CONTROLLED INITIAL TARGET-DEPENDENT PRODUCTION OF TEMPLATES FOR LIGASE CHAIN REACTION

摘要

An improvement to the ligase chain reaction (LCR) method of selectively and geometrically ligating nucleic acid probes; before the LCR, a template-dependent controlled reaction increases the population of templates capable of supporting template-dependent ligation in LCR selectively in the presence of target nucleic acid, while substantially avoiding creation of any molecules capable of supporting template-dependent ligation in the absence of target nucleic acid. One way to control the initial reaction is to weight the reaction to create templates corresponding to only one of the two target strands, using e.g. template dependent ligation or polymerization. Another way to control the initial amplification is to create templates corresponding to both strands, but to prevent spurious creation of ligation templates by using a probe concentration low enough to avoid blunt end ligation or by using a method of amplification such as PCR that does not generate spurious ligation templates. 23