Multiplex detection of nucleic acids

Abstract

Methods of detecting nucleic acids, including methods of detecting two or more nucleic acids in multiplex branched-chain DNA assays, are provided. Nucleic acids captured on a solid support are detected, for example, through cooperative hybridization events that result in specific association of a label with the nucleic acids. Compositions, kits, and systems related to the methods are also described.

What is claimed is: 
     
         1 . A method of detecting two or more nucleic acids of interest, the method comprising:
 a) providing a sample comprising or suspected of comprising the nucleic acids of interest;   b) capturing those nucleic acids of interest present in the sample on a solid support;   c) providing two or more subsets of m label extenders, wherein m is at least two, wherein each subset of m label extenders is capable of hybridizing to one of the nucleic acids of interest;   d) providing a label probe system comprising a label, wherein a component of the label probe system is capable of hybridizing simultaneously to at least two of the m label extenders in a subset;   e) hybridizing each nucleic acid of interest captured on the solid support to its corresponding subset of m label extenders;   f) hybridizing the label probe system to the m label extenders; and   g) detecting the presence or absence of the label on the solid support.   
     
     
         2 . The method of  claim 1 , wherein capturing the nucleic acids of interest present in the sample on the solid support comprises:
 providing a pooled population of particles which constitute the solid support, the population comprising two or more subsets of particles, a plurality of the particles in each subset being distinguishable from a plurality of the particles in every other subset, and the particles in each subset having associated therewith a different capture probe;   providing two or more subsets of n capture extenders, wherein n is at least two, wherein each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and wherein the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected subset of the particles; and   hybridizing each of the nucleic acids of interest present in the sample to its corresponding subset of n capture extenders and hybridizing the subset of n capture extenders to its corresponding capture probe, whereby the hybridizing the nucleic acid of interest to the n capture extenders and the n capture extenders to the corresponding capture probe captures the nucleic acid on the subset of particles with which the capture extenders are associated.   
     
     
         3 . The method of  claim 2 , wherein detecting the presence or absence of the label on the solid support comprises identifying at least a portion of the particles from each subset and detecting the presence or absence of the label on those particles, thereby determining which subsets of particles have a nucleic acid of interest captured on the particles and indicating which of the nucleic acids of interest were present in the sample. 
     
     
         4 . The method of  claim 1 , wherein the solid support comprises two or more capture probes, wherein each capture probe is provided at a selected position on the solid support; and wherein capturing the nucleic acids of interest present in the sample on the solid support comprises:
 providing two or more subsets of n capture extenders, wherein n is at least two, wherein each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and wherein the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected position on the solid support; and   hybridizing each of the nucleic acids of interest present in the sample to its corresponding subset of n capture extenders and hybridizing the subset of n capture extenders to its corresponding capture probe, whereby the hybridizing the nucleic acid of interest to the n capture extenders and the n capture extenders to the corresponding capture probe captures the nucleic acid on the solid support at the selected position with which the capture extenders are associated.   
     
     
         5 . The method of  claim 4 , wherein detecting the presence or absence of the label on the solid support comprises detecting the presence or absence of the label at the selected positions on the solid support, thereby determining which selected positions have a nucleic acid of interest captured at that position and indicating which of the nucleic acids of interest were present in the sample. 
     
     
         6 . The method of  claim 1 , wherein the two or more nucleic acids of interest comprise five or more, 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more nucleic acids of interest; and wherein the two or more subsets of m label extenders comprise five or more, 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more subsets of m label extenders. 
     
     
         7 . The method of  claim 1 , wherein the label is a fluorescent label, and wherein detecting the presence of the label comprises detecting a fluorescent signal from the label. 
     
     
         8 . The method of  claim 1 , wherein detecting the presence of the label comprises measuring an intensity of a signal from the label, the method comprising correlating the intensity of the signal with a quantity of the nucleic acids of interest present. 
     
