Direct multiplex characterization of genomic DNA

Abstract

The invention is directed to novel methods of multiplexing nucleic acid reactions, including amplification, detection and genotyping. The invention relies on the use of precircle probes that are circularized in the presence of the corresponding target nucleic acids, cleaved, and then amplified.

1 - 47 . (canceled)  
     
     
         48 . A method of determining the identity of a nucleotide at a detection position in a target sequence comprising a first domain and a second domain, said method comprising the steps of: 
 a) hybridizing said first domain to a first end of a padlock probe wherein said padlock probe comprises: 
 i) a first end that is complementary to said first domain of said target sequence;  
 ii) a barcode sequence;  
 iii) a second end that is complementary to said second domain of said target sequence wherein said first end of said padlock probe and said second end of said padlock probe are located at the ends of said padlock probe; and  
 iv) two priming sites  
   b) hybridizing said second domain of said target sequence to said second end of said padlock probe;    c) covalently attaching said first end of said padlock probe and said second end of said padlock probe while hybridized to said target sequence;    d) amplifying a sequence comprising at least a portion of said padlock probe following said covalently attaching, thereby producing amplification products comprising said barcode sequence; and    e) binding said barcode sequence of said amplification product to a substrate wherein said substrate comprises an attached barcode capture probe and/or an attached complement of said barcode sequence;    thereby identifying said nucleotide at said detection position.    
     
     
         49 . A method according to  claim 48 , wherein said substrate is an array.  
     
     
         50 . A method according to  claim 49 , wherein said array is prepared by spotting.  
     
     
         51 . A method according to  claim 49 , wherein said array is prepared by photolithography.  
     
     
         52 . A method according to  claim 48 , wherein said array is prepared from beads on a solid support.  
     
     
         53 . The method according to  claim 48  wherein said first end and said second end of said padlock probe are hybridized to said target sequence and said covalently attaching comprises ligating said first and second ends of said padlock probe.  
     
     
         54 . The method according to  claim 48  wherein one of said first end or said second end of said padlock probe hybridized to said target sequence is extended using a polymerase, and said covalently attaching comprises ligating said first and second ends of said padlock probe after said first or second end is extended.  
     
     
         55 . The method according to  claim 48  wherein said detection position is an SNP.  
     
     
         56 . The method according to  claim 48  wherein after said covalently attaching said first end of said padlock probe and said second end of said padlock probe, a sequence comprising at least a portion of said padlock probe is amplified with a polymerase, then cleaved.  
     
     
         57 . The method according to  claim 48  wherein said detection position is comprised within either said first domain of said target sequence or said second domain of said target sequence.  
     
     
         58 . The method according to  claim 48  wherein said detection position is between said first domain of said target sequence and said second domain of said target sequence.  
     
     
         59 . A method of determining the identity of a nucleotide at a detection position in a target sequence comprising a first domain and a second domain, said method comprising the steps of: 
 a) hybridizing said first domain to a first end of a padlock probe wherein said padlock probe comprises: 
 i) a first end that is complementary to said first domain of said target sequence;  
 ii) a barcode sequence;  
 iii) a cleavage site;  
 iv) a second end that is complementary to said second domain of said target sequence wherein said first end of said padlock probe and said second end of said padlock probe are located at the ends of said padlock probe; and  
 v) two priming sites;  
   b) hybridizing said second domain of said target sequence to said second end of said padlock probe;    c) covalently attaching said first end of said padlock probe and said second end of said padlock probe while hybridized to said target sequence;    d) cleaving said covalently attached padlock probe at said cleavage site; and    e) binding said barcode sequence of said covalently attached padlock probe to a substrate wherein said substrate comprises a capture probe and/or an attached complement of said adapter sequence;    thereby identifying said nucleotide at said detection position.    
     
     
         60 . A method according to  claim 59 , wherein said substrate is an array.  
     
     
         61 . A method according to  claim 60 , wherein said array is prepared by spotting.  
     
     
         62 . A method according to  claim 60 , wherein said array is prepared by photolithography.  
     
     
         63 . A method according to  claim 60 , wherein said array is prepared from beads on a solid support.  
     
     
         64 . The method according to  claim 59  wherein said first end and said second end of said padlock probe are hybridized to said target sequence and said covalently attaching comprises ligating said first and second ends of said padlock probe.  
     
     
         65 . The method according to  claim 59  wherein one of said first end or said second end of said padlock probe hybridized to said target sequence is first extended using a polymerase, and said covalently attaching comprises ligating said first and second ends of said padlock probe after said first or second end is extended.  
     
     
         66 . The method according to  claim 59  wherein said detection position is an SNP.  
     
     
         67 . The method according to  claim 59  further comprising amplifying a sequence comprising at least a portion of said padlock probe with a polymerase following said covalently attaching, thereby producing amplification products comprising said barcode sequence.  
     
     
         68 . The method according to  claim 59  wherein said detection position is comprised within either said first domain of said target sequence or said second domain of said target sequence.  
     
     
         69 . The method according to  claim 59  wherein said detection position is between said first domain of said target sequence and said second domain of said target sequence.  
     
     
         70 . The method according to  claim 67  further comprising binding said barcode sequence of said amplification products to said substrate that comprises an attached capture probe and/or an attached complement of said adapter sequence.  
     
     
         71 . The method according to  claim 67  wherein said amplifying comprises polymerase chain reaction.  
     
     
         72 . The method according to  claim 48  wherein said amplifying comprises polymerase chain reaction.