Patent ID: 6599703
Filing Date: 2003-07-29
Classification: C12Q

Abstract:
A method for automated sequencing of double-strande DNA segments with nested single strand overhang templates, such method compromising the steps of:i) providing a support array having a plurality of sample holders arrayed in a matrix of positions on the support, and wherein the array is on a stage; ii) immobilizing a plurality of double-stranded DNA segments at respective sample holders of said array, each DNA segment having an end compromising a single-strand overhang template sequence no longer than about twenty nucleotides in length, wherein said overhang sequence is created by a process including ligation of a strand of a recognition domain to each template and digestion by an enzyme that cuts at a site at least one nucleotide away from the recognition domain, and includes ligation of a DNA sequence that can be used to generate a primer annealing site during DNA amplification in vitro following ligation of the recognition domain and prior to generation of the DNA template; iii) simultaneously treating all sample holders with one or more reagents which selectively react with at least one nucleotide of said single-strand overhang template to effectively label the material at each holder; iv) reading said array by automated scan detection to thereby determine at least one nucleotide of said single-strand overhang template; and v) reducing length of each strand of said DNA segment at each holder by a fixed number n>1 at said overhang end to produce a homologously ordered array of shorter and nested DNA segments, each with a single-strand overhang template sequence, and further performing steps iii) and iv) to determine at least one nucleotide at each single-strand overhang sequence, wherein the steps of treating, reading and reducing the length of the strands of the DNA segment at each holder by a number of n>1 nucleotides are iteratively performed as automated process steps to produce nested and progressively shorter DNA segments and to sequence the plurality of DNA segments immobilized at the array of sample holders in situ.