Enzymatic ligation of nucleic acids

The method of any one of the preceding claims, wherein ligation is performed by a ligase listed in Table 1A, 1B or 1C.

The method of any one of the preceding claims, wherein the probe and the primer are not hybridized to the same polynucleotide (“template”) if they are different polynucleotides,

The method of any one of the preceding claims, wherein ligation is achieved with a small footprint ligase (“SFL”) at substantial efficiency, and optionally

• (a) at least 10% of the probe or primer is ligated, or
• (b) the SFL ligates the proximal termini of the primer and the probe at least 50% as efficiently as the SFL can ligate the proximal termini of the primer and a corresponding octanucleotide whose N proximal residues are identical to the probe, and whose distal 12-N residues are perfectly complementary to the template.

The method of any one of the preceding claims, wherein the SFL is CV ligase, or alternatively the SFL is not CV ligase or a derivative thereof.

The method of any one of the preceding claims, wherein the primer is not more than 6 nucleotides in length, for example not more 5, 4, 3 or 2 nucleotides.

The method of any one of the preceding claims, wherein the template, if present, is not more than 11 nucleotides in length, for example not more 10, 9, 8, 7, 6, 5, 4, 3 or 2 nucleotides.

The method of any one of the preceding claims, wherein the proximal terminus of the probe is its 3′ terminus and the proximal terminus of the primer is its 5′ terminus.

The method of any one of the preceding claims, wherein the probe is labeled.

The method of any one of the preceding claims, wherein the primer or template is labeled.

The method of any one of the preceding claims, wherein at least two of the probe, primer and template are labeled. The method of any one of the preceding claims, wherein at least one of the probe, the template and/or the primer is immobilized.

The method of any one of the preceding claims, wherein the ligation is repeated at least once.

A method of template-dependent ligation of an oligonucleotide (“probe”) with a polynucleotide (“primer”), where the length of the probe is less than 8 nucleotides.

A kit comprising a small footprint ligase (“SFL”) and an oligonucleotide probe less than N nucleotides in length, where N is from 2 to 7.

A mutant Hin (Haemophilus Influenzae) DNA ligase comprising a sequence of DLX, DLXd or DLXd2 shown in Table 1C.

A mutant Hin DNA ligase consisting essentially of a sequence of DLX, DLXd or DLXd2 shown in Table 1C.

A mutant Hin DNA ligase that has at least 70% identity to Hin D ligase sequence provided in Table 1C or in GenBank Accession No. P44121, which ligase comprises an amino acid mutation at position 193 of Hin D ligase sequence provided in Table 1C or in GenBank Accession No. P44121, optionally wherein said amino acid mutation consists of changing the glycine at position 193 to aspartic acid or glutamic acid.