Methods of detecting nucleic acids in individual cells and of identifying rare cells from large heterogeneous cell populations

Abstract

Methods of detecting multiple nucleic acid targets in single cells through indirect capture of labels to the nucleic acids are provided. Methods of assaying the relative levels of nucleic acid targets through normalization to levels of reference nucleic acids are also provided. Methods of detecting individual cells, particularly rare cells from large heterogeneous cell populations, through detection of nucleic acids are described. Related compositions, systems, and kits are also provided.

1 - 97 . (cancelled) 
     
     
         98 . A method of detecting two or more nucleic acid targets within an individual cell, the method comprising:
 providing a sample comprising the cell, which cell comprises or is suspected of comprising two or more nucleic acid targets;   providing for each nucleic acid target a label probe system comprising two or more identical label probe complexes, each of which comprises a nucleic acid component and one or more labels, wherein the labels of the label probe systems are distinct for each target nucleic acid;   providing for each nucleic acid target a capture probe system comprising two or more sets of capture probes, wherein each set of capture probes comprises two or more different capture probes, wherein all capture probes in the capture probe system comprise a T section and an L section, wherein the T section is a nucleic acid sequence complementary to a section on the nucleic acid target and the L section is a nucleic acid sequence complementary to a section on the nucleic acid component of the label probe complex, and wherein the T sections of the different capture probes are complementary to non-overlapping regions of the nucleic acid target, and the L sections of the different capture probes are complementary to non-overlapping regions of the nucleic acid component of the label probe complex;   hybridizing, in the cell and in the presence of cellular non-target nucleic acids, the capture probe system to the two or more nucleic acid targets, when present in the cell, thereby providing hybridization of two or more different capture probe sets to a single copy of the nucleic acid target molecules in the presence of cellular non-target nucleic acids;   capturing, in the cell and in the presence of cellular non-target nucleic acids, the label probe system to the capture probe system hybridized to the nucleic acid target molecules, wherein the capturing occurs by simultaneously hybridizing the at least two different capture probes in each capture probe set to a single molecule of the nucleic acid component of the label probe complex that is complementary to the L sections of the capture probes, thereby capturing the two or more label probe complexes to the nucleic acid target in the presence of cellular non-target nucleic acids; and   detecting a signal from the two or more label probe complexes captured on the nucleic acid targets.   
     
     
         99 . The method of  claim 98 , wherein hybridizing the at least two different capture probes of the sets of capture probes to the single molecule of the nucleic acid component of the label probe complex that is complementary to the capture probes is performed at a hybridization temperature that is greater than a melting temperature T m  of a complex between each individual capture probe and the nucleic acid component of the label probe complex. 
     
     
         100 . The method of  claim 98 , wherein the at least two different capture probes of the sets of capture probes hybridize to adjacent sections on the nucleic acid target. 
     
     
         101 . The method of  claim 98 , wherein hybridizing the two or more capture probes of the sets of capture probes to the single molecule of the nucleic acid target is performed at a hybridization temperature that is greater than a melting temperature T m  of a complex between each individual capture probe and the nucleic acid target. 
     
     
         102 . The method of  claim 98 , further comprising providing one or more blocking probes capable of hybridizing to regions of the nucleic acid targets not occupied by the capture probes. 
     
     
         103 . The method of  claim 98 , wherein each hybridizing or capturing step is accomplished for each of multiple nucleic acid targets at the same time. 
     
     
         104 . The method of  claim 98 , wherein the two or more nucleic acid targets are independently selected from the group consisting of a DNA, a chromosomal DNA, an RNA, an mRNA, a microRNA, a ribosomal RNA, a nucleic acid endogenous to the cell, and a nucleic acid introduced to or expressed in the cell by infection of the cell with a pathogen. 
     
     
         105 . The method of  claim 98 , wherein the two or more nucleic acid targets comprise a first nucleic acid target comprising a first region of an mRNA and a second nucleic acid target comprising a second region of the same mRNA. 
     
