）-A等位基因2601、2902和0206的质粒模板。现在有可能将这种简单、快速、灵敏且可靠的碱基堆积杂交技术应用于临床诊断和其他应用中微阵列上DNA序列变异的检测。
