Multiplex Detection of Nucleic Acids

Abstract

Described herein is a new approach in which a nucleic acid species of interest (e.g. a chromosome) containing multiple unique target sequences is detected using multiple specific probes that are amplified by rolling circle amplification and detected. Multiple probes are used to provide a detectable signal, where the magnitude of the signal is proportional to the number of probes recognising their target sequences. Individual signals from the plurality of probes are converted into a single cumulative detectable signal, amplifying the individual signals through the multiplex probing. Ten or more probes produce a signal amplification of ten-fold or more. The generated signals depend on correctly reacted probes upon target recognition, using sequence specific hybridisation and enzymatic catalysis to generate specific products from which the signal is obtained.

1 - 24 . (canceled) 
     
     
         25 . A method of quantifying labelled rolling circle amplification (RCA) products, comprising:
 (a) spreading a mixture comprising at least a first set of labelled RCA products and a second set of labelled RCA products across the surface of a support, wherein the first and second sets of labelled RCA products are distinguishably labelled;   (b) counting the number of RCA products in the first set on the support, to provide a first count;   (c) counting the number of RCA products in the second set on the support, to provide a second count; and   (d) comparing the first and second counts, thereby determining the relative quantities of the RCA products in the first and second sets of RCA products.   
     
     
         26 . The method of  claim 25 , wherein the counting of (b) and (c) comprises:
 (i) imaging the first and second sets of labeled RCA products on the support to produce one or more images, wherein the labeled RCA products of the first set and the second set are spatially separated in the one or more images; and   (ii) determining the number of labeled RCA products of the first set and the second set, in the image.   
     
     
         27 . The method of  claim 25 , wherein at least some of the labeled RCA products comprise a sequence of a fragment of human genomic DNA. 
     
     
         28 . The method of  claim 27 , wherein the fragment is in a range of 10 to 30 nucleotides in length. 
     
     
         29 . The method of  claim 28 , wherein the fragment is a fragment of human chromosome 21. 
     
     
         30 . The method of  claim 29 , wherein the fragment is a restriction fragment. 
     
     
         31 . The method of  claim 25 , wherein the first and second sets of labelled RCA products are fluorescently labelled. 
     
     
         32 . The method of  claim 31 , wherein the fluorescently labelled RCA products are made by hybridizing distinguishably labelled oligonucleotides to a population of unlabelled RCA products, wherein the hybridizing is done in solution and wherein the labelled oligonucleotides hybridize to sequences that are repeated in the RCA products. 
     
     
         33 . The method of  claim 25 , wherein the first and second sets of labelled RCA products each comprise at least 1,000 labelled RCA products. 
     
     
         34 . The method of  claim 25 , wherein the support is a planar support.