     
         106 . The method of  claim 98 , wherein the two or more nucleic acid targets comprise a reference nucleic acid, and wherein the method comprises normalizing the signal of one or more of the nucleic acid targets to the signal of the reference nucleic acid. 
     
     
         107 . The method of  claim 98 , further comprising the step of correlating the intensity of the signal of each nucleic acid target with a quantity of the corresponding nucleic acid target present in the cell. 
     
     
         108 . The method of  claim 98 , wherein the sample comprises a tissue section, or wherein the sample comprising the cell is derived from a bodily fluid, from blood, from a swab, or from a cell culture. 
     
     
         109 . The method of  claim 98 , wherein the cell is in suspension during the hybridizing, capturing, and/or detecting steps. 
     
     
         110 . The method of  claim 98 , wherein the cell is a circulating tumor cell. 
     
     
         111 . The method of  claim 98 , wherein each capture probe comprises the T section of the capture probe at the 5′ end and the L section of the capture probe at the 3′ end, or the T section of the capture probe at the 3′ end and the L section of the capture probe at the 5′ end. 
     
     
         112 . The method of  claim 98 , wherein the label probe system comprises:
 (A) the two or more identical label probe complexes each comprising the nucleic acid component and the one or more labels, wherein the nucleic acid component of the label probe complexes comprises one or more label probes capable of hybridizing to the two or more different capture probes of the sets of capture probes of the capture probe system;   (B) the two or more identical label probe complexes each comprising the nucleic acid component and the one or more labels, wherein the nucleic acid component of the label probe complexes comprises one or more label probes and an amplifier capable of hybridizing to the one or more label probes and to the two or more different capture probes of the sets of capture probes of the capture probe system; or   (C) the two or more identical label probe complexes each comprising the nucleic acid component and the one or more labels, wherein the nucleic acid component of the label probe complexes comprises one or more label probes, one or more amplifiers capable of hybridizing to the one or more label probes, and a preamplifier capable of hybridizing to the one or more amplifiers and to the two or more different capture probes of the sets of capture probes of the capture probe system.   
     
     
         113 . A method of detecting an individual cell of a specified type by detecting two or more nucleic acid targets within the individual cell, the method comprising:
 providing a sample comprising a mixture of cell types, which mixture comprises or is suspected of comprising at least one cell of the specified type which comprises two or more nucleic acid targets;   providing for each nucleic acid target a label probe system comprising two or more identical label probe complexes, each of which comprises a nucleic acid component and one or more labels, wherein the labels of the label probe systems are distinct for each target nucleic acid;   providing for each nucleic acid target a capture probe system comprising two or more sets of capture probes, wherein each set of capture probes comprises two or more different capture probes, wherein all capture probes in the capture probe system comprise a T section and an L section, wherein the T section is a nucleic acid sequence complementary to a section on the nucleic acid target and the L section is a nucleic acid sequence complementary to a section on the nucleic acid component of the label probe complex, and wherein the T sections of the different capture probes are complementary to non-overlapping regions of the nucleic acid target, and the L sections of the different capture probes are complementary to non-overlapping regions of the nucleic acid component of the label probe complex;   hybridizing, in the cell and in the presence of cellular non-target nucleic acids, the capture probe system to the two or more nucleic acid targets, when present in the cell, thereby providing hybridization of two or more different capture probe sets to a single copy of the nucleic acid target molecules in the presence of cellular non-target nucleic acids;   capturing, in the cell and in the presence of cellular non-target nucleic acids, the label probe system to the capture probe system hybridized to the nucleic acid target molecule, wherein the capturing occurs by simultaneously hybridizing the at least two different capture probes in each capture probe set to a single molecule of the nucleic acid component of the label probe complex that is complementary to the L sections of the capture probes, thereby capturing the two or more label probe complexes to the nucleic acid targets in the presence of cellular non-target nucleic acids;   detecting a signal from the two or more label probe complexes captured on each of the two or more nucleic acid targets;   correlating the signal detected from the cell with the presence, absence, or amount of the two or more nucleic acid targets in the cell; and   identifying the cell as being of the specified type based on detection of the presence, absence, or amount of the two or more nucleic acid targets within the cell.   
     
