Evaluating single-base-pair discriminating capability of planar oligonucleotide microchips using a non-equilibrium dissociation approach
Evaluating single-base-pair discriminating capability of planar oligonucleotide microchips using a non-equilibrium dissociation approach
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DOI：
10.1111/j.1462-2920.2004.00648.x
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发表时间：
2004-11-01
影响因子：
5.1
通讯作者：
Liu, WT
中科院分区：
文献类型：
--
作者：
Li, ESY;Ng, JKK;Liu, WT
使用同时比较所有探针-靶双链体的非平衡解离曲线和解离温度（T（d）s）的方法评价基于平面rRNA的寡核苷酸微阵列用于单碱基对辨别的能力。使用三组不同特异性水平的16 S rRNA基因特异性探针沿着其对应探针，用于在特定外部（靠近末端）和各种内部位置与其靶具有一个或两个错配（MM）的各组。基于T-d方法和鉴别指数（DI）的标准被证明能够区分PM与内部MM双链体，但并不总是用于外部MM双链体。分离PM双链体与具有两个和一个内部MM的双链体的最大DI通常分别发生在约5- 10 ℃和10- 15 ℃的温度下，高于PM双链体的T（d）s。洗涤缓冲液类型和盐浓度，MM数量和位置在统计学上影响解离曲线，T-d和单碱基对辨别能力。进一步证明了平面微芯片的可重复使用潜力。
The capability of planar rRNA-based oligonucleotide microarrays for single-base-pair discrimination was evaluated using an approach that compares the non-equilibrium dissociation profiles and dissociation temperatures (T(d)s) of all probe-target duplexes simultaneously. Three sets of 16S rRNA gene specific probes at different levels of specificity were used along with their counter probes for individual sets having either one or two mismatches (MM) to their targets at specific external (next to terminus) and various internal positions. Criteria based on the T-d approach and a discrimination index (DI) were proven to be competent in discriminating PM from internal MM duplexes, but not always for external MM duplexes. Maximal DI for separating PM duplexes from ones with two and one internal MM usually occurred at temperatures approximately 5-10degreesC and 10-15degreesC, respectively, higher than the T(d)s of the PM duplexes. Washing buffer type and salt concentration, and MM number and position were shown statistically to affect dissociation profiles, T-d, and single-base-pair discriminating capability. The reusability potential of the planar microchip was further demonstrated.