Systems, methods, and compositions for enhancing the specificity of nucleic acid hybridization

Abstract

Systems, methods and compositions of matter according to the present invention, can be used in capture/enrichment, gene expression profiling and targeted sequencing. Provided are systems, methods and compositions concerning the enhancement of nucleic acid hybridization specificity and controlling the shapes of melting curves revealed by nucleic acid hybrid pairs to optimize nucleic acid analysis. These systems, methods and compositions comprise producing a positively charged surface or surface coating, on the surface of microarray slides or other types of surfaces similarly purposed, which enhances melting curve analysis to the point of allowing detection or differentiation of small changes in sequences between nucleic acid binding partners. The accuracy or resolution of melting curve analysis was to be sufficient to distinguish between the melting of perfect matched dsDNA and dsDNA with the smallest possible change in sequence, a one base pair mismatch.

What is claimed is: 
     
         1 . A kit for nucleic acid hybridization, comprising:
 a solid support comprising a positively charged surface capable of binding a first nucleic acid; and,   an ionic buffer;   wherein the first nucleic acid when bound to the positively charged surface, is capable of hybridizing to a second nucleic acid to produce a hybridized nucleic acid pair;   the hybridized nucleic acid pair when heated sufficiently, is capable of revealing a bi-phasic melting curve shape;   whereby the ionic buffer comprises an ion concentration sufficient to provide a shielding effect and modulates hydrogen bond attraction between the first nucleic acid bound positively charged surface and the second nucleic acid;   wherein the positively charged surface of the solid support changes the kinetics by lowering melting temperature (Tm), narrowing the temperature range of melting between one basepair mismatch and perfect match.   
     
     
         2 . The kit of  claim 1 , wherein the positively charged surface comprises a chemical selected from the group consisting of polyethyleneimine, epoxide, amine and any chemical compound with a positive charge. 
     
     
         3 . The kit of  claim 2 , wherein the positively charged chemical is polyethyleneimine. 
     
     
         4 . The kit of  claim 3 , wherein the polyethyleneimine is present in the amount from about 1% to about 10% vol/vol. 
     
     
         5 . The kit of  claim 1 , wherein the first nucleic acid is a segment of DNA or RNA. 
     
     
         6 . The kit of  claim 1 , wherein the first nucleic acid is a DNA or RNA fragment. 
     
     
         7 . The kit of  claim 1 , wherein the second nucleic acid comprises a target nucleotide. 
     
     
         8 . The kit of  claim 1 , wherein the solid is selected from the group consisting of polystyrene, microbeads, glass, metal, charcoal, colloidal gold, bentonite, polypropylene, plastics and silica. 
     
     
         9 . The kit of  claim 8 , wherein the solid is a glass. 
     
     
         10 . The kit of  claim 9 , wherein the glass is a micro array slide. 
     
     
         11 . The kit of  claim 10 , wherein the micro array slide comprises from about 10 to about 4.2 million probes. 
     
     
         12 . The kit of  claim 1 , wherein the first nucleic acid includes a label. 
     
     
         13 . The kit of  claim 1 , further comprising reagents for PCR amplification. 
     
     
         14 . The kit of  claim 1 , wherein the first nucleic acid comprises a primer. 
     
     
         15 . A kit for detecting a target nucleotide of interest, the kit comprising:
 a solid support comprising a positively charged surface capable of binding a first nucleic acid, the positively charged surface comprising polyethyleneimine; and,   an ionic buffer;   wherein the first nucleic acid when bound to the positively charged surface, is capable of hybridizing to the target nucleotide to produce a hybridized nucleic acid pair;   the hybridized nucleic acid pair when heated sufficiently, is capable of revealing a bi-phasic melting curve shape;   whereby the ionic buffer comprises an ion concentration sufficient to provide a shielding effect and modulates hydrogen bond attraction between the first nucleic acid bound positively charged surface and the target nucleotide;   wherein the positively charged surface of the solid support changes the kinetics by lowering melting temperature (Tm), narrowing the temperature range of melting between one basepair mismatch and perfect match to detect the presence of the target nucleotide.   
     
     
         16 . The kit of  claim 15 , wherein the target nucleotide comprises nucleic acid from a disease or condition. 
     
     
         17 . A kit for detecting the presence of wild type and mutations of the KRAS gene comprising:
 a solid support comprising a positively charged surface capable of binding a first nucleic acid;   wherein the first nucleic acid comprises at least two probes, the a first probe comprising wild type KRAS genes and a second probe comprising mutant KRAS genes;   whereby the mutant KRAS gene comprises at least one of 12 different activating mutations in KRAS codons 12 and 13;   wherein the first nucleic acid when bound to the positively charged surface, is capable of hybridizing to a second nucleic acid to produce a hybridized nucleic acid pair;   whereby the second nucleic acid comprises a test sample from a patient;   the hybridized nucleic acid pair when heated sufficiently, is capable of revealing a bi-phasic melting curve shape;   whereby the ionic buffer comprises an ion concentration sufficient to provide a shielding effect and modulates hydrogen bond attraction between the first nucleic acid bound positively charged surface and the target nucleotide;   wherein the positively charged surface of the solid support changes the kinetics by lowering melting temperature (Tm), narrowing the temperature range of melting between one basepair mismatch and perfect match to detect the presence of wild type and mutations of the KRAS gene in the test sample.   
     
     
         18 . The kit of  claim 17 , wherein the first solution comprises a positively charged chemical selected from the group consisting of polyethyleneimine, epoxide, amine and any chemical compound with a positive charge. 
     
     
         19 . The kit of  claim 18 , wherein the positively charged chemical is polyethyleneimine. 
     
     
         20 . The kit of  claim 17 , further comprising reagents for PCR amplification.