Gene sequencer and method for determining the nucleotide sequence of a chromosome

• US 6,566,069 B2
• Filed: 05/21/2001
• Issued: 05/20/2003
• Est. Priority Date: 02/21/1997
• Status: Expired due to Fees

• Current Assignee: Nagaoka & Co., Ltd.
• Original Assignee: Burstein Technologies, Inc.
• Inventors: Virtanen, Jorma
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/861,787
• Publication Number:
  US 20020045174A1
• Time in Patent Office: 729 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 435/287.2, 536/22.1, 536/23.1, 536/24-- 2
• US Class Current: 435/6.12
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00536 in the shape of disks
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00635 by reactive plasma treatment
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/501 : Ligase
• CPC Class Codes: C12Q 2525/197 : incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2565/501 : being an array of oligonucleotides
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: G01N 35/00069 : whereby the sample substrate is of the bio-disk type, i.e. having the format of an optical disk

Abstract

A method for determining the nucleotide sequence of a chromosome. The chromosome is hydrolyzed to form a mixture of oligonucleotides having unknown nucleotide sequences. The mixture is exposed to a library of oligonucleotide probe pairs having known sequences. The probe pairs are located so that they hybridize to outer opposed sections of the chromosomal oligonucleotides. The use of oligonucleotide probe pairs provides for sequencing of relatively long unknown DNA sequences using relatively short oligonucleotide probes.

Claims

1. A method for determining the nucleotide sequence of a chromosome comprising the steps of:

providing a library of oligonucleotide probes, each of said probes including a distal and a proximal oligonucleotide side arm linked together by a cleavable spacer such that when the cleavable spacer is cleaved said side arms are separated from each other;

attaching a label onto said distal side arm;

attaching an array of said library of oligonucleotide probes through said proximal side arm to a solid support;

providing a sample containing a mixture of single stranded DNA fragments having unknown nucleotide sequences having a first outer section with p nucleofides and a second outer section with r nucleotides where r and p are each from 4 to 26, and a center section located between said first and second outer sections, said center section having q nucleotides where q is 0 or from 4 to 26;

allowing said first outer section of said single stranded DNA fragments to bind to its complementary distal side arm on said oligonucleotide probe and said second outer section to bind to its complimentary proximal side arm on said oligonucleotide probe thereby linking both side arms such that when said cleavable spacer is cut said side arms are not separated and said label remains tethered to said solid support;

cleaving all of said cleavable spacers on each of said oligonucleotide probes; and

determining which oligonucleotide probe sequences have single stranded DNA fragments bound thereto by detecting the presence or absence of said label.

2. The method according to claim 1 further including the step of processing the sequence information obtained from the determining step to deduce the nucleotide sequence of said sample.

3. The method according to claim 1 wherein said cleavable spacer is selected from the group comprising double stranded DNA, homoduplexed single stranded DNA, and chemically cleavable molecules.

4. The method according to claim 1 wherein said cleavable spacer is a double stranded DNA having a restriction site.

5. The method according to either claim 3 or 4 wherein said cleaving step includes the use of a restriction enzyme to cleave said spacer.

6. The method according to claim 1 further comprising the step of exposing said sample of single stranded DNA fragments to a mixture of soluble center section probes prior to exposing said sample to said library of oligonucleotide probes, said mixture of soluble probes including all possible combinations of oligonucleotides having q nucleotides when q is from 4 to 26.

7. The method according to claim 6 in which said library of oligonucleotide probes includes a sufficient number of oligonucleotide probes to provide all possible combinations of known nucleotide sequences for said distal and proximal side arms.

8. The method according to claim 7 in which if q is 0, the sum of p and r is at least 16.

9. The method according to claim 7 in which the sum of p, q and r is 60 or less.

10. The method according to claim 9 in which q is greater than p.

11. The method according to claim 10 in which r is equal to p.

12. The method according to claim 11 in which r is 8 and q is 11.

13. The method according to claim 7 in which if q is 0, p and r are at least 16.

14. The method according to claim 7 in which q is greater than p.

15. The method according to claim 14 in which r is equal to p.

16. The method according to claim 15 in which r is 8 and q is 11.

17. The method according to claim 1 wherein said label is selected from the group comprising microspheres, fluorophores, and chromophores.

18. The method according to claim 1 wherein said solid support is selected from the group comprising CDs, CD-Rs, DVDs, and DVD-Rs.