Methods and Compositions for the Analysis of Biological Molecules

Abstract

Provided herein are compositions and methods for analysis of nucleic acids, including, methods and compositions for genotyping, haplotyping, sequencing and performing other genetic and epigenetic analyses on nucleic acids, for example. In some embodiments, methods and compositions suitable for whole-genome sequencing on single molecules of nucleic acid are provided. In some embodiments, analysis of single molecules of nucleic acid are performed in conjunction with nanopores and/or nanopore devices.

1 . A method for analyzing a nucleic acid, which comprises:
 (a) contacting a target nucleic acid with a plurality of nucleic acid probes under conditions in which probes having a nucleotide sequence substantially complementary to a subsequence of the target nucleic acid can hybridize to the target nucleic acid (“complementary nucleotide sequence”), wherein:
 (i) the complementary nucleotide sequence is located at the 5′ or 3′ end of each probe; 
 (ii) the ends of each complementary nucleotide sequence of each probe in probe pairs are (1) adjacent to one another when hybridized to the target nucleic acid, (2) are adjacent to an intervening linker probe, (3) do not abut one another and intervening nucleotides are added by an enzyme capable of polymerizing nucleotides complementary to the target nucleic acid sequence; 
 (iii) each probe comprises a detector region having a polynucleotide sequence not substantially complementary to a subsequence of the target nucleic acid; 
 (iv) each detector region is located at the 5′ or 3′ end of each probe and on the end of the probe opposite the complementary nucleotide sequence; 
 (v) the end of a detector region of a first probe of a probe pair is in proximity to the end of a detector region of a second probe in another probe pair, with the proviso that the end of a detector region of one probe in two probe pairs is not in proximity to the end of the detector region of another probe; and 
 (vi) the first probe flanks the second probe when the first probe and second probe are hybridized to the target nucleic acid; 
   (b) ligating the ends of the of the complementary nucleotide sequences of probe pairs that are adjacent to one another when hybridized to the target nucleic acid;   (c) joining the ends of detector regions in proximity to one another, thereby forming a linked probe molecule; and   (d) determining the base sequence of the linked probe molecule, whereby the target nucleic acid is analyzed.   
     
     
         2 . The method of  claim 1 , wherein the target nucleic acid is analyzed by a method selected from the group consisting of: nucleic acid sequencing; copy number determination; methylation analysis; genotyping; haplotyping; detection of specific sequences; identification of insertions, deletions, or translocations; identification of single nucleotide polymorphisms and sequence variations; detection of allelic variance; measurement of phenotypic variance, and combinations thereof. 
     
     
         3 . The method of  claim 1 , wherein the target nucleic acid comprises DNA or RNA, or PNA. 
     
     
         4 . The method of  claim 1 , wherein the target nucleic acid is from a single sample. 
     
     
         5 . The method of  claim 1 , wherein the target nucleic acid comprises pooled samples. 
     
     
         6 . The method of  claim 1 , wherein the target nucleic acid is methylated DNA. 
     
     
         7 . The method of  claim 6 , wherein the target nucleic acid is treated with an agent that converts non-methylated nucleotide, or methylated nucleotide, in the target nucleic acid to a detectable entity. 
     
     
         8 . The method of  claim 1 , wherein the detector region of the probe comprises a detectable moiety. 
     
     
         9 . The method of  claim 1 , wherein the detectable moiety is selected from the group consisting of: a radioactive agent, a fluorescent agent, a light scattering agent, a molecular beacon, an affinity capture agent, a chemiluminescent agent, a protein agent, a peptide agent, a chromogenic agent, a biomolecule, and a combination thereof. 
     
     
         10 . The method of  claim 1 , wherein the detector portions of probes in proximity to one another are joined by chemical ligation or crosslinking. 
     
     
         11 . The method of  claim 10 , wherein the cross-linking is effected by UV light, a nonspecific cross-linking agent, a sequence specific cross-linking agent, or a combination thereof. 
     
     
         12 . The method of  claim 1 , wherein the base sequence of linked probes is determined by a nucleotide sequencing method. 
     
     
         13 . A kit for analyzing a nucleic acid, wherein the kit comprises:
 (a) a plurality of nucleic acid probes having a nucleotide sequence substantially complementary to a subsequence of the target nucleic acid can hybridize to the target nucleic acid (“complementary nucleotide sequence”), wherein:   (i) the complementary nucleotide sequence is located at the 5′ or 3′ end of each probe;   (ii) the ends of each complementary nucleotide sequence of each probe in probe pairs are (1) adjacent to one another when hybridized to the target nucleic acid, (2) are adjacent to an intervening linker probe, (3) do not abut one another and intervening nucleotides are added by an enzyme capable of polymerizing nucleotides complementary to the target nucleic acid sequence;   (iii) each probe comprises a detector region having a polynucleotide sequence not substantially complementary to a subsequence of the target nucleic acid;   (iv) each detector region is located at the 5′ or 3′ end of each probe and on the end of the probe opposite the complementary nucleotide sequence;   (v) when hybridized to the target nucleic acid, the end of a detector region of a first probe of a probe pair is in proximity to the end of a detector region of a second probe in another probe pair, with the proviso that the end of a detector region of one probe in two probe pairs is not in proximity to the end of the detector region of another probe; and   (vi) the first probe flanks the second probe when the first probe and second probe are hybridized to the target nucleic acid;   wherein the ends of detector regions in proximity to one another can be joined to form a linked probe molecule.   
     
     
         14 . A composition comprising a target nucleic acid and a plurality of nucleic acid probes, wherein probes having a nucleotide sequence substantially complementary to a subsequence of the target nucleic acid can hybridize to the target nucleic acid (“complementary nucleotide sequence”), wherein:
 (i) the complementary nucleotide sequence is located at the 5′ or 3′ end of each probe; 
 (ii) the ends of each complementary nucleotide sequence of each probe in probe pairs are (1) adjacent to one another when hybridized to the target nucleic acid, (2) are adjacent to an intervening linker probe, (3) do not abut one another and intervening nucleotides are added by an enzyme capable of polymerizing nucleotides complementary to the target nucleic acid sequence; 
 (iii) each probe comprises a detector region having a polynucleotide sequence not substantially complementary to a subsequence of the target nucleic acid; 
 (iv) each detector region is located at the 5′ or 3′ end of each probe and on the end of the probe opposite the complementary nucleotide sequence; 
 (v) the end of a detector region of a first probe of a probe pair is in proximity to the end of a detector region of a second probe in another probe pair, with the proviso that the end of a detector region of one probe in two probe pairs is not in proximity to the end of the detector region of another probe; and 
 (vi) the first probe flanks the second probe when the first probe and second probe are hybridized to the target nucleic acid; 
 wherein the ends of detector regions in proximity to one another are joined to form a linked probe molecule.