Co-operativity of hexamer ligation

被引:15
作者
Kaczorowski, T
Szybalski, W
机构
[1] UNIV WISCONSIN,MCARDLE LAB CANC RES,SCH MED,MADISON,WI 53706
[2] UNIV GDANSK,DEPT MICROBIOL,PL-80822 GDANSK,POLAND
关键词
SPEL-6; T4 DNA ligase; point mutant detection; Rhodothermus marinus DNA ligase;
D O I
10.1016/S0378-1119(96)00324-1
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
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.
引用
收藏
页码:189 / 193
页数:5
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