Oligonucleotide tags for sorting and identification

• US 6,235,475 B1
• Filed: 08/07/1998
• Issued: 05/22/2001
• Est. Priority Date: 10/13/1994
• Status: Expired due to Term

• Current Assignee: Solexa Incorporated (Illumina Incorporated)
• Original Assignee: Lynx Therapeutics, Inc. (Illumina Incorporated)
• Inventors: Brenner, Sydney, Macevicz, Stephen C., Albrecht, Glenn
• Primary Examiner(s): Schwartzman, Robert A.
• Assistant Examiner(s): SHIBUYA, MARK LANCE
• Application Number:
  US09/130,862
• Time in Patent Office: 1,019 Days
• Field of Search: 435/6, 435/91.1, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32, 536/25.6
• US Class Current: 506/41
• 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 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• 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 2219/00722 Nucleotides
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/161 : incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/191 : incorporating an adaptor
• CPC Class Codes: C12Q 2525/204 : specific length of the oligonucleotides
• CPC Class Codes: C12Q 2563/107 : fluorescence
• CPC Class Codes: C12Q 2563/185 : Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• CPC Class Codes: C12Q 2565/501 : being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/518 : characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/519 : characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 70/00 : Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• CPC Class Codes: C40B 80/00 : Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers

Abstract

The invention provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention comprise oligonucleotides selected from a minimally cross-hybridizing set. Preferably, such oligonucleotides each consist of a plurality of subunits 3 to 9 nucleotides in length. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports. This embodiment provides a readily automated system for manipulating and sorting polynucleotides, particularly useful in large-scale parallel operations, such as large-scale DNA sequencing, mRNA fingerprinting, and the like, wherein many target polynucleotides or many segments of a single target polynucleotide are sequenced simultaneously.

Claims

1. A method of generating a minimally cross-hybridizing set of sequences for synthesizing oligonucleotide tags, the method comprising the steps of:

(i) generating a list of sequences having a selected length,

(ii) selecting a first sequence from the list for inclusion in the minimally cross-hybridizing set,

(iii) selecting a second sequence from the list,

(iv) comparing the second sequence with every sequence in the minimally cross-hybridizing set,

(v) retaining the second sequence for inclusion in the minimally cross-hybridizing set if its sequence differs from every sequence in the minimally cross-hybridizing set by at least two bases and if it forms a duplex with its complement having a stability equivalent to those of the duplexes of every other sequence in the minimally cross-hybridizing set and its complement, or otherwise discarding the second sequence;

(vi) repeating steps (iii)-(v) until all sequences in the list have been selected, compared and retained or discarded; and

(vii) synthesizing one or more oligonucleotide tags each having one or more oligonucleotide sequences of the plurality.

2. The method of claim 1 wherein said selected length is between 4 and 18 nucleotides, inclusive.