Solution-based methods and materials for sequence analysis by hybridization

Abstract

Novel solution-based methods and materials, including apparatus, for sequence analysis by hybridization are provided.

What is claimed is:  
     
         1 . A method of detecting a sequence of a target nucleic acid, comprising: 
 (a) contacting a target nucleic acid with one or more mixtures of a plurality of oligonucleotide probe molecules of predetermined length and predetermined sequence, wherein each probe molecule comprises an information region and at least two probe molecules have different information regions, under conditions which produce, on average, more probe:target hybridization with probe molecules which are perfectly complementary to the target nucleic acid in the information region of the probe molecules than with probe molecules which are mismatched in the information region, wherein the target nucleic acid is not attached to a support, and wherein the probe molecules are not attached to a support;    (b) detecting probe molecules that hybridize with the target nucleic acid, using a reader capable of detecting an individual probe molecule; and    (c) detecting a sequence of the target nucleic acid by overlapping sequences of the information regions of at least two of the probe molecules contacted with the target in step (a).    
     
     
         2 . The method of  claim 1  wherein at least two mixtures are contacted simultaneously.  
     
     
         3 . The method of  claim 1  wherein at least two mixtures are contacted sequentially.  
     
     
         4 . The method of  claim 1  wherein the mixture of probe molecules comprises at least about 10 probe molecules distinct in their information regions.  
     
     
         5 . The method of  claim 1  wherein the mixture of probe molecules comprises at least about 100 probe molecules distinct in their information regions.  
     
     
         6 . The method of  claim 1  wherein the mixture of probe molecules comprises at least about 1,000 probe molecules distinct in their information regions.  
     
     
         7 . The method of  claim 1  wherein the mixture of probe molecules comprises at least about 10,000 probe molecules distinct in their information regions.  
     
     
         8 . The method of  claim 1  wherein the probe molecules comprise modified bases.  
     
     
         9 . The method of  claim 1  wherein multiple probe molecules are associated with identification tags.  
     
     
         10 . The method of  claim 9  wherein multiple probe molecules each have two identification tags.  
     
     
         11 . The method of  claim 9  wherein multiple probe molecules having the same information region are each associated with the same identification tag.  
     
     
         12 . The method of  claim 9  wherein at least two probe molecules having different information regions are associated with different identification tags.  
     
     
         13 . The method of  claim 9  wherein the probe molecules are divided into pools, wherein each pool comprises at least two probe molecules having different information regions, and all probe molecules within each pool are associated with the same identification tag which is unique to the pool.  
     
     
         14 . The method of  claim 9  wherein at least one identification tag is a bar code.  
     
     
         15 . The method of  claim 14  wherein the bar code is based on a property selected from the group consisting of size, shape, electrical properties, magnetic properties, optical properties, and chemical properties.  
     
     
         16 . The method of  claim 14  wherein the identification tag is a DNA bar code comprising modified bases.  
     
     
         17 . The method of  claim 14  wherein the identification tag is a molecular bar code.  
     
     
         18 . The method of  claim 14  wherein the identification tag is a nanoparticle bar code.  
     
     
         19 . The method of  claim 14  wherein the bar code comprises elements of varying length, each element comprising a preset number of unit tags.  
     
     
         20 . The method of  claim 1  wherein the target nucleic acid is associated with a separator tag.  
     
     
         21 . The method of  claim 1  wherein the probe molecules are associated with separator tags.  
     
     
         22 . The method of  claim 1  wherein before detection step (b), probe molecules that hybridize to the target nucleic acid are separated from probe molecules that do not hybridize to the target nucleic acid.  
     
     
         23 . The method of  claim 22  wherein probe molecules that do not hybridize to the target nucleic acid are eliminated by enzymatic digestion.  
     
     
         24 . The method of  claim 1  wherein step (b) further comprises counting the number of times probe molecules having the same information region are detected.  
     
     
         25 . The method of  claim 1  wherein a reader comprising a nanopore channel is used to detect probe molecules in step (b).  
     
