Broude N E, Sano T, Smith C L, Cantor C R
Center for Advanced Biotechnology, Boston University, MA 02215.
Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3072-6. doi: 10.1073/pnas.91.8.3072.
An enhanced version of DNA sequencing by hybridization (SBH), termed positional SBH (PSBH), has been developed. PSBH uses duplex probes containing single-stranded 3' overhangs, instead of simple single-stranded probes. Stacking interactions between the duplex probe and a single-stranded target should provide enhanced stringency in distinguishing perfectly matched 3' sequences. A second enhancement is the use of enzyme-catalyzed steps, instead of pure physical hybridization. The feasibility of this scheme has been investigated using biotinylated duplex probes containing single-stranded 5-base 3' overhangs, immobilized on streptavidin-coated magnetic beads. Ligation of a single-stranded target, hybridized to the single-stranded region of the duplex probes, provided enhanced discrimination of perfectly matched targets from those containing mismatches. In distinction to the serious complications caused by base composition effects in ordinary SBH, there was little effect of base composition in PSBH. The hardest mismatch to discriminate was the one furthest from the phosphodiester bond formed by ligation. However, mismatches in this position were efficiently discriminated by 3' extension of the duplex probe using a template-dependent DNA polymerase. These results demonstrate that PSBH offers considerable promise to facilitate actual implementations of SBH.
已经开发出一种称为位置杂交测序（PSBH）的杂交测序（SBH）增强版本。PSBH使用含有单链3'突出端的双链体探针，而不是简单的单链探针。双链体探针与单链靶标之间的堆积相互作用应能在区分完全匹配的3'序列时提供更高的严谨性。第二个改进是使用酶催化步骤，而不是单纯的物理杂交。已使用固定在链霉亲和素包被的磁珠上的含有单链5碱基3'突出端的生物素化双链体探针研究了该方案的可行性。与双链体探针单链区域杂交的单链靶标的连接，提高了对完全匹配靶标与含有错配靶标的区分能力。与普通SBH中碱基组成效应引起的严重并发症不同，PSBH中碱基组成的影响很小。最难区分的错配是离连接形成的磷酸二酯键最远的错配。然而，使用模板依赖性DNA聚合酶对双链体探针进行3'延伸可有效区分该位置的错配。这些结果表明，PSBH为促进SBH的实际应用提供了很大的前景。
