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.