Method for amplification of targeted segments of nucleic acid using nested polymerase chain reaction

Claim 1

An improved method for performing a nested polymerase chain reaction to selectively amplify a target segment of nucleic acid from a sample nucleic acid reaction mixture, said method amplifying in the first stage a nucleic acid segment flanked by an outer primer pair and in the second stage a nucleic acid target segment flanked by an inner nested primer pair; the improvement comprising wherein by controlling the concentrations and annealing times of the outer and inner primer pairs in the first and second stages selective amplification of the target segment is accomplished during the second stage by the inner primer pair, wherein the entire volume of the first stage reaction mixture is used in the second stage without depletion or removal of the outer primers from the reaction mixture; the improved method comprises the steps of: adding a pair of outer primers to a sample nucleic acid reaction mixture to achieve a concentration of said outer primers which is described by P 011 ; repetitively performing the polymerase chain reaction utilizing an annealing time at each cycle which is described by t 1 ; adding a pair of inner primers to the sample nucleic acid reaction mixture to achieve a concentration of said inner primer which is described by P 022 ; and repetitively performing the polymerase chain reaction utilizing an annealing time at each cycle which is described by t 2 ; wherein P 011 , t 1 , P 022 and t 2 are selected according to the formulas ε.sub.max 1 (1-exp-(k.sub.1 P.sub.011 t.sub.1))>0.4 ε.sub.max 2 (1-exp-(k.sub.2 P.sub.022 t.sub.2))>0.4 ε.sub.max 1 (1-exp(-k.sub.1 P.sub.012 t.sub.2))<1/5 ε.sub.max 2 (1-exp(-k.sub.2 P.sub.022 t.sub.2)) wherein P 011 is the concentration of each of the outer primers in the first stage; P 022 is the concentration of each of the inner primers in the second stage; P 012 is the concentration of each of the outer primers in the second stage; t 1 is the annealing time in the first stage; t 2 is the annealing time in the second stage; k 1 is the second order rate constant for the formation of extension product from the outer primers; k 2 is the second order rate constant for the formation of extension product from the inner primers; ε max 1 is the maximum per cycle primer extension of the outer primer; and ε max 2 is the maximum per cycle extension of the inner primers.