Primer extension detection methods on active electronic microarrays

Abstract

This invention relates to methods for detecting the extent of hybridization of a nucleic acid in a sample to a probe nucleic acid sequence by electronically hybridizing the nucleic acid in the sample to the probe, utilizing the hybridized nucleic acid as a template in a nucleic acid polymerase reaction to extend the bound probe and incorporate a labeled nucleotide, and detecting the labeled product. This invention also relates to methods of detecting the extent of hybridization of a plurality of nucleic acids in a sample to a plurality of nucleic acid probes using similar methods.

1 . A method of detecting the extent of hybridization of a nucleic acid in a sample to a probe nucleic acid sequence, the method comprising: 
 (a) electronically hybridizing the nucleic acid in a sample to a nucleic acid probe bound to a support at a predetermined location;    (b) utilizing the hybridized nucleic acid as a template in a nucleic acid polymerase reaction to extend the bound probe, whereby a labeled nucleotide is incorporated into the extended probe; and    (c) detecting the labeled nucleotide incorporated into the extended bound probe at the predetermined location.    
     
     
         2 . The method of  claim 1 , wherein the labeled nucleotide comprises a labeling moiety selected from the group consisting of fluorescent moieties, colorigenic moieties, chemiluminescent moieties, and affinity moieties.  
     
     
         3 . The method of  claim 2 , wherein the labeled nucleotide comprises a fluorescent moiety.  
     
     
         4 . The method of  claim 1 , wherein the nucleic acid polymerase reaction is a DNA polymerase reaction.  
     
     
         5 . The method of  claim 1 , wherein the nucleic acid polymerase reaction is a reverse-transcriptase reaction.  
     
     
         6 . The method of  claim 1 , wherein a control sequence probe is also bound to the support at the location.  
     
     
         7 . The method of  claim 3 , wherein the fluorescent moiety is selected from the group consisting of cyanine dye moieties, Bodipy Texas Red moieties, rhodamine moieties, fluorescein moieties, and cumarin moieties.  
     
     
         8 . A method of detecting the extent of hybridization of a plurality of nucleic acids in a sample to a plurality of nucleic acid probes, the method comprising: 
 (a) electronically hybridizing the nucleic acid in the sample to a plurality of nucleic acid probes bound to a support at a two or more predetermined locations, wherein: 
 i) the nucleic acids in the sample are electronically hybridized to nucleic acid probes at two or more locations on the support, and  
 ii) the sequence of at least one nucleic acid probe on a first location is different from the sequence of the nucleic acid probes on a second location.  
   (b) utilizing the hybridized nucleic acids as a templates in a nucleic acid polymerase reaction to extend the bound probes, whereby a labeled nucleotide is incorporated into the extended probes; and    (c) detecting the labeled nucleotide incorporated into the extended bound probes at the predetermined location.    
     
     
         9 . The method of  claim 8 , wherein the labeled nucleotide comprises a labeling moiety selected from the group consisting of fluorescent moieties, colorigenic moieties, chemiluminescent moieties, and affinity moieties.  
     
     
         10 . The method of  claim 9 , wherein the labeled nucleotide comprises a fluorescent moiety.  
     
     
         11 . The method of  claim 8 , wherein the fluorescent moiety is selected from the group consisting of cyanine dye moieties, Bodipy Texas Red moieties, rhodamine moieties, fluorescein moieties, and cumarin moieties.  
     
     
         12 . The method of  claim 11 , wherein the fluorescent moiety is a cyanine dye moiety selected from the group consisting of Cy5 and Cy3.  
     
     
         13 . The method of  claim 8 , wherein the nucleic acid polymerase reaction is a DNA polymerase reaction.  
     
     
         14 . The method of  claim 8 , wherein the nucleic acid polymerase reaction is a reverse-transcriptase reaction.  
     
     
         15 . The method of  claim 8 , wherein the two or more locations each comprise at least one nucleic acid probe with the same sequence.  
     
     
         16 . The method of  claim 15 , wherein the nucleic acid probe with the same sequence is a control sequence probe.  
     
     
         17 . The method of  claim 8 , wherein the nucleic acids in the sample are electronically hybridized to nucleic acid probes at five or more locations on the support, and the sequence of at least one nucleic acid probe at each of the five locations is different from the sequences of the nucleic acid probes at the other locations.  
     
     
         18 . The method of  claim 8 , wherein the nucleic acids in the sample are electronically hybridized to nucleic acid probes at ten or more locations on the support, and the sequence of at least one nucleic acid probe at each of the ten locations is different from the sequences of the nucleic acid probes at the other locations.  
     
     
         19 . The method of  claim 8 , wherein the nucleic acids in the sample are electronically hybridized to nucleic acid probes at twenty or more locations on the support, and the sequence of at least one nucleic acid probe at each of the twenty locations is different from the sequences of the nucleic acid probes at the other locations.  
     
     
         20 . The method of  claim 8 , wherein the nucleic acids in the sample are electronically hybridized to nucleic acid probes at forty or more locations on the support, and the sequence of at least one nucleic acid probe at each of the forty locations is different from the sequences of the nucleic acid probes at the other locations.  
     
     
         21 . The method of  claim 10 , wherein the fluorescent moiety is a cyanine dye moiety selected from the group consisting of Cy5 and Cy3.