Random Array DNA Analysis by Hybridization

Abstract

The invention relates to methods and devices for analyzing single molecules, i.e., nucleic acids. Such single molecules may be derived from natural samples, such as cells, tissues, soil, air and water without separating or enriching individual components. In certain aspects of the invention, the methods and devices are useful in performing nucleic acid sequence analysis by probe hybridization.

1 . A system for analyzing a target nucleic acid, comprising:
 (a) a reaction platform;   (b) an array on a surface of the platform, wherein the array comprises a solid substrate comprising a plurality of areas, each area configured for immobilization of a polynucleotide comprising a fragment of the target nucleic acid;   (c) a light source configured to excite fluorescent molecules at or near the surface;   (d) a megapixel camera positioned above the reaction platform; and   (e) a lens configured to focus areas of the platform such that each area of the array is focused on an individual pixel of the camera.   
     
     
         2 . The system of  claim 1 , wherein each area is 1 μm 2 . 
     
     
         3 . The system of  claim 1 , wherein the array comprises one million or more of the areas. 
     
     
         4 . The system of  claim 1 , wherein the light source is a laser, and the system further comprises galvanometers to control light from the laser. 
     
     
         5 . The system of  claim 1 , comprising fragments of the target nucleic acid immobilized on the surface at an average density of approximately one polynucleotide per pixel. 
     
     
         6 . The system of  claim 5 , comprising fluorescently labeled probes hybridized to the fragments of the target nucleic acid. 
     
     
         7 . The system of  claim 1 , wherein the camera is a CCD camera. 
     
     
         8 . The system of  claim 1 , wherein the polynucleotide comprises a fragment of the target nucleic acid and an adapter sequence at each end of the fragment. 
     
     
         9 . The system of  claim 8 , wherein each area comprises an attached oligonucleotide, wherein the oligonucleotide is complementary to the adapter sequence. 
     
     
         10 . A method for analyzing a target nucleic acid, comprising:
 (a) arraying polynucleotides comprising fragments of the target nucleic acid on the reaction platform of a system according to  claim 1  to form an array having an average density of one polynucleotide per pixel;   (b) performing a sequencing reaction on the array;   (c) recording signals from each pixel; and   (d) repeating steps (b) to (c) to produce a sequence of the target nucleic acid.   
     
     
         11 . An apparatus for determining sequence information for a target nucleic acid by probe hybridization, comprising:
 a sample integration module configured for mixing, introducing, and/or removing reagents;   a reaction cartridge configured for contacting an array of target nucleic acid fragments with probe pools;   a subsystem configured for illuminating fluorophores on the array; and   a subsystem configured for detecting fluorophores on the array.   
     
     
         12 . The apparatus of  claim 11 , wherein the sample integration module is configured for arraying fragments of a target nucleic acid in a substrate. 
     
     
         13 . The apparatus of  claim 11 , wherein the reaction cartridge is a plug-in cartridge. 
     
     
         14 . The apparatus of  claim 11 , wherein the reaction cartridge comprises a mixing chamber connected to a plurality of probe pool reservoirs by means of a single microfluidic channel. 
     
     
         15 . The apparatus of  claim 11 , wherein the illuminating subsystem is configured to create a 100 to 500 nm thick evanescent field at the interface of two optically different materials. 
     
     
         16 . The apparatus of  claim 11 , wherein the detecting subsystem is a sensitive electron multiplying charge-coupled device (CCD) configured for detection of fluorophores on the array. 
     
     
         17 . The apparatus of  claim 11 , further comprising probe modules that are configured for delivering fluorescently labeled probes to the apparatus. 
     
     
         18 . A system for determining sequence information for a target nucleic acid comprising an apparatus according to  claim 11 , and a plurality of probe pools. 
     
     
         19 . The system of  claim 18 , wherein the probes in the probe pools contain a label and a nucleotide sequence comprising the formula N x B y N z , the formula N x B y  or the formula B y N z , wherein:
 (i) each N is independently a degenerate base;   (ii) each B is independently an informative base;   (iii) x and z are each at least one.   
     
     
         20 . The system of  claim 19 , further comprising a computer programmed for parallel processing of data from the detecting subsystem. 
     
     
         21 . The system of  claim 19 , further comprising an array of fragments of a target nucleic acid configured for hybridizing with the probe pools. 
     
     
         22 - 24 . (canceled)