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 composition comprising a plurality of padlock probes for detecting a plurality of target sequences in a sample, wherein each target sequence comprises first and second target domains, and each of the padlock probes comprises: 
 a) a first probe sequence complementary to the first target domain;    b) a second probe sequence complementary to the second target domain;    c) a priming site that is identical for each of the plurality of probes; and    d) a restriction endonuclease site that is identical for each of the plurality of probes, wherein the restriction site occurs in a sequence intervening between the first probe sequence and the second probe sequence.    
     
     
         49 . The composition of  claim 48 , wherein each padlock probe also comprises an adapter sequence.  
     
     
         50 . The composition of  claim 49 , wherein the adapter sequence is unique to a specific combination of first and second probe sequences in a padlock probe.  
     
     
         51 . The composition of  claim 49 , wherein the adapter sequence occurs in a sequence intervening between the first probe sequence and the second probe sequence.  
     
     
         52 . The composition of  claim 48 , wherein the padlock probe has a first terminus comprising the first probe sequence and a second terminus comprising the second probe sequence.  
     
     
         53 . The composition of  claim 52 , wherein the first terminus is hybridized to the first target domain and the second terminus is hybridized to the second target domain.  
     
     
         54 . The composition of  claim 53 , wherein a circularized probe can be formed by ligation of the padlock probe.  
     
     
         55 . The composition of  claim 53 , wherein a circularized probe can be formed by extension and ligation of the padlock probe.  
     
     
         56 . The composition of  claim 48 , wherein the padlock probe is circularized.  
     
     
         57 . The composition of  claim 56 , wherein the padlock probe has no terminus.  
     
     
         58 . The composition of  claim 48 , wherein the first target domain comprises a single nucleotide polymorphism (SNP).