     
         26 . The method of  claim 25  wherein sensing of electrical responses within or around the nanopore channel is used to detect probe molecules in step (b).  
     
     
         27 . The method of  claim 25  wherein the reader detects molecular bar codes in step (b).  
     
     
         28 . The method of  claim 1  wherein the probe molecules are associated with one or more tags that allow identification of 5′/3′ orientation of probe molecules during detection step (b).  
     
     
         29 . The method of  claim 1  wherein the sequence of the probe molecule(s) is detected in step (b).  
     
     
         30 . The method of  claim 29  wherein at least two probe molecules are associated with identification tags and the identification tags are also detected in step (b).  
     
     
         31 . A method of sequencing a target nucleic acid, comprising: 
 (a) contacting a target nucleic acid with one or more mixtures of a plurality of oligonucleotide probe molecules of predetermined length and predetermined sequence, wherein each probe molecule comprises an information region and at least two probe molecules have different information regions, under conditions which produce, on average, more probe:target hybridization with probe molecules which are perfectly complementary to the target nucleic acid in the information region of the probe molecules than with probe molecules which are mismatched in the information region, wherein the target nucleic acid is not attached to a support, and wherein the probe molecules are not attached to a support;    (b) covalently joining probe molecules that form contiguous probe:target hybrids that are perfectly complementary to the target in the information region of the probe molecules; and    (c) detecting covalently joined probe molecules, using a reader capable of detecting an individual probe molecule.    
     
     
         32 . The method of  claim 31  further comprising the step of: 
 (d) detecting a sequence of the target nucleic acid by overlapping at least two sequences generated by combining sequences of the information region of two probe molecules contacted with target nucleic acid in step (a).  
 
     
     
         33 . The method of  claim 31  wherein before detection step (c), covalently joined probe molecules are separated from probe molecules that have not been covalently joined.  
     
     
         34 . The method of  claim 1  or  18  wherein at least one nucleotide is added to the end of one or more probe molecules that hybridize to target nucleic acid using a polymerase or active fragment thereof.  
     
     
         35 . The method of  claim 34  wherein the probe molecules are contacted with a mixture of four different uniquely labeled nucleotides.  
     
     
         36 . The method of  claim 1  wherein target nucleic acids comprising an entire human genome are contacted with probe molecules.  
     
     
         37 . The method of any one of claims  1 ,  18  or  20  wherein a single nucleotide polymorphism is detected.  
     
     
         38 . A kit comprising a mixture of probe molecules, wherein about 100 or more probe molecules each have distinct information regions, wherein two or more of the sequences of said distinct information regions within the mixture overlap  
     
     
         39 . The kit of  claim 38  wherein about 10 5  or less probe molecules each have the same information region.  
     
     
         40 . The kit of  claim 38  wherein about 10 4  or less probe molecules each have the same information region.  
     
     
         41 . The kit of  claim 38  wherein each information region is represented by 10 4  or more probe molecules having the same information region.  
     
     
         42 . The kit of  claim 38  wherein at least two probe molecules having the same information region have the same identification tag.  
     
     
         43 . A kit comprising a set of mixtures of probe molecules, wherein about 100 or more probe molecules each have distinct information regions, wherein two or more of the sequences of said distinct information regions within the set overlap  
     
     
         44 . The kit of  claim 43  wherein about 10 5  or less probe molecules each have the same information region.  
     
     
         45 . The kit of  claim 43  wherein at least two probe molecules having different information regions are in the same pool and have the same identification tag.  
     
     
         46 . The kit of  claim 38  or  43  wherein about 5000 or more probe molecules each have the same information region.  
     
     
         47 . A tag which is a bar code comprising an alternating arrangement of elements of varying detectable properties, wherein consecutive elements have a difference in at least one of their detectable properties.  
     
     
         48 . The tag of  claim 47  wherein said elements comprise multiple unit tags of varying detectable properties and said elements vary in length.