| Nucleic acid isothermal amplification technology has been widely applied in life science research and related areas. Our group developed new isothermal amplification technology, target circle-rolling circle amplification. To laid a solid foundation in pathogen detection, immunoassay fields, here we optimize the TC-RCA technology. Otherwise, the ligation effects of adaptor and template was crucial to TC-RCA specificity, thus the ligation mechanism of Thermus aquaticus (Taq) DNA ligase need to be further investigated, to provide theoretical basis for application of TC-RCA technology. The content and results are as follows.(1) In the study, adaptors and templates were specifically ligated to form circle double-stranded DNA structure, followed by the addition of specific primers to perform rolling circle amplification (RCA). The effects of adaptors mismatch, adaptor template concentration ratio, primer length and various restriction enzymes on TC-RCA were evaluated. The results in TC-RCA showed that non-specific amplification was eliminated only when the mismatch lead to unligation of template and adaptor; 100:1 adaptor template concentration ratio,12-15 mer length primer and restriction enzyme Hgal could meet the basic requirements for sensitivity and specificity.(2) The content and results of the mechanism of Taq DNA ligase are as follows: ① here we used thermal stable DNA hairpin of which containing a 11-nt-long overhang and another short single stranded DNA (5-10 nt) as substrate to evaluate the ligation effects of various parameters, including fragments length, temperature, structure of ligation sites, polyethylene glycol (PEG) concentrations, etc. The ligation results showed that the ligation efficiency of the 3’single-stranded DNA with hairpin Ⅰ is much higher than that of 5’single-stranded DNA with hairpin Ⅱ; the minimum length of single stranded DNA that could ligate to hairpin was 6 nt. The ligation efficiency increased gradually with the increasement of temperature between 20℃ and 65℃. The thermal stability of complex which formed by substrate and single stranded DNA was of little importance to ligation, since the ligation efficiency was still very high when the reaction temperature was much higher than the melting temperature (Tm) of the complex. The results also revealed that PEG 6000 improved the ligation efficiency of short fragments (6-7 nt) but did not with longer ones (8-10 nt), even suppress the ligation efficiency. ②More thermo stable hairpin structure was designed to ligate to longer fragments (15～20 nt). Taq DNA ligase still have a high efficiency at high temperatures (80℃). ③Design 3’-OH the end of stable hairpin structures with two bases mismatch (with the same sticky ends of the restriction endonuclease TspRI) as substrates for the ligation, to study the ability to distinguish different mismatches. The results show that the ability to discriminate mismatch at 3’-OH end was stronger than that at 5’end.In summary, through optimization of experimental condition, in the TC-RCA reaction process adaptors and templates ligated at high temperatures can increase efficiency and ability to recognize mismatches. To overcome the low ligation specificity of two adapters ligated to the template.we planned to use single stranded DNA ligate to the template to form DNA circle followed by RCA at relative high temperature. Besides, the applications of TC-RCA technique including detection of various pathogens and clinical diagnosis are still need to be further explored. |
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