Multiplex amplification and separation of nucleic acid sequences on a bioelectronic microchip using asymmetric structures
AssigneeNanogen/Becton Dickinson Partnership
InventorsCarl F. Edman, Michael I. Nerenberg, Lorelei P. Westin, John J. Carrino
Life of this patent
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Abstract
A method for amplifying nucleic acids is provided wherein detection of amplified species is enhanced by the use of asymmetric amplification. Such amplification is made asymmetric by using divergent ratios of amplification primers or by using non-extending and/or non-cleavable amplification primers. Detection of the amplicons is improved because maintenance of single stranded species of amplicons during amplification facilitates their direct capture by immobilized probes without having to include denaturing steps.
Claims47
A method for the strand displacement amplification, multiplex assaying, and detection of target nucleic acids of interest using a bioelectronic microchip comprising the steps of:
- a. introducing at least one of the target nucleic acids of interest onto a bioclectronic microchip having a plurality of electronically addressable capture sites;
- b. asymmetrically amplifying the target nucleic acids to form amplicons, wherein unequal populations of complementary first and second amplified strands of the target nucleic acids are formed, and wherein the target nucleic acids are amplified using at least one set of amplification primers that includes an unequal effective concentration ratio of extendable and cleavable upstroam primer to extendable and cleavable downstream primer, wherein the upstream and downstream primers are specific for the first and the second stand of the target nucleic acids, respectively;
- c. capturing the amplicons of the target nucleic acids onto specified capture sites of the microchip by a capture probe that has specificity for the amplicons;
- and
- d. detecting the presence of the captured amplicons.
A method for the strand displacement amplification, multiplex assaying, and detection of target nucleic acids of interest using a bioelectronic microhip comprising the steps of:
- a introducing at least one of the target nucleic acids of interest onto a bioelectronic microchip having a plurality of electronically addressable capture sites;
- b. addressing the target nucleic acids of interest to at least one of the capture sites;
- c. asymmetrically amplifying the target nucleic acids to form amplicons, wherein unequal populations of complementary first and second amplified strands of the target nucleic acids are formed, and wherein the target nucleic acids are amplified using at least one set of amplification primers that includes an unequal effective concentration ratio of extendable and cleavable upstream primer to extendable and cleavable downstream primer, wherein the upstream and downstream primers are specific for the first and the second strand of the target nucleic acids, respectively;
- d. capturing the amplicons of the target nucleic acids onto specified capture sites of the microchip by a capture probe that has specificity for the amplicons;
- and
- e. detecting the presence of the captured amplicons.
Dependencies are inferred from the claim text.