Nucleic acid detection methods using universal priming

Abstract

The present invention is directed to providing sensitive and accurate assays for genotyping with a minimum or absence of target-specific amplification.

We claim:  
     
         1 . A method of determining the identification of a nucleotide at a detection position in a target sequence comprising: 
 a) providing a first probe comprising: 
 i) an upstream universal priming site (UUP);  
 ii) an adapter sequence;  
 iii) a first target-specific sequence comprising a first base at a readout position; and  
 iv) a downstream universal priming site (DUP);  
   b) contacting said first probe with said target sequence under conditions whereby only if said first base is perfectly complementary to a nucleotide at said detection position is a first hybridization complex formed;    c) removing non-hybridized first probes;    d) denaturing said hybridization complex;    e) amplifying said first probe to generate a plurality of amplicons;    f) contacting said amplicons with an array of capture probes; and    g) determining the nucleotide at said detection position.    
     
     
         2 . A method according to  claim 1  wherein said amplicons comprise a label.  
     
     
         3 . A method according to  claim 1  further comprising: 
 a) providing a second probe comprising: 
 i) an upstream universal priming site (UUP);  
 ii) an adapter sequence;  
 iii) a second target-specific sequence comprising a second base at said readout position; and  
 iv) a downstream universal priming site (DUP);  
 
 b) contacting said second probe with said target sequence under conditions whereby only if said second base is perfectly complementary to a nucleotide at said detection position is a second hybridization complex formed;  
 c) removing non-hybridized second probes;  
 d) denaturing said second hybridization complex;  
 e) amplifying said second probe to generate a plurality of amplicons;  
 f) contacting said amplicons with an array of capture probes; and  
 g) determining the nucleotide at said detection position.  
 
     
     
         4 . A method of determining the identification of a nucleotide at a detection position in a target sequence comprising: 
 a) providing a plurality of readout probes each comprising: 
 i) an upstream universal priming site (UUP);  
 ii) an adapter sequence;  
 iii) a target-specific sequence comprising a unique base at a readout position; and  
 iv) a downstream universal priming site (DUP);  
   b) contacting said detection probes with said target sequence under conditions whereby only if said base at said readout position is perfectly complementary to a nucleotide at said detection position is a first hybridization complex formed;    c) removing non-hybridized first probes;    d) denaturing said first hybridization complex;    e) amplifying said detection probes to generate a plurality of amplicons;    f) contacting said amplicons with an array of capture probes; and    g) determining the nucleotide at said detection position.    
     
     
         5 . A method of determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain comprising said detection position and a second target domain adjacent to said detection position, wherein said method comprises: 
 a) hybridizing a first ligation probe to said first target domain, said first ligation probe comprising: 
 i) an upstream universal priming site (UUP); and  
 ii) a first target-specific sequence; and  
   b) hybridizing a second ligation probe to said second target domain, said second ligation probe comprising: 
 i) a downstream universal priming site (DUP); and  
 ii) a second target-specific sequence comprising a first base at an interrogation position;  
   wherein if said first base is perfectly complementary to said nucleotide at said detection position a ligation complex is formed and wherein at least one of said first and second ligation probes comprises an adapter sequence;    c) removing non-hybridized first probes;    d) providing a ligase that ligates said first and second ligation probes to form a ligated probe;    e) amplifying said ligated probe to generate a plurality of amplicons;    f) contacting said amplicons with an array of capture probes; and    g) determining the nucleotide at said detection position.    
     
     
         6 . A method of determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain comprising said detection position and a second target domain adjacent to said detection position, wherein said method comprises: 
 a) hybridizing a first ligation probe to said first target domain, said first ligation probe comprising: 
 i) an upstream universal priming site (UUP); and  
 ii) a first target-specific sequence; and  
   b) hybridizing a second ligation probe to said second target domain, said second ligation probe comprising: 
 i) a downstream universal priming site (DUP); and  
 ii) a second target-specific sequence comprising a first base at an interrogation position;  
   wherein if said first base is perfectly complementary to said nucleotide at said detection position a ligation complex is formed and wherein at least one of said first and second ligation probes comprises an adapter sequence;    c) removing non-hybridized first probes;    d) providing a ligase that ligates said first and second ligation probes to form a ligated probe;    e) hybridizing said ligated probe to a rolling circle (RC) sequence comprising: 
 i) an upstream priming sequence; and  
 ii) a downstream priming sequence;  
   f)providing a ligase that ligates said upstream and downstream priming sites to form a circular ligated probe;    g) amplifying said circular ligated probe to generate a plurality of amplicons;    f) contacting said amplicons with an array of capture probes; and    g) determining the nucleotide at said detection position.    
     
