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.

What is claimed is:  
     
         1 . A method for determining the identification of a nucleotide at a detection position in a target sequence, comprising: 
 (a) hybridizing a target sequence with a probe set to form a hybridization complex, said probe set comprising a first probe having a first portion containing an upstream universal priming site (UUP), a second portion containing a first target specific sequence and a detection position, and a second probe having a first portion containing a downsteam universal priming site (DUP) and a second portion containing a second target specific sequence, wherein said probe set further comprises at least one adapter sequence;    (b) contacting said hybridization complex with a ligase to form a ligated hybridization complex;    (c) removing non-hybridized probe sets;    (d) amplifying said ligated hybridization complex with a universal primer pair to form amplicons;    (e) immobilizing said amplicons to a solid support;    (f) contacting said immobilized amplicons with a detection label, wherein the mass of said detection label is indicative of said nucleotide at said detection position, and    (g) determining a nucleotide at said detection position.    
     
     
         2 . The method of  claim 1 , wherein said probe set further comprises a plurality of first and second probes.  
     
     
         3 . The method of  claim 1 , wherein said first or second probe further comprises a partially double stranded nucleic acid.  
     
     
         4 . The method of  claim 1 , wherein said first or second probe further comprises a linker.  
     
     
         5 . The method of  claim 1 , wherein said first or second probe further comprises a nucleotide modification.  
     
     
         6 . The method of  claim 5 , wherein said nucleotide modification comprises a nuclease inhibitor.  
     
     
         7 . The method of  claim 5 , wherein said nucleotide modification comprises a nucleotide analog.  
     
     
         8 . The method of  claim 1 , further comprising removing said non-hybridized probes by contacting said hybridization complex with a nuclease.  
     
     
         9 . The method of  claim 8 , wherein said nuclease comprises an exonuclease.  
     
     
         10 . The method of  claim 1 , wherein said removing of said non-hybridized probes further comprises the steps: 
 (a) immobilizing said ligated hybridization complex to a solid support, and    (b) washing said ligated hybridization complex-immobilized to said solid support.    
     
     
         11 . The method of  claim 1 , wherein at least one primer of said universal primer pair further comprises biotin.  
     
     
         12 . The method of  claim 1 , wherein said solid support is selected from the group consisting of a bead, a reaction vessel and an array.  
     
     
         13 . The method of  claim 12 , wherein said reaction vessel further comprises a microtiter well.  
     
     
         14 . The method of  claim 12 , wherein said array further comprises a plurality of capture probes.  
     
     
         15 . The method of  claim 1 , wherein said detection label further comprises a secondary label.  
     
     
         16 . The method of  claim 15 , wherein said secondary label allows detection of said amplicons by separation.  
     
     
         17 . The method of  claim 1 , wherein said detection label comprises a primer.  
     
     
         18 . The method of  claim 17 , wherein said primer comprises a universal primer.  
     
     
         19 . The method of  claim 1 , wherein said detection label comprises one or more nucleotides.  
     
     
         20 . The method of  claim 1 , further comprising the step: 
 (a) extending one or more probes within said hybridization complex with a polymerase.    
     
     
         21 . The method of  claim 1 , further comprising immobilizing said target sequence to a solid support.  
     
     
         22 . The method of  claim 1 , further comprising amplifying said target sequence.  
     
     
         23 . A method for determining the identification of nucleotides at detection positions in a plurality of target sequences, comprising: 
 (a) hybridizing a plurality of target sequences with a plurality of probe sets to form a plurality of hybridization complexes, each of said probe sets within said plurality comprising a first probe having a first portion containing an upstream universal priming site (UUP), a second portion containing a first target specific sequence and a detection position, and a second probe having a first portion containing a downsteam universal priming site (DUP) and a second portion containing a second target specific sequence, wherein each of said probe sets within said plurality of probe sets further comprises at least one adapter sequence;    (b) contacting said plurality of hybridization complexes with a ligase to form ligated hybridization complexes;    (c) removing non-hybridized probe sets;    (d) amplifying said plurality of ligated hybridization complexes with a plurality of universal primer pairs to form a plurality of amplicons;    (e) immobilizing said plurality of amplicons to a solid support;    (f) contacting said plurality of immobilized amplicons with a plurality of detection labels, wherein the mass of said detection labels is indicative of a nucleotide at said detection position, and    (g) determining a nucleotide at said detection position.    
     
     
         24 . The method of  claim 23 , wherein said plurality of probe sets further comprise a plurality of first and second probes.  
     
     
         25 . The method of  claim 23 , wherein said first or second probes further comprise a partially double stranded nucleic acid.  
     
     
         26 . The method of  claim 23 , wherein said first or second probes further comprise a linker.  
     
     
         27 . The method of  claim 23 , wherein said first or second probes further comprise a nucleotide modification.  
     
     
         28 . The method of  claim 27 , wherein said nucleotide modification comprises a nuclease inhibitor.  
     
     
         29 . The method of  claim 27 , wherein said nucleotide modification comprises a nucleotide analog.  
     
     
         30 . The method of  claim 23 , further comprising removing said non-hybridized probe sets by contacting said ligated hybridization complexes with a nuclease.  
     
