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Template‐independent ligation of single‐stranded DNA by T4 DNA ligase
Author(s) -
Kuhn Heiko,
FrankKamenetskii Maxim D.
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04954.x
Subject(s) - dna ligase , sequencing by ligation , ligation , ligase chain reaction , dna , microbiology and biotechnology , oligonucleotide , biology , chemistry , polymerase chain reaction , biochemistry , genomic library , gene , base sequence , multiplex polymerase chain reaction
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 0.1–10 n m , 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.