Single nucleotide polymorphism discrimination assisted by improved base stacking hybridization using oligonucleotide microarrays
Single nucleotide polymorphism discrimination assisted by improved base stacking hybridization using oligonucleotide microarrays
复制标题
DOI：
10.2144/03352st02
复制
发表时间：
2003-08-01
期刊：
影响因子：
2.7
通讯作者：
Cheng, J
中科院分区：
文献类型：
--
作者：
Wang, D;Gao, H;Cheng, J
在不同的杂交温度下，研究了不同尺寸捕获探针的错配识别效率。探针长度分别为17、15、13、11、9和7个核苷酸，在它们的3'端含有单碱基错配。在熔点温度（T-m）为36°c的捕获探针检测40°c的DNA序列变异时，观察到碱基堆叠杂交对错配识别的最佳信号强度和效率。我们采用非对称PCR方法制备单链靶DNA，用荧光染料标记，PCR产物在DNA芯片上杂交，无需进一步纯化。我们的工作提高了基堆杂交对错配判别的灵敏度，简化了程序。作为模型实验，利用该改进技术在寡核苷酸芯片上鉴定人白细胞抗原(HLA)-A等位基因2601、2902和0206的质粒模板。现在可以应用这种简单、快速、敏感和可靠的碱基堆叠杂交技术来检测临床诊断和其他应用中微阵列上的DNA序列变化。
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 (T-m) value of 36degreesC, in the detection of DNA sequence variations at 40degreesC. 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.