Methods and Compositions for Efficient Nucleic Acid Sequencing

Abstract

Disclosed are novel methods and compositions for rapid and highly efficient nucleic acid sequencing based upon hybridization with two sets of small oligonucleotide probes of known sequences. Extremely large nucleic acid molecules, including chromosomes and non-amplified RNA, may be sequenced without prior cloning or subcloning steps. The methods of the invention also solve various current problems associated with sequencing technology such as, for example, high noise to signal ratios and difficult discrimination, attaching many nucleic acid fragments to a surface, preparing many, longer or more complex probes and labelling more species.

1 . A nucleic acid assay comprising: 
 a) hybridizing two sets of oligonucleotides of known length and sequence with nucleic acid fragments substantially longer than each oligonucleotide    b) ligating two oligonucleotides one from each set when contiguously hybridized on a nucleic acid fragment.    
     
     
         2 . The assay of  claim 1  wherein one oligo set is labeled and comprises substantially all possible oligonucleotides of a predetermined base length.  
     
     
         3 . The assay of  claim 1  wherein degenerate bases are positioned on one or both ends of at least one oligonucleotide set.  
     
     
         4 . The assay of  claim 1  which is performed on a solid support in the form of an array.  
     
     
         5 . The assay of  claim 2  wherein the label from ligated labeled probes is detected on a solid support.  
     
     
         6 . The assay of  claim 2  wherein each labeled oligonucleotide has a degradable nucleotide bond.  
     
     
         7 . A method of detecting nucleotide sequences in a target nucleic acid comprising: 
 testing ligation of a majority, substantially all or all probe pairs between two sets of oligonucleotide probes on a target nucleic acid template, wherein one set of oligonucleotide probes provides only 3′ OH group and the other set of oligonucleotide probes provides only 5′ PO 4  group for said probe pair ligation, thereby allowing only direction specific ligation, and wherein one or both of said sets of oligonucleotide probes comprises all or substantially all possible oligonucleotides of a predetermined base length, and    detecting ligated oligonucleotide probes to determine nucleotide sequences in said target nucleic acid that are complementary to specified bases in ligated oligonucleotide probes.    
     
     
         8 . The method of  claim 7  wherein one set of oligonucleotide probes is labeled and probes labeled with different labels are used simultaneously.  
     
     
         9 . The method of  claim 8  wherein label from the ligated labeled oligonucleotide probes is detected on a solid support.  
     
     
         10 . A method of analyzing a nucleic acid target comprising the step of testing ligation of all or substantially all pairs of probes from two sets of oligonucleotide probes using a target nucleic acid as template, wherein either one of said sets has all or substantially all possible probes of a given informative length or both sets of oligonucleotide probes have all or substantially all possible probes of a given informative length that may be the same or different between two sets, wherein one set of oligonucleotide probes is labeled and wherein at least two oligonucleotide probes having different labels are tested together.  
     
     
         11 . A method of determining a nucleotide sequence of a target nucleic acid, the method comprising the steps of: 
 providing an array of probe:fragment complexes, wherein each fragment in each of such complexes is a portion of the target nucleic acid and is longer than the probe in the complex, said probe of known specified sequence;    covalently joining a labeled probe of known specified sequence to probes of the probe:fragment complexes to form probe-probe:fragment complexes whenever said labeled probe hybridizes adjacent a probe of a probe:fragment complex; and    detecting the label in the bound probe-probe:fragment complexes to identify at least one nucleotide sequence in each of said fragments on the bases of specified probe sequences.    
     
     
         12 . The method of  claim 11  wherein the set of labeled probes comprises all or substantially all sequences of a given informative length and labeled probes have either one or more specified or non-specified or universal base positions at a terminus that participates in covalent joining or a terminus that does not participate in covalent joining.  
     
     
         13 . The method of  claim 11  wherein label from the covalently joined labeled probes is detected on a solid support.  
     
     
         14 . The method of  claim 11  further comprising the step of washing under conditions appropriate for discriminating detection of covalently joined oligonucleotides.  
     
     
         15 . The method of  claim 11  wherein at least two probes having different labels are hybridized in the same reaction.  
     
     
         16 . A method of detecting one or more sequences in a plurality of nucleic acid fragments comprising: 
 performing ligation of a first set of labeled oligonucleotides to second set of oligonucleotide molecules present in support bound or arrayed hybridization complexes of target nucleic acid fragments and said second set of oligonucleotide molecules, wherein the first set of labeled oligonucleotide comprises all or substantially all possible oligonucleotides of a given specified length, and none, some or all of labeled oligonucleotides in said first set are mixed if labeled with different labels, and wherein labeled oligonucleotide in said first set have either one or more specified or non-specified or universal bases at positions at the ligated terminus of said oligonucleotide, and    detecting the label of ligated oligonucleotides present on the support to identify at least one nucleotide sequence in a majority of said target nucleic acid fragments on the bases of specified portions of ligated oligonucleotides.    
     
     
         17 . The method of  claim 16  wherein each labeled oligonucleotide has a degradable nucleotide bond.  
     
     
         18 . A method for identifying nucleic acid sequences in nucleic acid fragments comprising 
 a) providing an array of hybridization complexes of nucleic acid fragments and first oligonucleotide wherein said nucleic acid fragments are longer than said first oligonucleotide and include adjacent non-hybridized sequences;    b) ligating a second oligonucleotide to said first oligonucleotide, wherein said second oligonucleotide hybridizes with no or tolerable mismatches adjacent to said first oligonucleotide    c) detecting the presence of ligated first and second oligonucleotides in the array; and    d) identifying nucleic acid sequences in the nucleic acid fragment from the sequences of ligated oligonucleotides    
     
     
         19 . The method of  claim 18  wherein the second oligonucleotide is labeled.  
     
