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 . 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 disposable plug-in reaction cartridge configured for contacting an array of target nucleic acid fragments with probe pools, wherein the reaction cartridge comprises a slot for securing an array of single DNA molecules or amplicons, and quick connect ports for flow-through connection to the sample integration module;   a subsystem configured for illuminating fluorophores on an array in the reaction cartridge; and   a subsystem configured for detecting fluorophores on an array in the reaction cartridge.   
     
     
         2 . The apparatus of  claim 1 , wherein the sample integration module is configured for arraying fragments of a target nucleic acid in a substrate. 
     
     
         3 . The apparatus of  claim 1 , wherein the reaction cartridge comprises a mixing chamber connected to a plurality of probe pool reservoirs by means of a single microfluidic channel. 
     
     
         4 . The apparatus of  claim 1 , wherein the illuminating subsystem is configured to create a 100 to 500 nm thick evanescent field at the interface of two optically different materials. 
     
     
         5 . The apparatus of  claim 1 , wherein the detecting subsystem is a sensitive electron multiplying charge-coupled device (CCD) configured for detection of fluorophores on the array. 
     
     
         6 . The apparatus of  claim 1 , further comprising probe modules that are configured for delivering fluorescently labeled probes to the apparatus. 
     
     
         7 . A system for determining sequence information for a target nucleic acid comprising an apparatus according to  claim 1 , and a plurality of probe pools. 
     
     
         8 . The system of  claim 7 , 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 wherein N represents any of the four nucleotide bases and varies between probes in each of said probe pools;   (ii) each B is independently an informative base, wherein B is the same base for probes in each of said probe pools;   (iii) x and z are each at least one.   
     
     
         9 . The system of  claim 8 , further comprising a computer programmed for parallel processing of data from the subsystem configured for detecting fluorophores on an array in the reaction cartridge. 
     
     
         10 . The system of  claim 8 , further comprising an array of fragments of a target nucleic acid configured for hybridizing with the probe pools.