Methods of enzymatic discrimination enhancement and surface bound double-stranded DNA

Abstract

Methods for discriminating between fully complementary hybrids and those that differ by one or more base pairs and libraries of unimolecular, double-stranded oligonucleotides on a solid support. In one embodiment, the present invention provides methods of using nuclease treatment to improve the quality of hybridization signals on high density oligonucleotide arrays. In another embodiment, the present invention provides methods of using ligation reactions to improve the quality of hybridization signals on high density oligonucleotide arrays. In yet another embodiment, the present invention provides libraries of unimolecular or intermolecular, double-stranded oligonucleotides on a solid support. These libraries are useful in pharmaceutical discovery for the screening of numerous biological samples for specific interactions between the double-stranded oligonucleotides, and peptides, proteins, drugs and RNA. In a related aspect, the present invention provides libraries of conformationally restricted probes on a solid support. The probes are restricted in their movement and flexibility using double-stranded oligonucleotides as scaffolding. The probes are also useful in various screening procedures associated with drug discovery and diagnosis. The present invention further provides methods for the preparation and screening of the above libraries.

1 - 16 . (canceled)  
     
     
         17 . A method for sequencing a target nucleic acid, said method comprising: 
 (a) combining: 
 (i) a substrate comprising an array of chemically synthesized and positionally distinguishable oligonucleotides each of which is complementary to a defined subsequence of preselected length;  
   and 
 (ii) a target nucleic acid which is longer than each of said probes; thereby forming target-oligonucleotide hybrid complexes of complementary subsequences of known sequence with a 3′ target overhang;  
   (b) contacting said target-oligonucleotide hybrid complexes with a ligase and a labeled, ligatable oligonucleotide probe;    (c) removing unbound target nucleic acid and labeled, unligated oligonucleotide probes; and    (d) determining which of said oligonucleotides contain said labeled, ligatable oligonucleotide probe as an indication of a subsequence which is complementary to a subsequence of said target nucleic acid.    
     
     
         18 . The method as recited in  claim 17  wherein said array of oligonucleotides recognizes substantially all possible subsequences of preselected length found in said target nucleic acid.  
     
     
         19 . The method as recited in  claim 17  wherein each oligonucleotide is of a length between about 6 and 20 bases.  
     
     
         20 . The method as recited in  claim 17  wherein each oligonucleotide is of a length between about 8 and 15 bases.  
     
     
         21 . The method as recited in  claim 17  wherein said array of oligonucleotides comprises about 1,000 different oligonucleotides.  
     
     
         22 . The method as recited in  claim 17  wherein said array of oligonucleotides comprises about 3,000 different oligonucleotides.  
     
     
         23 . The method as recited in  claim 17  wherein said array of oligonucleotides comprises about 10 4  different oligonucleotides.  
     
     
         24 . The method as recited in  claim 17  wherein said array of oligonucleotides comprises about 10 5  different oligonucleotides.  
     
     
         25 . The method as recited in  claim 17  wherein said array of oligonucleotides comprises about 10 6  different oligonucleotides.  
     
     
         26 . The method as recited in  claim 17  wherein said target nucleic acid is deoxyribonucleic acid (DNA).  
     
     
         27 . The method as recited in  claim 17  wherein said ligase is a member selected from the group consisting of T4 DNA ligase, ligases isolated from  E. coli  and ligases isolated from bacteriophages.  
     
     
         28 . The method as recited in  claim 17  wherein said ligase is T4 DNA ligase.  
     
     
         29 . A method for sequencing an unlabeled target oligonucleotide, said method comprising: 
 (a) combining: 
 (i) a substrate comprising an array of positionally distinguishable oligonucleotide probes each of which has a constant region and a variable region, said variable region capable of binding to a defined subsequence of preselected length;  
 (ii) a constant oligonucleotide having a sequence which is complementary to said constant region of said oligonucleotide probes;  
 (iii) a target oligonucleotide to be sequenced; and  
 (iv) a ligase, thereby forming target-oligonucleotide hybrid complexes of complementary subsequences of known sequence;  
   (b) contacting said target oligonucleotide-oligonucleotide probe hybrid complexes with a ligase and a pool of labeled, ligatable oligonucleotide probes of a preselected length, said pool of labeled, ligatable oligonucleotide probes representing all possible sequences of said preselected length;    (c) removing unbound target nucleic acid and labeled, unligated oligonucleotide probes; and    (d) determining which of said oligonucleotide probes contain said labeled, ligatable oligonucleotide probe as an indication of a subsequence which is complementary to a subsequence of said target oligonucleotide.    
     
     
         30 . A method for sequencing an unlabeled target oligonucleotide, said method comprising; 
 (a) contacting an unlabeled target oligonucleotide with a library of labeled oligonucleotide probes, each of said oligonucleotide probes having a known sequence and being attached to a solid support at a known position, to hybridize said target oligonucleotide to at least one member of said library of probes, thereby forming a hybridized library;    (b) contacting said hybridized library with a nuclease capable of cleaving double-stranded oligonucleotides to release from said hybridized library a portion of said labeled oliogonucleotide probes or fragments thereof; and    (c) identifying said positions of said hybridized library from which labeled probes or fragments thereof have been removed, to determine the sequence of said unlabeled target oligonucleotide.    
     
     
         31 - 59 . (canceled)  
     
     
         60 . A method of analyzing a target nucleic acid, comprising 
 providing an array of target-oligonucleotide hybrid complexes, wherein said array includes at least one target-oligonucleotide hybrid complex comprising an oligonucleotide hybridized to the target nucleic acid to be analyzed, said target nucleic acid having a suitable 3′ overhang to serve as a template for hybridization;    contacting the array with a pool of labeled oligonucleotides comprising all oligonucleotides of a preselected length;    contacting the array with a ligase whereby a labeled oligonucleotide complementary to the target nucleic acid is ligated to the at least one oligonucleotide hybridized to the target nucleic acid; and    detecting the labeled oligonucleotide.    
     
     
         61 . The method of  claim 60 , wherein the array of target:oligonucleotide hybrid complexes has been formed on a substrate comprising beads.  
     
     
         62 . The method of  claim 60 , wherein said ligase is selected from the group consisting of T4 DNA ligase, a ligase isolated from  E. coli , and a ligase isolated from a bacteriophage.  
     
     
         63 . The method of  claim 62 , wherein said ligase is T4 DNA ligase.  
     
     
         64 . The method of  claim 60 , wherein the labeled oligonucleotides are 6 nucleotides in length.  
     
     
         65 . The method of  claim 60 , wherein each of the labeled oligonucleotides comprises of fluorescent label.  
     
     
         66 . The method of  claim 64 , wherein the fluorescent label is covalently attached to the 5′ end of the oligonucleotide.