Method of aligning high-density biochemical array chips with asynchronous tracks by moiré averaging

Abstract

An array chip useful for biochemical assays is provided wherein the chip includes a field region arranged with attachment sites according to a first pitch and at least one track region having a one-dimensional spot pattern arranged according to a second pitch that is less dense and is a non-integer multiple of the first pitch so that one-dimensional Moiré averaging may be applied in the track region, thereby to attain alignment of the chip to the optical instrumentation with a higher density of attachment sites.

What is claimed is: 
     
         1 . A method of sequencing nucleic acid macromolecules that are arrayed on a solid planar substrate in a two-dimensional array such that there is a uniform distance between adjacent nucleic acid molecules in each row and between adjacent nucleic acid molecules in each column of the array;
 wherein the planar substrate also includes a linear array of alignment sites characterized as having a uniform distance between adjacent alignment sites and separated from the two-dimensional array by a site-free band;   the method comprising:   obtaining a plurality of images of the arrayed macromolecules by iteratively correcting for alignment errors in sub-pixel X-Y alignment by Moiré averaging-based alignment of the array of nucleic acid macromolecules in relation to the alignment sites, imaging the patterned array using the digital camera, and optically moving the digital camera across the array chip for taking another image; then   determining nucleic acid sequences of a plurality of the macromolecules in the two-dimensional array from the images.   
     
     
         2 . The method of  claim 1 , wherein selected alignment sites are deleted in the linear array, and the method further comprises using the deletion sites as registration markers in a pattern matching scheme for absolute location identification. 
     
     
         3 . The method of  claim 1 , wherein the nucleic acid macromolecules have fluorophore-labeled probes hybridized thereto. 
     
     
         4 . The method of  claim 1 , wherein nucleic acid macromolecules on the substrate are labeled with one of at least four different fluorophores, each fluorophore indicating a different nucleotide base. 
     
     
         5 . The method of  claim 1 , comprising aligning the two-dimensional array with a digital camera such that each experiment site on the array chip is aligned with a single pixel of the digital camera. 
     
     
         6 . The method of  claim 1 , comprising a plurality of said two-dimensional arrays arranged on the planar substrate in a plurality of columns and rows, separated from each other by a dividing space that includes a site free band having a width that is greater than the uniform distance between adjacent experiment sites in the two dimensional arrays. 
     
     
         7 . The method of  claim 6 , wherein the dividing space includes a linear array of alignment sites with a site free band on either side. 
     
     
         8 . A method of obtaining multiple images of nucleic acid macromolecules on an array, comprising:
 (a) obtaining a solid planar substrate having
 (i) a two-dimensional array of experiment sites on the substrate, characterized as having a uniform distance between adjacent experiment sites in each row and between adjacent experiment sites in each column; and 
   
       (ii) a linear array of alignment sites on the substrate, characterized as having a uniform distance between adjacent alignment sites, and separated on the substrate from the two-dimensional array;
 wherein experiment sites in the two dimensional array and alignment sites in the linear array have a surface that is functionalized to bind and retain nucleic acid macromolecules; 
 (b) attaching a nucleic acid macromolecule to each of a plurality of the experiment sites and to each of a plurality of the alignment sites: then 
 (c) obtaining an image of the nucleic acid macromolecules on the substrate by monitoring optical alignment of a digital camera with the patterned array using the alignment region(s) to determine alignment errors, correcting the alignment errors, and imaging the patterned array using the digital camera. 
 
     
     
         9 . The method of  claim 8 , comprising correcting for errors in sub-pixel X-Y alignment by Moiré averaging-based alignment of the two-dimensional array in relation to the linear array before obtaining the image. 
     
     
         10 . The method of  claim 8 , comprising aligning the two-dimensional array with a digital camera such that each experiment site on the array chip is aligned with a single pixel of the digital camera. 
     
     
         11 . The method of  claim 8 , wherein selected alignment sites are deleted in the linear array, and the method further comprises using the deletion sites as registration markers in a pattern matching scheme for absolute location identification. 
     
     
         12 . The method of  claim 8 , comprising obtaining a plurality of images of the nucleic acid macromolecules on the substrate by an iterative process that includes:
 monitoring optical alignment of a digital camera with the patterned array using the alignment region(s) to determine alignment errors;   correcting the alignment errors;   imaging the patterned array using the digital camera; and then   optically moving the digital camera across the array chip to position it for taking another image.   
     
     
         13 . The method of  claim 12 , wherein selected alignment sites are deleted in the linear array, and the iterative process includes using the deletion sites as registration markers in a pattern matching scheme for absolute location identification. 
     
     
         14 . The method of  claim 8 , further comprising determining nucleic acid sequences of the macromolecules from the images. 
     
     
         15 . The method of  claim 8 , wherein the nucleic acid macromolecules are nucleic acid amplicons. 
     
     
         16 . The method of  claim 8 , wherein nucleic acid macromolecules on the substrate are labeled with one of at least four different fluorophores, each fluorophore indicating a different nucleotide base. 
     
     
         17 . The method of  claim 8 , whereby at least 90% of the nucleic acid macromolecules in the two-dimensional array are aligned and imaged for discrete analysis. 
     
     
         18 . The method of  claim 8 , wherein the substrate further comprises a second linear array of alignment sites separated from the two dimensional array by a site-free band, wherein the second linear array is perpendicular to the first linear array. 
     
     
         19 . The method of  claim 8 , comprising a plurality of said two-dimensional arrays arranged on the planar substrate in a plurality of columns and rows, separated from each other by a dividing space that includes a site free band having a width that is greater than the uniform distance between adjacent experiment sites in the two dimensional arrays. 
     
     
         20 . The method of  claim 19 , wherein the dividing space includes a linear array of alignment sites with a site free band on either s