US2007264642A1PendingUtilityA1

Direct multiplex characterization of genomic DNA

67
Individually held — no corporate assignee on recordPriority: Oct 24, 2000Filed: Jun 30, 2006Published: Nov 15, 2007
Est. expiryOct 24, 2020(expired)· nominal 20-yr term from priority
Inventors:Thomas WillisPaul HardenbolManeesh JainViktor StolcMostafa RonaghiRonald W. Davis
C12Q 1/6827C12Q 1/6858C12Q 1/686
67
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Claims

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.

Claims

exact text as granted — not AI-modified
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).  
     
     
         59 . 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 padlock probe comprises: 
 a) a first terminus comprising a first probe sequence complementary to the first target domain, wherein said first probe sequence is unique for each of the plurality of probes;    b) a second terminus comprising a second probe sequence complementary to the second target domain, wherein said second probe sequence is unique for each of the plurality of probes;    c) two priming sites that are 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.    
     
     
         60 . The composition of  claim 59 , wherein each padlock probe also comprises a barcode sequence.  
     
     
         61 . The composition of  claim 60 , wherein the barcode sequence is unique to a specific combination of first and second probe sequences in a padlock probe.  
     
     
         62 . The composition of  claim 60 , wherein the barcode sequence occurs in a sequence intervening between the first probe sequence and the second probe sequence.  
     
     
         63 . The composition of  claim 59 , wherein the first terminus is hybridized to the first target domain and the second terminus is hybridized to the second target domain.  
     
     
         64 . The composition of  claim 59 , wherein a circularized probe can be formed by ligation of the first terminus and the second terminus of the padlock probe when the first terminus is hybridized to the first target domain and the second terminus is hybridized to the second target domain.  
     
     
         65 . The composition of  claim 59 , wherein a circularized probe can be formed by extension of at least one terminus and ligation of the padlock probe when the first terminus is hybridized to the first target domain and the second terminus is hybridized to the second target domain.  
     
     
         66 . The composition of  claim 59 , wherein the padlock probe is circularized.  
     
     
         67 . The composition of  claim 66 , wherein the padlock probe has no terminus.  
     
     
         68 . The composition of  claim 59 , wherein the first target domain comprises a single nucleotide polymorphism (SNP).  
     
     
         69 . 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.    
     
     
         70 . A method according to  claim 69 , wherein said substrate is an array.  
     
     
         71 . A method according to  claim 70 , wherein said array is prepared by spotting.  
     
     
         72 . A method according to  claim 70 , wherein said array is prepared by photolithography.  
     
     
         73 . A method according to  claim 69 , wherein said array is prepared from beads on a solid support.  
     
     
         74 . The method according to  claim 69  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.  
     
     
         75 . The method according to  claim 69  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.  
     
     
         76 . The method according to  claim 69  wherein said detection position is an SNP.  
     
     
         77 . The method according to  claim 69  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.  
     
     
         78 . The method according to  claim 69  wherein said detection position is comprised within either said first domain of said target sequence or said second domain of said target sequence.  
     
     
         79 . The method according to  claim 69  wherein said detection position is between said first domain of said target sequence and said second domain of said target sequence.  
     
     
         80 . 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.    
     
     
         81 . A method according to  claim 80 , wherein said substrate is an array.  
     
     
         82 . A method according to  claim 81 , wherein said array is prepared by spotting.  
     
     
         83 . A method according to  claim 81 , wherein said array is prepared by photolithography.  
     
     
         84 . A method according to  claim 81 , wherein said array is prepared from beads on a solid support.  
     
     
         85 . The method according to  claim 80  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.  
     
     
         86 . The method according to  claim 80  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.  
     
     
         87 . The method according to  claim 80  wherein said detection position is an SNP.  
     
     
         88 . The method according to  claim 80  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.  
     
     
         89 . The method according to  claim 80  wherein said detection position is comprised within either said first domain of said target sequence or said second domain of said target sequence.  
     
     
         90 . The method according to  claim 80  wherein said detection position is between said first domain of said target sequence and said second domain of said target sequence.  
     
     
         91 . The method according to  claim 88  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.  
     
     
         92 . The method according to  claim 88  wherein said amplifying comprises polymerase chain reaction.  
     
     
         93 . The method according to  claim 69  wherein said amplifying comprises polymerase chain reaction.

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