Methods and apparatus for DNA sequencing and DNA identification

Abstract

Sequencing by Hybridization (SBH) methods and apparatus employing subdivided filters for discrete multiple probe analysis of multiple samples may be used for DNA identification and for DNA sequencing. Partitioned filters are prepared. Samples are affixed to sections of partitioned filters and each sector is probed with a single probe or a multiplexed probe for hybridization scoring. Hybridization data is analyzed for probe complementarity, partial sequencing by SBH or complete sequencing by SBH.

1 . A method for analyzing nucleic acids by hybridization, comprising the steps of: 
 arraying a first plurality of nucleic acid segments on a first sector of a substrate;    disposing a second plurality of nucleic acid segments on a second sector of said substrate;    exposing, under conditions discriminating between full complementarity and a one base mismatch, said first plurality of nucleic acid segments to a first hybridization probe in said first sector, said first hybridization probe being shorter than one from among said first plurality of nucleic acid segments, to said plurality of nucleic acid segments;    incubating under conditions discriminating between full complementarity and a one base mismatch, a second hybridization probe in said second sector, said second hybridization probe being shorter than a segment from among said second plurality of nucleic acid segments and said second hybridization probe being different in sequence from said first hybridization probe;    detecting hybridization of a hybridization probe to a nucleic acid segment; and    analyzing the result.    
     
     
         2 . The method as recited in  claim 1 , further comprising, prior to said disposing step, the step of introducing a barrier to movement of a nucleic acid.  
     
     
         3 . The method as recited in  claim 1  further comprising, after said arraying and said disposing step but before said incubating step, the step of introducing a barrier to movement of a nucleic acid.  
     
     
         4 . The method as recited in  claim 3  wherein said introducing step comprises pressing a physical barrier against said substrate.  
     
     
         5 . The method as recited in  claim 2  wherein said introducing step comprises the step of applying a direction-switching electrical field perpendicular to said support to prevent the mixing of probes between sectors.  
     
     
         6 . The method as recited in  claim 3  wherein said introducing step comprises the step of applying a direction-switching electrical field perpendicular to said support to prevent the mixing of probes between sectors.  
     
     
         7 . The method as recited in  claim 1  wherein said arraying step comprises the step of spotting nucleic acid samples by means of a pin array.  
     
     
         8 . The method as recited in  claim 1  wherein said arraying step comprises the step of dispensing nucleic acid samples by an array of tubes.  
     
     
         9 . The method as recited in  claim 1  wherein said arraying step comprises the step of jet printing nucleic acid samples.  
     
     
         10 . The method as recited in  claim 1  wherein said exposing step comprises the step of applying a plurality of contiguously hybridizing probes.  
     
     
         11 . The method as recited in  claim 1  wherein said incubating step comprises the step of applying a plurality of contiguously hybridizing probes.  
     
     
         12 . The method as recited in  claim 10  further comprising the step of ligating at least two of said plurality of contiguously hybridizing probes.  
     
     
         13 . The method as recited in  claim 11  further comprising the step of ligating at least two of said plurality of contiguously hybridizing probes.  
     
     
         14 . The method as recited in  claim 1  wherein said exposing step comprises the step of applying a plurality of competitively hybridizing probes having overlapping nucleic acid sequences.  
     
     
         15 . The method as recited in  claim 1  wherein said incubating step comprises the step of applying a plurality of competitively hybridizing probes having overlapping nucleic acid sequences.  
     
     
         16 . The method as recited in  claim 1  wherein a least two of said first plurality of nucleic acid segments are arrayed as a mixture.  
     
     
         17 . The method as recited in  claim 1  wherein a least two of said second plurality of nucleic acid segments are disposed as a mixture.  
     
     
         18 . The method as recited in  claim 1  further comprising the steps of preparing samples by digestion with an Hga I type restriction enzyme and ligating the resulting restriction fragments with an anchor.  
     
     
         19 . The method as recited in  claim 1  further comprising the step of selecting probes from a universal set of probes of a given length.  
     
     
         20 . The method as recited in  claim 1  further comprising the step of selecting probes from an incomplete set of probes of a given length.  
     
     
         21 . The method as recited in  claim 1  further comprising the step of selecting deoxyribonucleotide probes.  
     
     
         22 . The method as recited in  claim 1  further comprising the step of selecting ribonucleotide probes.  
     
     
         23 . The method as recited in  claim 1  further comprising the step of selecting a nucleic acid analog selected from the group consisting of protein nucleic acid probes and probes containing base analogs.  
     
     
         24 . The method as recited in  claim 1  further comprising the step of multiplex labelling of probes.  
     
     
         25 . The method as recited in  claim 1  further comprising the step of degrading a label on an unhybridized probe.  
     
     
         26 . The method as recited in  claim 19  wherein said exposing or said incubating step comprises the step of assembling a set of universal probes 6, 7, 8, 9 or 10 bases in length.  
     
     
         27 . The method as recited in  claim 19  wherein said exposing or said incubating step comprises the step of assembling a set of universal probes 6, 7, 8, 9 or 10 bases in length.  
     
     
         28 . The method as recited in  claim 20  wherein said exposing or said incubating step comprises the step of assembling an incomplete set of probes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases in length.  
     
     
         29 . Apparatus analyzing nucleic acids by hybridization comprising a substrate having points of attachment for nucleic acid fragments, said substrate being segmented by hydrophobic regions.  
     
     
         30 . The method as recited in  claim 20  wherein said disposing step comprises the step of assembling an incomplete set of probes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases in length.  
     
     
         31 . The method of  claim 1  further comprising the step of confirming the relative order of at least two bases in a segment by detecting hybridization of two or more probes having overlapping nucleic acid sequences including said at least two bases.  
     
     
         32 . A method for nucleotide sequence analysis comprising the steps of: 
 introducing a sample to an array of probes;    adjusting the temperature to be one at which a majority of sample molecules are unassociated with ligated probes at any given time;    adding a labelled probe to the mixture;    incubating the mixture with ligase;    removing free probes; and    detecting ligation products.    
     
     
         33 . The method as recited in  claim 1  further comprising the steps of defining additional probes for improving a desired result and repeating said exposing, incubating, detecting and analyzing steps.  
     
     
         34 . The method as recited in  claim 1  further comprising the step of stripping the substrate of probes for reuse of said pluralities of nucleic acid segments.