Anchored strand displacement amplification on an electronically addressable microchip

Abstract

Methods and compositions of matter are provided for the strand displacement amplification of target nucleic acids of interest using primer pair sets that are anchored to electronically addressable capture sites on a microarray. The primer pair sets may be individually bound to the capture sites or may comprise a unique branched primer pair moiety. The anchored primers allow for the simultaneous multiplex capture, amplification and detection of a target nucleic acid derived from any sample source.

What is claimed is:  
     
         1 . A strand displacement amplification primer set comprising: 
 a. at least one pair of single stranded nucleic acid sequences, a first portion of said sequences having sequence complementary with a target nucleic acid of interest, the portion of said single stranded nucleic acid sequences of said pair having sequence complementary with a target nucleic acid of interest further having complementary sequence to said target nucleic acid wherein one of said pair is complementary to a plus strand of said target and the other of said pair is complementary to a minus strand of said target;    b. a second portion of said sequences encoding either a plus or a minus strand of at least one endonuclease restriction site; and    c. a molecular structure having covalent attachment moieties for binding a 5′ terminus of each single stranded nucleic acid sequence of said pair either covalently or non-covalently to a solid support.    
     
     
         2 . A primer set according to  claim 1  wherein said solid support is an electronically addressable microchip.  
     
     
         3 . A primer set according to  claim 1  used in association with an electronically addressable microchip for multiplex assaying a plurality of target nucleic acids of interest.  
     
     
         4 . A plurality of said set of primers according to  claim 3  wherein any one primer set of said plurality having sequence complementary to any one said target is electronically addressed to a separate capture pad site on said microchip.  
     
     
         5 . A primer set according to  claim 1  wherein said molecular structure of (c) is selected from the group consisting of Biotin, SH-bromoacetyl, Prolinx, amide bonds, a covalent linkage molecular element, and a non-covalent macromolecule attachment linking said primer to said solid support.  
     
     
         6 . A branched strand displacement amplification primer set comprising: 
 a. at least one pair of single stranded nucleic acid sequences, a first portion of said sequences having sequence complementary with a target nucleic acid of interest, the portion of said single stranded nucleic acid sequences of said pair having sequence complementary with a target nucleic acid of interest further having complementary sequence to said target nucleic acid wherein one of said pair is complementary to a plus strand of said target and the other of said pair is complementary to a minus strand of said target;    b. a second portion of said sequences encoding either a plus or a minus strand of at least one endonuclease restriction site; and    c. a branched molecular structure having covalent attachment moieties for binding a 5′ terminus of each single stranded nucleic acid sequence of said pair and attachments for binding either covalently or non-covalently to a solid support.    
     
     
         7 . A primer set according to  claim 6  wherein said solid support is an electronically addressable microchip.  
     
     
         8 . A primer set according to  claim 6  used in association with an electronically addressable microchip for multiplex assaying a plurality of target nucleic acids of interest.  
     
     
         9 . A plurality of said set of primers according to  claim 8  wherein any one primer set of said plurality having sequence complementary to any one said target is electronically addressed to a separate capture pad site on said microchip.  
     
     
         10 . A primer pair according to  claim 6  wherein said branched molecular structure of (c) comprises moieties selected from the group consisting of lysine, polyethylene glycol-derivatives as space elements, polyamino acids, and nucleic acids.  
     
     
         11 . A kit for carrying out anchored SDA reactions for use on a bioelectronic microchip comprising: 
 a. one or more oligonucleotides for use on a bioelectronic microchip specific for Factor V, Hemochromotosis, or a bacterium, which oligonucleotides comprise amplification primers, bumper primers, capture probes, and/or signal probes selected from the group consisting of Seq. Id. Nos. 20, 21, 31, 32, 45, 46, 51, 52, 57, and 58.    
     
     
         12 . A kit according to  claim 11  wherein said signal probes are labeled with a detectable label.