     
         7 . A method of determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain comprising said detection position and a second target domain adjacent to said detection position, wherein said method comprises: 
 a) hybridizing a rolling circle (RC) probe to said target sequence, said RC probe comprising: 
 i) an upstream universal priming site (UUP); and  
 ii) a first target-specific sequence;  
 iii) a second target-specific sequence comprising a first base at an interrogation position; and  
 iv) an adapter sequence;  
   wherein if said first base is perfectly complementary to said nucleotide at said detection position a ligation complex is formed;    c) providing a ligase that ligates said first and second ligation probes to form a ligated probe;    d) amplifying said ligated probe to generate a plurality of amplicons;    e) contacting said amplicons with an array of capture probes; and    f) determining the nucleotide at said detection position.    
     
     
         8 . A method according to  claim 7 , further comprising removing non-hybridized RC probe.  
     
     
         9 . A method according to  claim 1 ,  4 ,  5 ,  6  or  8  wherein said removing comprises: 
 a) enzymatically adding a binding ligand to said target sequence;  
 b) binding a hybridization complex comprising said target sequence comprising said binding ligand to a binding partner immobilized on a solid support;  
 c) washing away unhybridized probes; and  
 d) eluting said probe off said solid support.  
 
     
     
         10 . A method according to  claim 1 ,  4 ,  5 ,  6  or  8  wherein said removing is done using a double-stranded specific moiety.  
     
     
         11 . A method according to  claim 10  wherein said double-stranded specific moiety is an intercalator attached to a support.  
     
     
         12 . A method according to  claim 9  wherein said support is a bead.  
     
     
         13 . A method according to  claim 1 ,  4 ,  5 ,  6  or  7  wherein said amplifying is done by: 
 a) hybridizing a first universal primer to said UUP;  
 b) providing a polymerase and dNTPs such that said first universal primer is extended;  
 c) hybridizing a second universal primer to said DUP;  
 d) providing a polymerase and dNTPs such that said second universal primer is extended; and  
 e) repeating steps a) through d).  
 
     
     
         14 . A method according to  claim 1 ,  4 ,  5 ,  6  or  7  wherein said array comprises: 
 a) a substrate with a patterned surface comprising discrete sites; and  
 b) a population of microspheres comprising at least a first subpopulation comprising a first capture probe and a second subpopulation comprising a second capture probe.  
 
     
     
         15 . A method according to  claim 14  wherein said discrete sites comprise wells.  
     
     
         16 . A method according to  claim 14  or  15  wherein said substrate comprises a fiber optic bundle.  
     
     
         17 . A method of determining the identification of a nucleotide at a detection position in a genomic target sequence comprising: 
 a) attaching a library of genomic target sequences to a solid support;    b) adding at least one probe and an enzyme to form an extended primer;    c) denaturing said extended primer from said target sequence;    d) hybridizing said extended primer to an array comprising capture probes; and    e) determining said nucleotide at said detection position.    
     
     
         18 . A method according to  claim 17 , further comprising removing unhybridized probes.  
     
     
         19 . A method according to  claim 1 ,  4 ,  5 ,  6  or  7 , further comprising providing a support on which the target sequence is immobilized.  
     
     
         20 . A method according to  claim 19 , wherein said non-hybridized first probes are removed without removing said target sequence from said support.  
     
     
         21 . A method according to  claim 1 ,  4 ,  5 ,  6  or  7 , further comprising attaching said target sequence to a support.  
     
     
         22 . A method according to  claim 21 , wherein said target sequence is attached to said support by a method selected from the group consisting of labeling said target sequence with a functional attachment moiety, absorption of said target sequence on a charged support, direct chemical attachment of said target sequence to said support and photocrosslinking said target sequence to said support.  
     
     
         23 . A method according to  claim 1 ,  4 ,  5 ,  6  or  7 , wherein said support is selected from the group consisting of paper, plastic and tubes.  
     
