) The specificity of the thermostable Tth DNA ligase plays a vital role in determining the sensitivity of PCR/LDR and PCR/RE/LDR detection systems. Increased specificity has been acquired through detailed analysis of mutant ligases and modified substrates. We are developing a biochemical and molecular biological approach to study the fidelity of Thermus ligases.
Our specific aims are: (i) Constructing mutant thermostable ligases and testing for greater specificity. Site-directed mutagenesis of Tth ligase will be conducted on sites identified by the peptide sequence data obtained from affinity cleavage and protein sequence alignment of Thermus ligases from four homology groups. (ii) Testing the specificity of wild- type and mutant thermostable ligases for discriminating substrates containing different length mono-nucleotide repeats. Wild-type and mutant ligases will be screened for their ability to detect changes in mono- nucleotide repeats. (iii) Testing modified oligonucleotides for greater specificity during ligation. The effect of various near-3'-end Q analogues on ligation fidelity will be evaluated and action conditions optimized. (iv) Determining the sequence and specificity of additional thermostable ligases. Four geographic homology groups among Thermus species were proposed based on sequence comparisons of nine TagI isoschizomers. Ligase genes from three homology groups (USA, Portugal, New Zealand) will be sequenced and compared with the Tth DNA ligase which represents the Japan group. The corresponding new """"""""iso-ligases"""""""" will be over-expressed and characterized.
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