Methods for multiplexing amplification reactions

Abstract

A two-step multiplex amplification reaction includes a first step which truncates the standard initial multiplex amplification round to “boost” the sample copy number by only a 100-1000 fold increase in the target. Following the first step the product is divided into optimized secondary single amplification reactions, each containing one of the primer sets that were used previously in the first or multiplexed booster step. The booster step can occur using an aqueous target nucleic acid or using a solid phase archived nucleic acid. In particular, nucleic acid sequences that uniquely identify E. Coli were identified using the multiplex amplification method.

1 .- 24 . (canceled) 
     
     
         25 . A method for amplifying multiple nucleic acid targets from a low abundant nucleic acid sample, the method comprising:
 a) performing a first amplification step, wherein the nucleic acid sample comprising the target nucleic acids is amplified such that the target nucleic acids are increased by 100-1000 fold, and wherein the first amplification step comprises a multiplex amplification reaction using a first set of at least five primer pairs, and wherein the first amplification step results in a pre-amplified nucleic acid sample comprising a plurality of first amplification products;   b) diluting the pre-amplified nucleic acid sample by at least 1:5; and,   c) performing a second amplification step on the pre-amplified nucleic acid sample from step (a) using at least two primer pairs used in step (a).   
     
     
         26 . The method of  claim 25 , wherein the nucleic acid sample is irreversibly bound to a solid phase material. 
     
     
         27 . The method of  claim 25 , wherein the nucleic acid sample is in aqueous solution. 
     
     
         28 . The method of  claim 25 , wherein the concentration of the primers used in the first amplification step is at a concentration that is at least 50× less than the concentration of the primer concentration of the primers used in the second amplification step. 
     
     
         29 . The method of  claim 25 , wherein the number of amplification cycles of the first amplification step is less than ten cycles. 
     
     
         30 . The method of  claim 25 , wherein the nucleic acid sample is DNA. 
     
     
         31 . The method of  claim 25 , wherein the nucleic acid sample is RNA. 
     
     
         32 . The method of  claim 25 , wherein the first amplification step is accomplished using a polymerase chain reaction. 
     
     
         33 . The method of  claim 25 , wherein the second amplification step is accomplished using a polymerase chain reaction. 
     
     
         34 . The method of  claim 25 , wherein a first set of 15-40 primer pairs are used in step (a). 
     
     
         35 . The method of  claim 25 , wherein the method further comprises a step for dividing the pre-amplified nucleic acid sample into at least two distinct amplification reactions prior to the second amplification step. 
     
     
         36 . The method of  claim 35 , wherein, for the second amplification step, the at least two distinct amplification reactions each comprise a different primer pair. 
     
     
         37 . The method of  claim 25 , wherein the at least two primers of the first set of at least five primer pairs of step (a) have melting temperatures that differ by at least 5 degrees. 
     
     
         38 . The method of  claim 25 , wherein each of the at least five primer pairs in step (a) is at a concentration that is about between 2-6 nM.