     
         24 . A method of determining the identification of a nucleotide at a detection position in a target sequence comprising: 
 a) providing a support on which the target sequence is immobilized;    b) providing a first probe comprising: 
 i) an upstream universal priming site (UUP);  
 ii) an adapter sequence;  
 iii) a first target-specific sequence comprising a first base at a readout position; and  
 iv) a downstream universal priming site (DUP);  
   c) contacting said first probe with said target sequence under conditions whereby only if said first base is perfectly complementary to a nucleotide at said detection position is a first hybridization complex formed;    d) removing non-hybridized first probes;    e) denaturing said hybridization complex;    f) amplifying said first probe to generate a plurality of amplicons;    g) contacting said amplicons with an array of capture probes; and    h) determining the nucleotide at said detection position    
     
     
         25 . A method of determining the identification of a nucleotide at a detection position in a target sequence comprising: 
 a) providing a support on which the target sequence is immobilized;    b) providing a plurality of readout probes each comprising: 
 i) an upstream universal priming site (UUP);  
 ii) an adapter sequence;  
 iii) a target-specific sequence comprising a unique base at a readout position; and  
 iv) a downstream universal priming site (DUP);  
   c) contacting said detection probes with said target sequence under conditions whereby only if said base at said readout position is perfectly complementary to a nucleotide at said detection position is a first hybridization complex formed;    d) removing non-hybridized first probes;    e) denaturing said first hybridization complex;    f) amplifying said detection probes to generate a plurality of amplicons;    g) contacting said amplicons with an array of capture probes; and    h) determining the nucleotide at said detection position.    
     
     
         26 . A method of determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain comprising said detection position and a second target domain adjacent to said detection position, wherein said method comprises: 
 a) providing a support on which the target sequence is immobilized;    b) hybridizing a first ligation probe to said first target domain, said first ligation probe comprising: 
 i) an upstream universal priming site (UUP); and  
 ii) a first target-specific sequence; and  
   c) hybridizing a second ligation probe to said second target domain, said second ligation probe comprising: 
 i) a downstream universal priming site (DUP), and  
 ii) a second target-specific sequence comprising a first base at an interrogation position;  
   wherein if said first base is perfectly complementary to said nucleotide at said detection position a ligation complex is formed and wherein at least one of said first and second ligation probes comprises an adapter sequence;    d) removing non-hybridized first probes;    e) providing a ligase that ligates said first and second ligation probes to form a ligated probe;    f) amplifying said ligated probe to generate a plurality of amplicons;    g) contacting said amplicons with an array of capture probes; and    h) determining the nucleotide at said detection position.    
     
     
         27 . A method of determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain comprising said detection position and a second target domain adjacent to said detection position, wherein said method comprises: 
 a) providing a support on which the target sequence is immobilized;    b) hybridizing a first ligation probe to said first target domain, said first ligation probe comprising: 
 i) an upstream universal priming site (UUP); and  
 ii) a first target-specific sequence; and  
   c) hybridizing a second ligation probe to said second target domain, said second ligation probe comprising: 
 i) a downstream universal priming site (DUP); and  
 ii) a second target-specific sequence comprising a first base at an interrogation position;  
   wherein if said first base is perfectly complementary to said nucleotide at said detection position a ligation complex is formed and wherein at least one of said first and second ligation probes comprises an adapter sequence;    d) removing non-hybridized first probes;    e) providing a ligase that ligates said first and second ligation probes to form a ligated probe;    f) hybridizing said ligated probe to a rolling circle (RC) sequence comprising: 
 i) an upstream priming sequence; and  
 ii) a downstream priming sequence;  
   g) providing a ligase that ligates said upstream and downstream priming sites to form a circular ligated probe;    h) amplifying said circular ligated probe to generate a plurality of amplicons;    i) contacting said amplicons with an array of capture probes; and    j) determining the nucleotide at said detection position.    
     
     
         28 . A method of determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain comprising said detection position and a second target domain adjacent to said detection position, wherein said method comprises: 
 a) providing a support on which the target sequence is immobilized;    b) hybridizing a rolling circle (RC) probe to said target sequence, said RC probe comprising: 
 i) an upstream universal priming site (UUP); and  
 ii) a first target-specific sequence;  
 iii) a second target-specific sequence comprising a first base at an interrogation position; and  
 iv) an adapter sequence;  
   wherein if said first base is perfectly complementary to said nucleotide at said detection position a ligation complex is formed;    c) providing a ligase that ligates said first and second ligation probes to form a ligated probe;    d) amplifying said ligated probe to generate a plurality of amplicons;    e) contacting said amplicons with an array of capture probes; and    f) determining the nucleotide at said detection position.    
     
     
         29 . A method according to  claim 28 , further comprising removing unhybridized RC probe.