     
         9 . The method of  claim 1 , wherein the label probe system comprises an amplification multimer or preamplifier, which amplification multimer or preamplifier is capable of hybridizing to the at least two label extenders. 
     
     
         10 . The method of  claim 9 , wherein the hybridizing the label probe system to the m label extenders is performed at a hybridization temperature, which hybridization temperature is greater than a melting temperature T m  of a complex between each individual label extender and the amplification multimer or preamplifier. 
     
     
         11 . The method of  claim 10 , wherein the hybridization temperature is about 5° C. or more greater than the T m  of a complex between each individual label extender and the amplification multimer or preamplifier. 
     
     
         12 . The method of  claim 11 , wherein the hybridization temperature is about 7° C. or more, about 10° C. or more, about 12° C. or more, about 15° C. or more, about 17° C. or more, or about 20° C. or more greater than the T m  of a complex between each individual label extender and the amplification multimer or preamplifier. 
     
     
         13 . The method of  claim 1 , wherein the label probe system comprises a preamplifier, an amplification multimer and a label probe; wherein the preamplifier is capable of hybridizing simultaneously to the at least two of the m label extenders and to a plurality of amplification multimers; wherein the amplification multimer is capable of hybridizing simultaneously to the preamplifier and to a plurality of label probes; and wherein the label probe comprises the label. 
     
     
         14 . The method of  claim 1 , wherein each label extender comprises a polynucleotide sequence L-1 that is complementary to a polynucleotide sequence in the corresponding nucleic acid of interest and a polynucleotide sequence L-2 that is complementary to a polynucleotide sequence in the component of the label probe system; and wherein the m label extenders in a subset each have L-15′ of L-2 or wherein the m label extenders in a subset each have L-13′ of L-2. 
     
     
         15 . The method of  claim 1 , wherein each label extender comprises a polynucleotide sequence L-2 that is complementary to a polynucleotide sequence in the component of the label probe system, which sequence L-2 is 20 nucleotides or less in length. 
     
     
         16 . The method of  claim 15 , wherein L-2 is between 9 and 17 nucleotides in length. 
     
     
         17 . The method of  claim 16 , wherein L-2 is between 13 and IS nucleotides in length. 
     
     
         18 . The method of  claim 1 , comprising separating materials not captured on the solid support from the solid support. 
     
     
         19 - 23 . (canceled) 
     
     
         24 . A composition for detecting two or more nucleic acids of interest, comprising:
 a) i) a pooled population of particles, the population comprising two or more subsets of particles, a plurality of the particles in each subset being distinguishable from a plurality of the particles in every other subset, and the particles in each subset having associated therewith a different capture probe, or
 ii) a solid support comprising two or more capture probes, wherein each capture probe is provided at a selected position on the solid support; 
   b) two or more subsets of n capture extenders, wherein n is at least two, wherein each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and wherein the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected subset of the particles or with a selected position on the solid support;   c) two or more subsets of m label extenders, wherein m is at least two, wherein each subset of m label extenders is capable of hybridizing to one of the nucleic acids of interest; and   d) a label probe system comprising a label, wherein a component of the label probe system is capable of hybridizing simultaneously to at least two of the m label extenders in a subset.   
     
     
         25 - 81 . (canceled) 
     
     
         82 . A method of capturing a label to a first nucleic acid of interest, in a multiplex assay in which two or more nucleic acids of interest are to be detected, the method comprising:
 a) providing a sample comprising the first nucleic acid of interest and comprising or suspected of comprising one or more other nucleic acids of interest;   b) providing a first subset of m label extenders, wherein m is at least two, wherein the first subset of m label extenders is capable of hybridizing to the first nucleic acid of interest;   c) providing a label probe system comprising the label, wherein a component of the label probe system is capable of hybridizing simultaneously to at least two of the m label extenders in the first subset;   d) hybridizing the first nucleic acid of interest to the first subset of m label extenders; and   e) hybridizing the label probe system to the m label extenders, thereby capturing the label to the first nucleic acid of interest.   
     
     
         83 . (canceled)