Patent ID: 7851148

Claim:
A method for multiplex primer-based amplification of a target sequence from a plurality of agents, said target sequence being different for each agent, said method comprising: a. carrying out a first amplification reaction for each target sequence to be amplified using i) as a template, a nucleic acid from each of said plurality of agents, said nucleic acid containing said target sequence; ii) a first pair of target enrichment primers hybridizing to said nucleic acid and bracketing said target sequence; iii) a second pair of target enrichment primers hybridizing to said nucleic acid and bracketing said target sequence, said second pair of target enrichment primers being located proximate to said target sequence and one of the second pair of target enrichment primers comprising at its 5′ end a binding tag corresponding to the sequence of one of a pair of target amplification primers and the other of the second pair of target enrichment primers comprising at its 5′ end a binding tag corresponding to the sequence of the other of said pair of target amplification primers, wherein the second pair of target enrichment primers binds to the inside of the first set of target enrichment primers; and iv) amplification reagents and conditions for said first amplification reaction such that the first amplification reaction generates a plurality of first amplification products, wherein at least a portion of the first amplification products contain said target sequence and at least one complement of the binding tag for one of said target enrichment primers thereby forming at least one binding site for at least one of said target amplification primers; and b. carrying out a second amplification reaction for each target sequence to be amplified using i) as a template, said portion of the first amplification products containing said at least one binding site for at least one of said target amplification primers; ii) at least one of said first pair of target amplification primers binding to its corresponding binding sites on said portion of said first amplification products; and iii) amplification reagents and conditions for said second amplification reaction such that the second amplification reaction generates a plurality of second amplification products containing the target sequence.