Methods and compositions for detection or quantification of nucleic acid species

Abstract

The present invention provides a method for detecting a target nucleic acid species including the steps of providing an array of probes affixed to a substrate and a plurality of labeled probes wherein each labeled probe is selected to have a first nucleic acid sequence which is complementary to a first portion of a target nucleic acid and wherein the nucleic acid sequence of at least one probe affixed to the substrate is complementary to a second portion of the nucleic acid sequence of the target, the second portion being adjacent to the first portion; applying a target nucleic acid to the array under suitable conditions for hybridization of probe sequences to complementary sequences; introducing a labeled probe to the array; hybridizing a probe affixed to the substrate to the target nucleic acid; hybridizing the labeled probe to the target nucleic acid; affixing the labeled probe to an adjacently hybridized probe in the array; and detecting the labeled probe affixed to the probe in the array.

What is claimed is:  
     
         1 . A method for confirming sequencing results, comprising the steps of: 
 obtaining a sequence from a nucleic acid using SBH;    identifying a set of probes that are complementary and not exactly complementary to the sequence of the nucleic acid;    hybridizing the probes to the nucleic acid under conditions that allow the differentiation of perfect matches from one base mismatches;    confirming that the probes do not form perfect matches with the nucleic acid.    
     
     
         2 . The method of  claim 1 , wherein the SBH is format I SBH.  
     
     
         3 . The method of  claim 1 , wherein the SBH is format III SBH.  
     
     
         4 . The method of  claim 1 , wherein the set of probes are not exactly complementary to the sequence of the nucleic acid.  
     
     
         5 . A method for confirming sequencing results, comprising the steps of: 
 obtaining a sequence from a nucleic acid using SBH;    selecting at least one primer for the nucleic acid;    sequencing the nucleic acid with the primer using Sanger-sequencing;    comparing the sequence of the nucleic acid derived from SBH to the sequence for the nucleic acid derived from Sanger sequencing.    
     
     
         6 . A method for ordering a plurality of Sfs from a nucleic acid sequence, comprising the steps of: 
 obtaining the sequence from the nucleic acid using SBH;    identifying a plurality of primers from the sequence of the plurality of Sfs, whereby the primers can initiate a replication reaction on the nucleic acid that will read through a branch point;    sequencing the nucleic acid with the primers using Sanger-sequencing;    comparing the sequence derived by Sanger-sequencing of the nucleic acid around the branch point to the sequences of the Sfs, whereby the order of the Sfs is determined.    
     
     
         7 . A plurality of probes for analyzing a nucleic acid wherein the plurality of probes are used to interrogate the nucleic acid under conditions whereby the plurality of probes can be differentiated from each other.  
     
     
         8 . The probes of  claim 7 , wherein the nucleic acid has a known sequence and probes are labeled with a label.  
     
     
         9 . The plurality of probes of  claim 7 , wherein the plurality of probes are labeled with a plurality of different labels, whereby the probes can be differentiated from each other by the different labels attached to the probes.  
     
     
         10 . A set of probes for analyzing a nucleic acid, comprising a plurality of pools of probes wherein each pool is used to interrogate a nucleic acid, and wherein the plurality of probes are labeled with a plurality of different labels, whereby the probes in each pool can be differentiated from each other by the different labels attached to the probes.  
     
     
         11 . The set of probes in  claim 9 , wherein the plurality of different labels are a plurality of different radioisotopes.  
     
     
         12 . The set of probes of  claim 9 , wherein the plurality of different labels are a plurality of different flourescent molecules.  
     
     
         13 . The set of probes of  claim 9 , wherein the plurality of different labels are a plurality of different EMLs.  
     
     
         14 . The set of probes in  claim 10 , wherein the plurality of different labels are a plurality of different radioisotopes.  
     
     
         15 . The set of probes of  claim 10 , wherein the plurality of different labels are a plurality of different flourescent molecules.  
     
     
         16 . The set of probes of  claim 10 , wherein the plurality of different labels are a plurality of different EMLs.  
     
     
         17 . A method for analyzing a nucleic acid, comprising the steps of: 
 providing an array of oligonucleotide probes;    introducing a sample nucleic acid to the array;    adding a plurality of labeled probes to the array under conditions that allow the differentiation of perfect matches from one base mismatches,;    adding ligase to the array;    incubating the ligase, labeled probes, sample nucleic acid and array probes under conditions whereby labeled probe is ligated to array probes when the labeled probe is adjacent to the array probe on the sample nucleic acid; and    detecting the labeled probes that have been ligated to the array.    
     
     
         18 . The method of  claim 17 , further comprising the step of removing unligated labeled probe after the incubation step.  
     
     
         19 . The method of  claim 18 , wherein the nucleic acid has a known sequence and plurality of probes are labeled with a label.  
     
     
         20 . The method of  claim 19 , wherein the label is selected from the group consisting of a radioisotope, a flourescent molecule, and an EML.  
     
     
         21 . The method of  claim 18 , wherein the plurality of probes are labeled with a plurality of different labels, whereby the probes can be differentiated from each other by the different labels attached to the probes.  
     
