Study On The Dependence Of Non - Template Dependence And The Correlation Of High - Level Structure Of Template

Rolling Circle Amplification (RCA) with templates of circular DNA is a common replication way which is applied to whole genome amplification of single cell. But the template independent amplification(TIDA) of RCA usually occurs when template is omitted, or at low template concentrations. TIDA will confuse the analysis of results and bring about bad influence. Although some studies have also reported the related content, the sequence and source keep unclear.To obtain the sequences and make sure the source of TIDA, we make it by plasmid construction and DNA sequencing. We constructed the plasmid included the fragments of TIDA by the restriction endonuclease digestion of TIDA product. As a result, seven partial sequences from six positive clones showed high similarities with the genes from species of Rhodococcus equi, Rhodococcus erythropolis, Elizabethkingia, Phenylobacterium zucineum, Rubrivivax gelatinosus and Sordaria macrospora respectively. The sources of TIDA were dust in laboratory.To make sure the source of TIDA, we made use of persistent RCA. But the reaction kinetics curve of RCA indicated that the plateau emerged after more than four hours. As a result, the specific production would not become more and TIDA occurred. The reaction system was built to control TIDA and increase the output.At the same time, the relationship between reaction efficiency of RCA and molecular size of template was studied and it indicated that the reaction efficiency of RCA descended as the size become bigger and bigger.