     
         31 . The method of  claim 30 , wherein said nuclease comprises an exonuclease.  
     
     
         32 . The method of  claim 23 , wherein said removing of said non-hybridized probe sets further comprises the steps: 
 (a) immobilizing said ligated hybridization complexes to a solid support, and    (b) washing said ligated hybridization complexes immobilized to said solid support.    
     
     
         33 . The method of  claim 23 , wherein at least one primer of said universal primer pair further comprises biotin.  
     
     
         34 . The method of  claim 23 , wherein said solid support is selected from the group consisting of a bead, a reaction vessel and an array.  
     
     
         35 . The method of  claim 34 , wherein said reaction vessel further comprises a microtiter well.  
     
     
         36 . The method of  claim 34 , wherein said array further comprises a plurality of capture probes.  
     
     
         37 . The method of  claim 23 , wherein said plurality of detection labels further comprise a plurality of secondary labels.  
     
     
         38 . The method of  claim 37 , wherein said plurality of secondary labels allow detection of said plurality of amplicons by separation.  
     
     
         39 . The method of  claim 23 , wherein said plurality of detection labels comprise a plurality of primers.  
     
     
         40 . The method of  claim 39 , wherein said plurality of primers comprise a plurality of universal primers.  
     
     
         41 . The method of  claim 23 , wherein said plurality of detection labels comprise one or more nucleotides.  
     
     
         42 . The method of  claim 23 , further comprising the step: 
 (a) extending one or more probes within said plurality of hybridization complexes with a polymerase.    
     
     
         43 . The method of  claim 23 , further comprising immobilizing said plurality of target sequences to a solid support.  
     
     
         44 . The method of  claim 23 , further comprising amplifying said plurality of target sequences.  
     
     
         45 . A method of determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain adjacent to said detection position and a second target domain comprising said detection position, wherein said method comprises: 
 (a) hybridizing a first ligation probe to said first target domain, said first ligation probe comprising: 
 (i) a first portion comprising an upstream universal priming site (UUP); and  
 (ii) a second portion comprising a first target-specific sequence comprising a first base at an interrogation position; and  
   (b) hybridizing a second ligation probe to said second target domain, said second ligation probe comprising: 
 (i) a third portion comprising a downstream universal priming site (DUP); and  
 (ii) a fourth portion comprising a second target-specific sequence; wherein if said first base is 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 a fifth portion comprising a distinct adapter sequence;  
   (c) providing a ligase that ligates said first and second ligation probes to form a ligated probe;    (d) removing non-hybridized probes;    (e) amplifying said ligated probe using said UUP and said DUP to generate a plurality of amplicons;    (f) immobilizing said amplicons to a solid support;    (g) contacting said immobilized amplicons with a detection label, wherein the mass of said detection label is indicative of said nucleotide at said detection position, and    (h) determining the nucleotide at said detection position.    
     
     
         46 . The method of  claim 45 , wherein said first or second ligation probe further comprises a plurality of first or second probes.  
     
     
         47 . The method of  claim 45 , wherein said first or second ligation probe further comprises a partially double stranded nucleic acid.  
     
     
         48 . The method of  claim 45 , wherein said first or second ligation probe further comprises a linker.  
     
     
         49 . The method of  claim 45 , wherein said first or second ligation probe further comprise a nucleotide modification.  
     
     
         50 . The method of  claim 49 , wherein said nucleotide modification comprises a nuclease inhibitor.  
     
     
         51 . The method of  claim 49 , wherein said nucleotide modification comprises a nucleotide analog.  
     
     
         52 . The method of  claim 45 , further comprising removing said non-hybridized probes by contacting said hybridization complex with a nuclease.  
     
     
         53 . The method of  claim 52 , wherein said nuclease comprises an exonuclease.  
     
     
         54 . The method of  claim 45 , wherein said removing of said non-hybridized probes further comprises the steps: 
 (a) immobilizing said ligated probe to a solid support, and    (b) washing said ligated probe immobilized to said solid support.    
     
     
         55 . The method of  claim 45 , wherein at least on primer of said universal primer pair further comprises biotin.  
     
     
         56 . The method of  claim 45 , wherein said solid support is selected from the group consisting of a bead, a reaction vessel and an array.  
     
     
         57 . The method of  claim 56 , wherein said reaction vessel further comprises a microtiter well.  
     
     
         58 . The method of  claim 56 , wherein said array further comprises a plurality of capture probes.  
     
     
         59 . The method of  claim 45 , wherein said detection label further comprises a secondary label.  
     
     
         60 . The method of  claim 59 , wherein said secondary label allows detection of said amplicons by separation.  
     
     
         61 . The method of  claim 45 , wherein said detection label comprises a primer.  
     
     
         62 . The method of  claim 61 , wherein said primer comprises a universal primer.  
     
     
         63 . The method of  claim 45 , wherein said detection label comprises one or more nucleotides.  
     
     
         64 . The method of  claim 45 , further comprising the step: 
 (a) extending one or more probes within said ligation complex with a polymerase.    
     
     
         65 . The method of  claim 45 , further comprising immobilizing said target sequence to a solid support.  
     
     
         66 . The method of  claim 45 , further comprising amplifying said target sequence.