PMID:16302964

Citation	Kuhn, H and Frank-Kamenetskii, MD (2005) Template-independent ligation of single-stranded DNA by T4 DNA ligase. FEBS J. 272:5991-6000
Abstract	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 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.
Links	PubMed Online version:10.1111/j.1742-4658.2005.04954.x
Keywords	DNA Ligases/metabolism; DNA, Circular/chemistry; DNA, Circular/metabolism; DNA, Single-Stranded/chemistry; DNA, Single-Stranded/metabolism; Oligonucleotides/genetics; Oligonucleotides/metabolism; Polymerase Chain Reaction
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BPT4:DNLI		GO:0003909: DNA ligase activity	ECO:0000314:		F		In figure 2, single stranded DNA was mixed with T4 DNA ligase and oligonucleotides. In 2A, it was amplified using PCR. The resulting products were run through a gel. Lanes 1 and 6 were used as controls, either missing the ligase or the oligonucleotides. Lanes 3-5 showed the results from the PCR. The smear corresponds with the DNA ligase binding sequences of DNA to the experimental ssDNA, making the DNA longer and so the bands would have a larger masses. Figure 2B shows this better, as RCA was used to amplify the ssDNA, which is better to use for circular DNA. Distinct bands were seen due to this better fitting technique. The larger masses of the circular DNA also supports the fact that the DNA ligase can bind DNA ends together.	complete CACAO 13130

References

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