Multiplex amplification and separation of nucleic acid sequences on a bioelectronic microchip using asymmetric structures

An improved method for strand displacement amplification of one or more single-stranded target nucleic acids of interest, the method comprising:

• a) contacting each single-stranded target nucleic acid of interest with enzymes and reagents necessary to support strand displacement amplification, including at least one set of amplification primers that includes a first extendible and cleavable upstream SDA primer and a second extendable and cleavable downstream SDA primer, wherein both primers comprise an endonuclease recognition sequence, wherein the first SDA primer is specific for the target nucleic and the second SDA primer is specific for a nucleic acid sequence complementary to a portion of the target nucleic acid downstream from the first SDA primer, and wherein the effective concentration ratio of the first SDA primer to the second SDA primer is unequal;
• b) asymmetrically amplifying the target nucleic acids by strand displacement amplification, wherein unequal populations of complementary first and second amplicon species are formed;
• and
• c) producing single stranded amplicon species for detection, using asymmetrical amplification rather than heat or chemical denaturation.