Methods for sequencing repetitive sequences and for determining the order of sequence subfragments

• US 6,297,006 B1
• Filed: 03/04/1997
• Issued: 10/02/2001
• Est. Priority Date: 01/16/1997
• Status: Expired due to Fees

• Current Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Original Assignee: Hyseq, Inc. (Arca Biopharma, Inc.)
• Inventors: Drmanac, Snezana, Hou, Aaron, Drmanac, Radoje T., Hauser, Brian
• Primary Examiner(s): Myers, Carla J.
• Application Number:
  US08/812,951
• Time in Patent Office: 1,673 Days
• Field of Search: 435/6, 435/91.5, 435/91.52, 435/94, 435/91, 536/24.3, 436/501
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention provides a method for detecting a target nucleic acid species including the steps of providing an array of probes affixed to a substrate and a plurality of labeled probes wherein each labeled probe is selected to have a first nucleic acid sequence which is complementary to a first portion of a target nucleic acid and wherein the nucleic acid sequence of at least one probe affixed to the substrate is complementary to a second portion of the nucleic acid sequence of the target, the second portion being adjacent to the first portion; applying a target nucleic acid to the array under suitable conditions for hybridization of probe sequences to complementary sequences; introducing a labeled probe to the array; hybridizing a probe affixed to the substrate to the target nucleic acid; hybridizing the labeled probe to the target nucleic acid; affixing the labeled probe to an adjacently hybridized probe in the array; and detecting the labeled probe affixed to the probe in the array.

Claims

1. A method for sequencing a repetitive sequence in a target nucleic acid comprising the steps of:

providing a plurality of spacer oligonucleotides of varying lengths wherein each spacer oligonucleotide in said plurality of spacer oligonucleotides includes at least one repeat of said repetitive sequence;

providing a first oligonucleotide that is adjacent to a first end of the repetitive sequence;

providing a plurality of second oligonucleotides at least one of which is adjacent to a second end of the repetitive sequence, wherein each oligonucleotide in the plurality of second oligonucleotides is labeled;

hybridizing the first oligonucleotide, one of the plurality of second oligonucleotides, and one of the plurality of spacer oligonucleotides to the target nucleic acid under conditions that discriminate a perfect match hybrid from a base pair mismatch hybrid;

ligating the hybridized oligonucleotides;

separating ligated oligonucleotides from unligated oligonucleotides; and

determining a sequence of the repetitive sequence by identifying the spacer oligonucleotide and the second oligonucleotide in the ligated oligonucleotides.

2. A method for determining the order of a plurality of sequence subfragments comprising the steps of:

providing a first oligonucleotide that is complementary to a first portion of a the branch point sequence wherein the first oligonucleotide extends from a first end of the branch point sequence by at least one nucleotide,

providing a plurality of labeled oligonucleotides that are complementary to a second portion of the branch point sequence wherein each labeled oligonucleotide in said plurality of labeled oligonucleotides includes a sequence portion that extends from a second end of the branch point sequence by at least one nucleotide, the sequence portion including a sequence that is complementarty to one of a plurality of possible sequences that extend from the second end of the branch point sequence;

hybridizing the first oligonucleotide and at least one of the plurality of labeled oligonucleotides to a target nucleic acid;

ligating the hybridized first oligonucleotide to the hybridized labeled oligonucleotide;

detecting the labeled oligonucleotide that ligates to the first oligonucleotide; and

detemining the order of the sequence subfragments from the identity of the labeled oligonucleotide that ligates to the first oligonucleotide.

3. A method for sequencing a repetitive sequence in a target nucleic acid comprising the steps of:

providing a plurality of spacer oligonucleotides of varying lengths wherein each spacer oligonucleotide in said plurality of spacer oligonucleotides includes at least one repeat of said repetitive sequence;

providing a first oligonucleotide that is adjacent to a first end of the repetitive sequence;

providing a second oligonucleotide that is adjacent to a second end of the repetitive sequence, wherein the second oligonucleotide is labeled;

hybridizing the first oligonucleotide, the second oligonucleotide, and one of the plurality of spacer oligonucleotides to the target nucleic acid under conditions that discriminate between perfect matches and a base pair mismatch hybrid;

ligating the hybridized oligonucleotides;

separating ligated oligonucleotides from unligated oligonucleotides; and

determining a sequence of the repetitive sequence in the target nucleic acid by identifying the spacer oligonucleotide that ligated to the first and second oligonucleotide.