Method of detecting target nucleic acid molecules

Abstract

The present application provides methods for detecting a nucleic acid molecule involving the use of a signal code sequence which corresponds to said nucleic acid molecule and a plurality of labelled detection probes which yield signals which make up the signal code sequence. In particular, the invention provides a sequential barcoding and decoding scheme which utilises a sequencing-by-hybridisation (SBH) strategy to sequence and decode a nucleotide barcode sequence, and to differentiate the nucleotide barcode sequence from other nucleotide barcode sequences. In an extension of the method, the application also provides a new coding scheme for providing a target nucleic acid with a detectable “colour” (or similar signal)-based code.

1 - 50 . (canceled) 
     
     
         51 . A system for detecting a nucleic acid barcode sequence, the system comprising:
 (a) a padlock probe comprising target binding sites capable of hybridizing to a target sequence in a target nucleic acid molecule,
 wherein the padlock probe comprises a nucleotide barcode sequence that identifies the padlock probe, and wherein the nucleotide barcode sequence comprises a first domain sequence and a second domain sequence, wherein a portion of the first domain sequence overlaps with some but not all of the second domain sequence; 
   (b) a universal pool of reporter probes, wherein the universal pool of reporter probes comprises at least two different species of reporter probes, wherein the first species of reporter probe comprises a first optically detectable moiety, and the second species of reporter probe comprises a second optically detectable moiety;   (c) a first detection probe, wherein the first detection probe comprises (i) a recognition sequence comprising the first domain sequence, and (ii) a first overhang sequence comprising a reporter probe binding site complementary to a reporter probe of the universal pool of reporter probes;   (c) a second detection probe, wherein the second detection probe comprises (i) a recognition sequence complementary the second domain sequence, and (ii) a second overhang sequence comprising a reporter probe binding site complementary to a reporter probe of the universal pool of reporter probes;
 wherein the second detection probe is capable of initiating a strand displacement reaction to displace the first detection probe hybridized to the first domain. 
   
     
     
         52 . The system of  claim 51 , wherein the target sequence is in a native genomic DNA or in a naturally occurring RNA molecule, or in a cDNA, or in an amplification product generated from any of the foregoing nucleic acid molecules. 
     
     
         53 . The system of  claim 51 , wherein the target nucleic acid molecule is linked to an antibody. 
     
     
         54 . The system of  claim 51 , wherein each of the first optically detectable moiety and the second optically detectable moiety is a fluorophore. 
     
     
         55 . The system of  claim 54 , wherein the series of signal codes is a unique series of fluorescent colors. 
     
     
         56 . The system of  claim 51 , for detecting multiple different nucleic acid molecules present in the sample, wherein the system comprises a specific padlock probe with a different nucleotide barcode sequence assigned a different series of signal codes for each different nucleic acid molecule. 
     
     
         57 . The system of  claim 51 , wherein the number of different species of reporter probes in the universal pool of reporter probes is four. 
     
     
         58 . The system of  claim 55 , wherein the number of different barcode sequences is 50 or more. 
     
     
         59 . The system of  claim 51 , wherein the biological sample is a cell or a tissue sample on a solid substrate. 
     
     
         60 . The system of  claim 51 , wherein the first overhang sequence and the second overhang sequence are the same. 
     
     
         61 . The system of  claim 51 , wherein the first overhang sequence and the second overhang sequence are different. 
     
     
         62 . The system of  claim 51 , wherein the first domain is 5′ to the second domain in the nucleotide barcode sequence. 
     
     
         63 . The system of  claim 51 , wherein the first domain is 3′ to the second domain in the nucleotide barcode sequence. 
     
     
         64 . The system of  claim 51 , wherein the first domain and the second domain each comprises a first subunit and a second subunit, and wherein the second subunit from the first domain fully overlaps with the first subunit of the second domain. 
     
     
         65 . The system of  claim 51 , wherein the nucleotide barcode sequence comprises: (i) a first common region adjacent to the first subunit of the first domain, wherein the recognition sequence of the first detection probe comprises a sequence which is complementary to the first common region; and (ii) a second common region adjacent to the second subunit of the second domain, wherein the recognition sequence of the second detection probe comprises a sequence which is complementary to the second common region. 
     
     
         66 . The system of  claim 65 , wherein the first detection probe and the second detection probe each comprise a displacer toehold overhang region. 
     
     
         67 . The system of  claim 51 , wherein the first domain and the second domain each comprises a unique subunit. 
     
     
         68 . The system of  claim 51 , wherein each domain comprises a unique subunit. 
     
     
         69 . The system of  claim 51 , wherein the system comprises reagents for circularizing said padlock probe; and
 reagents for performing rolling circle amplification of said circularized padlock probe to generate a rolling circle product (RCP), the RCP comprising multiple complementary copies of the nucleotide barcode sequence.