Apparatus and system for nucleic acid detection

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 . (canceled) 
     
     
         98 . An apparatus comprising:
 a holder configured to accept a sample container, wherein the sample container comprises a plurality of cells;   a temperature controller configured to maintain the sample container at a selected temperature;   one or more fluid reservoirs that are fluidly connected to the sample container;   a fluid handling element fluidly connected to the sample container and configured to add fluid to and/or remove fluid from the sample container;   a mixing element configured to mix contents of the sample container; and   a detector for detecting one or more signals from within individual cells of the plurality of cells in the sample container.   
     
     
         99 . The apparatus of  claim 98 , wherein the temperature controller is configured to maintain the sample container at a temperature selected by a user of the apparatus. 
     
     
         100 . The apparatus of  claim 99 , wherein the temperature controller is configured to maintain the sample container at different temperatures during different steps in an assay procedure. 
     
     
         101 . The apparatus of  claim 98 , wherein at least one fluid reservoir comprises a capture probe system for detecting a target nucleic acid in the cells, wherein the capture probe system comprises 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 a label probe complex, and wherein the T sections of the two or more different capture probes are complementary to non-overlapping regions of the nucleic acid target, and the L sections of the two or more different capture probes are complementary to nonoverlapping regions of the nucleic acid component of the label probe complex. 
     
     
         102 . The apparatus of  claim 101 , wherein at least one fluid reservoir comprises a label probe system for the target nucleic acid, wherein the label probe system comprises two or more identical label probe complexes, each of which comprises a nucleic acid component and one or more labels. 
     
     
         103 . A system comprising:
 a sample container comprising a plurality of cells, wherein the cells comprise or are suspected of comprising two or more nucleic acid targets, and wherein the nucleic acid targets are immobilized in the sample container;   reagents for detecting nucleic acid targets in the sample container, wherein the reagents comprise:
 (i) a label probe system for each nucleic acid target, wherein each label probe system comprises 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; and 
 (ii) a capture probe system for each nucleic acid target, wherein the capture probe system comprises 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 two or more different capture probes are complementary to non-overlapping regions of the nucleic acid target, and the L sections of the two or more different capture probes are complementary to nonoverlapping regions of the nucleic acid component of the label probe complex; 
   a fluid handling system to add the reagents to the sample container, to hybridize the capture probe system to the two or more nucleic acid targets, and to capture the label probe system to the capture probe system hybridized to the two or more nucleic acid targets; and   a temperature control module coupled to the sample container.   
     
     
         104 . The system of  claim 103 , further comprising a detection element to detect a signal from the two or more label probe complexes captured on the nucleic acid targets, wherein the presence of a signal indicates the presence of the nucleic acid targets in the plurality of cells, and the absence of a signal indicates the absence of the nucleic acid targets in the plurality of cells. 
     
     
         105 . The system of  claim 103 , wherein the two or more different capture probes of the sets of capture probes hybridize to adjacent sections on the nucleic acid target. 
     
     
         106 . The method of  claim 103 , wherein, when the fluid handling subsystem adds the capture probe system to the sample container, the temperature control module maintains the sample at a temperature that is greater than a melting temperature T m  of a complex between each individual capture probe and the nucleic acid target. 
     
     
         107 . The system of  claim 103 , wherein, when the fluid handling subsystem adds the label probe system to the sample container, the temperature control module maintains the sample at a 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. 
     
     
         108 . The system of  claim 103 , wherein the fluid handling subsystem adds each capture probe system to the sample container at the same time. 
     
     
         109 . The system of  claim 103 , wherein the fluid handling subsystem adds each label probe system to the sample container at the same time. 
     
     
         110 . The system of  claim 103 , 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. 
     
     
         111 . The system of  claim 110 , wherein the two or more nucleic acid targets are RNA. 
     
     
         112 . The system of  claim 110 , wherein the two or more nucleic acid targets are mRNA. 
     
     
         113 . The system of  claim 103 , wherein the system correlates the intensity of the signal of each nucleic acid target with a quantity of the corresponding nucleic acid target present in the cell. 
     
     
         114 . The system of  claim 103 , wherein the sample comprises a tissue section. 
     
     
         115 . The system of  claim 103 , wherein the sample comprising the cell is derived from a bodily fluid, blood, a swab, or a cell culture. 
     
     
         116 . The system of  claim 103 , 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.   
     
     
         117 . The system of  claim 103 , 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.