Kaczorowski T, Szybalski W
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison 53706, USA.
Gene. 1996 Nov 7;179(1):189-93. doi: 10.1016/s0378-1119(96)00324-1.
The SPEL-6 (sequential rimer elongation by ligation of 6-mers) procedure is based on the assembly of DNA primers by ligation of three or more hexamers taken from a library of 4096 hexamers. In this way, the synthesized primers enable DNA sequencing by primer walking. Ligation by both T4 DNA ligase and Rhodothermus marinus thermophilic DNA ligase is highly cooperative. Sequencing ladders obtained with 18-60-nucleotide (nt) primers (produced by ligation of three to ten hexamers using T4 DNA ligase) were all of high quality, with no spurious bands. R. marinus DNA ligase requires at least seven hexamers for successful primer synthesis. Long primers (up to 60 nt), which are easy to obtain, especially by automated ligation, offer a definite advantage in DNA priming in regions with pronounced secondary structure. Moreover, the SPEL-6 procedure for DNA sequencing reduces the sequencing effort manifold. An additional application of hexamer ligation is the detection of point mutants, as described here.
SPEL-6（通过连接六聚体进行序列引物延伸）程序基于从4096个六聚体文库中选取的三个或更多六聚体的连接来组装DNA引物。通过这种方式，合成的引物能够通过引物步移进行DNA测序。T4 DNA连接酶和嗜热栖热放线菌嗜热DNA连接酶的连接具有高度协同性。使用18 - 60个核苷酸（nt）的引物（通过使用T4 DNA连接酶连接三到十个六聚体产生）获得的测序梯质量都很高，没有假带。嗜热栖热放线菌DNA连接酶成功合成引物至少需要七个六聚体。长引物（长达60 nt）易于获得，特别是通过自动连接，在具有明显二级结构的区域进行DNA引发时具有明显优势。此外，用于DNA测序的SPEL-6程序大大减少了测序工作量。如本文所述，六聚体连接的另一个应用是点突变的检测。
