tailieunhanh - Báo cáo khoa học: Template-independent ligation of single-stranded DNA by T4 DNA ligase

T4 DNA ligase is one of the workhorses of molecular biology and used in various biotechnological applications. Here we report that this ligase, unlikeEscherichia coliDNA ligase,TaqDNA ligase and Ampligase, is able to join the ends of single-stranded DNA in the absence of any duplex DNA structure at the ligation site. | ềFEBS Journal Template-independent ligation of single-stranded DNA by T4 DNA ligase Heiko Kuhn and Maxim D. Frank-Kamenetskii Center for Advanced Biotechnology and Department of BiomedicalEngineering Boston University MA USA Keywords blunt-end ligation circularization of oligonucleotides competitive PCR nontemplated ligation rolling-circle amplification Correspondence H. Kuhn Center for Advanced Biotechnology and Department of BiomedicalEngineering Boston University 36 Cummington St. Boston MA 02215 USA Fax 1 617 3538501 Tel 1 617 3538492 E-mail hkuhn@ Received 11 July 2005 revised 30 August 2005 accepted 2 September 2005 doi T4 DNA ligase is one of the workhorses of molecular biology and used in various biotechnological applications. Here we report that this ligase unlike Escherichia coli DNA ligase Taq DNA ligase and Ampligase is able to join the ends of single-stranded DNA in the absence of any duplex DNA structure at the ligation site. Such nontemplated ligation of DNA oligomers catalyzed by T4 DNA ligase occurs with a very low yield as assessed by quantitative competitive PCR between 10 6 and 10 4 at oligonucleotide concentrations in the range nM and thus is insignificant in many molecular biological applications of T4 DNA ligase. However this side reaction may be of paramount importance for diagnostic detection methods that rely on template-dependent or target-dependent DNA probe ligation in combination with amplification techniques such as PCR or rolling-circle amplification because it can lead to nonspecific background signals or false positives. Comparison of ligation yields obtained with substrates differing in their strandedness at the terminal segments involved in ligation shows that an acceptor duplex DNA segment bearing a 3 -hydroxy end but lacking a 5 -phosphate end is sufficient to play a role as a cofactor in blunt-end ligation. DNA ligases play a pivotal role in the replication repair and recombination