     
         20 . The method of  claim 18  where the detecting step is performed by observing location of the label on the array.  
     
     
         21 . The method of  claim 20  wherein two or more different labels are observed in locations in the array.  
     
     
         22 . A method of producing simultaneously hybridization complexes of nucleic acids on multiple arrays of immobilized oligonucleotides comprising the steps of 
 a) providing a support with multiple arrays of immobilized oligonucleotides;    b) applying nucleic acid samples the on multiple arrays under discriminating hybridization conditions,    c) forming complementary hybridization complexes simultaneously on the multiple arrays    
     
     
         23 . The method of  claim 22  further comprising the step of removing non-hybridized nucleic acid molecules by washing simultaneously multiple arrays on the said support.  
     
     
         24 . The method of  claim 22  wherein different nucleic acid samples are applied at almost every array.  
     
     
         25 . A method of determining a sequence in a target polynucleotide by assembling overlapping oligonucleotide sequences detected by ligation of two oligonucleotide probes hybridized to adjacent positions on said target polynucleotide  
     
     
         26 . The method of  claim 25  wherein ligation reactions are performed on a support.  
     
     
         27 . The method of  claim 26  where ligated labeled oligonucleotide probes are detected on a support.  
     
     
         28 . A method for making a support with multiple nucleic acid arrays comprising: 
 a) providing a solid support for making an array of nucleic acid arrays with space areas between arraying areas,    b) preparing nucleic acid arrays in arraying areas, and    c) creating, before or after step b, physical or hydrophobic barriers in the space areas between arraying areas to allow separate assay reactions for each array.    
     
     
         29 . The method of  claim 28  wherein space areas are less them 2 mm wide.  
     
     
         30 . A method for making a support with multiple nucleic acid arrays comprising: 
 a) providing a solid support for making nucleic acid arrays,    b) preparing multiple nucleic acid arrays on said support with space areas between arraying areas, and    c) creating, before or after step b, physical or hydrophobic barriers in the space areas between arraying areas to allow separate assay reactions for each array.    
     
     
         31 . An apparatus for identifying nucleic acid sequences comprising of: 
 a) a plurality of oligonucleotides with detectable labels, said oligonucleotide having all or substantially all base variants of a given number of specified bases, the given number being equal or smaller than 10;    b) a ligation agent that can ligate said labeled oligonucleotides with oligonucleotides present in hybridization complexes with nucleic acid fragments longer than said oligonucleotides in said complex;    c) means for delivery of labeled oligonucleotides and oligonucleotide ligation agent to said hybridization complexes; and    d) a reader that can detect the label from said labeled oligonucleotides after ligation of labeled oligonucleotides to oligonucleotides in said hybridization complexes.    
     
     
         32 . The apparatus of  claim 31  further comprising one or more computers with programs that control delivery of labeled oligonucleotides and the reader.  
     
     
         33 . The apparatus of  claim 31  wherein labeled oligonucleotides are delivered to an array of hybridization complexes is used and an array reader is used to detect labels.  
     
     
         34 . The apparatus of  claim 31  which allows for successive use of said array of hybridization complexes in different ligation reactions of labeled oligonucleotides.  
     
     
         35 . An apparatus for detecting sequences in target nucleic acids comprising of: 
 a) a set of labeled oligonucleotides that can detect 10 or less bases adjacent to oligonucleotides forming an array of hybridization complexes with nucleic acids fragments, wherein the fragments are longer than oligonucleotide in said hybridization complexes;    b) a ligation agent that can ligate said labeled oligonucleotides and oligonucleotides in said hybridization complexes;    c) means for delivery of labeled oligonucleotides and an oligonucleotide ligation agent to said hybridization complexes; and    c) a reader that can detect label from said labeled oligonucleotides after ligation of labeled oligonucleotides to oligonucleotides in said hybridization complexes.    
     
     
         36 . The apparatus of  claim 35  further comprising one or more computers with programs that control delivery of labeled oligonucleotides and the reader.  
     
     
         37 . The apparatus of  claim 35  wherein at least two subsets of labeled oligonucleotides each labeled with different label are used  
     
     
         38 . An apparatus for identifying nucleic acid sequences comprising of: 
 a) a plurality of oligonucleotides with detectable labels;    b) a plurality of unlabeled oligonucleotides;    c) a ligation agent that can ligate said labeled oligonucleotides and unlabeled oligonucleotides present in contiguous hybridization complexes with nucleic acid fragments longer than combined length of hybridized oligonucleotides;    d) means for delivery of labeled and unlabeled oligonucleotides and oligonucleotide ligation agent to react with nucleic acid fragments;    e) a reader that can detect the label from said labeled oligonucleotides after ligation of labeled and unlabeled oligonucleotides in said hybridization complexes.    
     
     
         39 . The apparatus of  claim 38  wherein plurality of labeled probes has all or substantially all possible sequences of a given number specified bases, the given number being equal or smaller than 10.  
     
     
         40 . The apparatus of  claim 38  wherein the reader comprises a CCD detector.  
     
     
         41 . The apparatus of  claim 38  further comprising one or more computers with programs that control delivery of labeled oligonucleotides and the reader.  
     
     
         42 . The apparatus of  claim 38  which allows for successive use of said array of hybridization complexes in different ligation reactions of labeled oligonucleotides.  
     
     
         43 . An apparatus for assaying target nucleic acids comprising 
 a) a substrate with an array of oligonucleotide arrays separated by barriers    b) means to deliver target nucleic acids in each of said oligonucleotide arrays and form hybridization complexes with said oligonucleotides    
     
     
         44 . The apparatus of  claim 43  further comprising means to wash said array of arrays of hybridization complexes and means that control delivery of said target nucleic acid or said washing.