     
         22 . The method of  claim 21 , wherein the plurality of different labels are a plurality of different radioisotopes.  
     
     
         23 . The method of  claim 21 , wherein the plurality of different labels are a plurality of different flourescent molecules.  
     
     
         24 . The method of  claim 21 , wherein the plurality of different labels are a plurality of different EMLs.  
     
     
         25 . A method for analyzing a plurality of nucleic acids, comprising the steps of: 
 obtaining a sample comprising the plurality of nucleic acids, wherein a target nucleic acid is present at least in a ratio of one part to ninety nine parts of a nucleic acid that is homologous to the target and differs by at least one nucleotide from the target;    selecting a set of probes that will identify the target nucleic acid;    mixing the sample and the probes under conditions that allow the differentiation of perfect matches from one base mismatches;    identifying whether the probes form a perfect match with a nucleic acid in the sample.    
     
     
         26 . An appartus for analyzing a nucleic acid, comprising: 
 a first array of nucleic acids;    a second array of nucleic acids;    a material disposed between the first and second arrays that prevents the mixing of the nucleic acids in the first array with the nucleic acids in the second array.    
     
     
         27 . The apparatus of  claim 26 , wherein the nucleic acids in the second array are labeled oligonucleotide probes.  
     
     
         28 . The apparatus of  claim 27 , wherein the nucleic acids in the first array are a plurality of sample nucleic acids.  
     
     
         29 . A method for analyzing a target nucleic acid, comprising the steps of: 
 providing an array of bound probes of known sequence fixed to a substrate;    providing an array of labeled probes of known sequence;    providing a material disposed between the arrays of bound and labeled probes that prevents the mixing of the probes in the bound and labeled probe arrays;    adding the target nucleic acid to the labeled probes;    removing the material between the bound and labeled probes so that the labeled probes bound probes and target nucleic acids are mixed together under conditions that allow the differentiation of perfect matches from one base mismatches;    joining the bound and labeled probes that are hybridized to adjacent sites in the target nucleic acid;    detecting the labeled probe that has been joined to the bound probe array.    
     
     
         30 . A method for analyzing a target nucleic acid, comprising the steps of: 
 providing an array of bound probes of known sequence fixed to a substrate;    providing an array of labeled probes of known sequence;    providing a material disposed between the arrays of bound and labeled probes that prevents the mixing of the probes in the bound and labeled probe arrays;    removing the material between the bound and labeled probes so that the labeled probes and bound probes are mixed together;    adding the target nucleic acid to the labeled and bound probes under conditions that allow the differentiation of perfect matches from one base mismatches;    ligating the bound and labeled probes that are hybridized to adjacent sites in the target nucleic acid;    detecting the labeled probe that has been ligated to the bound probe array.    
     
     
         31 . A method for analyzing a target nucleic acid, comprising the steps of: 
 providing an array of bound probes of known sequence fixed to a substrate, wherein some of the bound probes are complementary to a plurality of first portions of the target nucleic acid;    providing an array of labeled probes of known sequence, wherein some of the labeled probes are complementary to a plurality of second portions of the target nucleic acid and wherein specific second portions are adjacent to specific first portions;    providing a material disposed between the arrays of bound and labeled probes that prevents the mixing of the probes in the bound and labeled probe arrays;    adding the target nucleic acid to the labeled probes;    removing the material between the bound and labeled probes so that the labeled probes bound probes and target nucleic acids are mixed together under conditions that allow the differentiation of perfect matches from one base mismatches;    joining the bound and labeled probes that are bound at the specific first and second portions in the target nucleic acid;    detecting the labeled probe joined to the bound probe array.    
     
     
         32 . A method for analyzing a target nucleic acid, comprising the steps of: 
 providing an array of bound probes of known sequence fixed to a substrate, wherein some of the bound probes are complementary to a plurality of first portions of the target nucleic acid;    providing an array of labeled probes of known sequence, wherein some of the labeled probes are complementary to a plurality of second portions of the target nucleic acid and wherein specific second portions are adjacent to specific first portions;    providing a material disposed between the arrays of bound and labeled probes that prevents the mixing of the probes in the bound and labeled probe arrays;    removing the material between the bound and labeled probes so that the labeled probes and bound probes are mixed together;    adding the target nucleic acid to the labeled and bound probes under conditions that allow the differentiation of perfect matches from one base mismatches;    joining the bound and labeled probes that are bound at the specific first and second portions in the target nucleic acid;    detecting the labeled probe joined to the bound probe array.    
     
     
         33 . A method for analyzing a target nucleic acid, comprising the steps of: 
 providing an array of bound target nucleic acids;    providing an array of labeled probes of known sequence;    providing a material disposed between the arrays of bound target and labeled probes that prevents the mixing of the target nucleic acid and labeled probes;    removing the material between the bound target and labeled probes so that the labeled probes and bound target nucleic acids are mixed together under conditions that allow the differentiation of perfect matches from one base mismatches;    determining which labeled probes have formed perfect matches with the target DNA.