BUFFER, METHOD AND METHOD FOR EVALUATING OPTIMAL CONDITIONS, AMPLIFYING TARGET NUCLEIC ACID SEQUENCES, AND APPLICATIONS THEREOF

摘要

An application buffer, for facilitating nucleic acid denaturation while maintaining the structure of the enzymes used, and for significantly improving the performance of the polymerase chain reaction is described, as well as applications thereof, particularly in a method for amplifying nucleic acid target sequences using several temperatures and in a method for evaluating optimal conditions to be implemented in said amplification method. The buffer comprises, in addition to standard amplification buffer components, a mixture of at least one isodestabilising agent (AI) and at least one denaturing agent (AD), in concentrations capable of reducing the melting point (Tm) of the double-stranded nucleic acid target sequence, according to formula (4): Tm( DEG C)=81.5+0.41(% G+C)- DELTA TmAI-675/N+16.6.logM- DELTA TmAD, wherein DELTA TmAI=f[(% G+C), (molarity of AI)] (formula 1), DELTA TmAD=d(% denaturing agent) (formula 3), M: molarity of one or more monovalent cations present in the buffer, N: number of nucleotides, and d: efficiency coefficient of denaturing agent (AD), for reducing at least the denaturation temperature (Td) of said double-stranded nucleic acid target sequence, while allowing for the proper performance of the primer extension enzyme reaction.