     
         114 . The method of  claim 113 , wherein hybridizing the at least two different capture probes of the sets of capture probes to the single molecule of the nucleic acid component of the label probe complex is performed at a hybridization temperature that is greater than a melting temperature T m  of a complex between each individual capture probe and the nucleic acid component of the label probe complex. 
     
     
         115 . The method of  claim 113 , wherein the at least two different capture probes of the sets of capture probes hybridize to adjacent sections on the nucleic acid target. 
     
     
         116 . The method of  claim 113 , wherein hybridizing the two or more capture probes of the sets of capture probes to the single molecule of the nucleic acid target is performed at a hybridization temperature that is greater than a melting temperature T m  of a complex between each individual capture probe and the nucleic acid target. 
     
     
         117 . The method of  claim 113 , further comprising providing one or more blocking probes capable of hybridizing to regions of the nucleic acid targets not occupied by the capture probes. 
     
     
         118 . The method of  claim 113 , wherein each hybridizing or capturing step is accomplished for each of multiple nucleic acid targets at the same time. 
     
     
         119 . The method of  claim 113 , wherein the two or more nucleic acid targets are independently selected from the group consisting of a DNA, a chromosomal DNA, an RNA, an mRNA, a microRNA, a ribosomal RNA, a nucleic acid endogenous to the cell, and a nucleic acid introduced to or expressed in the cell by infection of the cell with a pathogen. 
     
     
         120 . The method of  claim 113 , wherein the two or more nucleic acid targets comprise a first nucleic acid target comprising a first region of an mRNA and a second nucleic acid target comprising a second region of the same mRNA. 
     
     
         121 . The method of  claim 113 , wherein the two or more nucleic acid targets comprise a reference nucleic acid, and wherein the method comprises normalizing the signal of one or more of the nucleic acid targets to the signal of the reference nucleic acid. 
     
     
         122 . The method of  claim 113 , further comprising the step of correlating the intensity of the signal of each nucleic acid target with a quantity of the corresponding nucleic acid target present in the cell. 
     
     
         123 . The method of  claim 113 , wherein the sample comprises a tissue section, or wherein the sample comprising the cell is derived from a bodily fluid, from blood, from a swab, or from a cell culture. 
     
     
         124 . The method of  claim 113 , wherein the cell is in suspension during the hybridizing, capturing, and/or detecting steps. 
     
     
         125 . The method of  claim 113 , wherein the cell is a circulating tumor cell. 
     
     
         126 . The method of  claim 113 , wherein each capture probe comprises the T section of the capture probe at the 5′ end and the L section of the capture probe at the 3′ end, or the T section of the capture probe at the 3′ end and the L section of the capture probe at the 5′ end. 
     
     
         127 . The method of  claim 113 , wherein the label probe system comprises:
 (A) the two or more identical label probe complexes each comprising the nucleic acid component and the one or more labels, wherein the nucleic acid component of the label probe complexes comprises one or more label probes capable of hybridizing to the two or more different capture probes of the sets of capture probes of the capture probe system;   (B) the two or more identical label probe complexes each comprising the nucleic acid component and the one or more labels, wherein the nucleic acid component of the label probe complexes comprises one or more label probes and an amplifier capable of hybridizing to the one or more label probes and to the two or more different capture probes of the sets of capture probes of the capture probe system; or   (C) the two or more identical label probe complexes each comprising the nucleic acid component and the one or more labels, wherein the nucleic acid component of the label probe complexes comprises one or more label probes, one or more amplifiers capable of hybridizing to the one or more label probes, and a preamplifier capable of hybridizing to the one or more amplifiers and to the two or more different capture probes of the sets of capture probes of the capture probe system.