Method of sorting polynucleotides

• US 5,863,722 A
• Filed: 06/07/1995
• Issued: 01/26/1999
• Est. Priority Date: 10/13/1994
• Status: Expired due to Term

• Current Assignee: Lynx Therapeutics, Inc. (Illumina Incorporated)
• Original Assignee: Lynx Therapeutics, Inc. (Illumina Incorporated)
• Inventors: Brenner, Sydney
• Primary Examiner(s): Chambers, Jasemine C.
• Assistant Examiner(s): PRIEBE, SCOTT DAVID
• Application Number:
  US08/485,105
• Time in Patent Office: 1,329 Days
• Field of Search: 435/6, 536/24.3
• US Class Current: 435/6.14
• CPC Class Codes: B01J 19/00 Chemical, physical or physico-chemical processes in general; Their relevant apparatus
• 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 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• 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 2219/00637 by coating it with another layer
• 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 15/09 Recombinant DNA-technology
• 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: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6813 : Hybridisation assays
• CPC Class Codes: C12Q 1/6827 : for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 : Enzymatic or biochemical coupling of nucleic acids to a solid phase
• 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 1/6869 : Methods for sequencing
• 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 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 20/04 : Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
• 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
• CPC Class Codes: G01N 37/00 : Details not covered by any other group of this subclass

Abstract

The invention provides a method and materials for sorting polynucleotides with oligonucleotide tags. Oligonucleotide tags of the invention are capable of hybridizing to complementary oligomeric compounds consisting of subunits having enhanced binding strength and specificity as compared to natural oligonucleotides. Such complementary oligomeric compounds are referred to herein as "tag complements." Subunits of tag complements may consist of monomers of non-natural nucleotide analogs, referred to herein as "antisense monomers" or they may comprise oligomers having lengths in the range of 3 to 6 nucleotides or analogs thereof, including antisense monomers, the oligomers being selected from a minimally cross-hybridizing set. In such a set, a duplex made up of an oligomer of the set and the complement of any other oligomer of the set contains at least two mismatches. Preferred antisense monomers include peptide nucleic acid monomers and nucleoside phosphoramidates having a 3'"'"'--NHP(O)(O--)O--5'"'"' linkage with its adjacent nucleoside.

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, or the like, wherein many target polynucleotides or many segments of a single target polynucleotide are sequenced simultaneously.

Claims

1. A method of characterizing a population of mRNA molecules, the method comprising the steps of:

forming a population of cDNA molecules from the population of mRNA molecules, the cDNA molecules being complementary to the mRNA molecules and each cDNA molecule having an oligonucleotide tag attached, (i) such that substantially all different cDNA molecules have different oligonucleotide tags attached and (ii) such that each oligonucleotide tag comprises a plurality of subunits and each subunit of the plurality consists of an oligonucleotide having a length from three to six nucleotides or from three to six basepairs, the subunits being selected from a minimally cross-hybridizing set wherein a subunit of the set and a complement of any other subunit of the set would have at least two mismatches,

sorting the cDNA molecules by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical complements in spatially discrete regions on one or more solid phase supports and the respective complements being oligodeoxyribonucleotide N3'"'"'→

P5'"'"' phosphoramidates or peptide nucleic acids;

determining the nucleotide sequence of a portion of each of the sorted cDNA molecules; and

characterizing the population of mRNA molecules by the frequency distribution of the portions of sequences of the cDNA molecules.

2. The method of claim 1 wherein said one or more solid phase supports are a plurality of microparticles.

3. The method of claim 2 wherein each said portion of each of said sorted cDNA molecules is in the range of from 12 to 50 nucleotides.

4. The method of claim 3 wherein each said portion of each of said sorted cDNA molecules is in the range of from 12 to 25 nucleotides.

5. The method of claim 1 wherein said one or more solid phase supports are a plurality of microparticles.

6. The method of claim 5 wherein after said step of sorting, said plurality of microparticles is fixed to a planar substrate.

7. The method of claim 6 wherein said plurality of microparticles are disposed randomly on the surface of said planar substrate at a density of between 1000 microparticles to 100 thousand microparticles per square centimeter.

8. The method of claim 1 wherein each of said oligonucleotide tags has a length the range of from 10 to 40 monomers.

9. The method of claim 1 wherein said population of cDNA molecules contains from 500 to 1000 cDNA molecules.

10. A method of characterizing a population of mRNA molecules, the method comprising the steps of:

forming a population of cDNA molecules from the population of mRNA molecules, the cDNA molecules being complementary to the mRNA molecules;

attaching an oligonucleotide tag from a repertoire of tags to each cDNA molecule of the population such that substantially all different cDNA molecules have different oligonucleotide tags attached;

sorting the cDNA molecules by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical complements in spatially discrete regions on one or more solid phase supports and the respective complements being oligodeoxyribonucleotide N3'"'"'→

P5'"'"' phosphoramidates or peptide nucleic acids;

determining the nucleotide sequence of a portion of each of the cDNA molecules of the population; and

characterizing the population of mRNA molecules by the frequency distribution of the portions of sequences of the cDNA molecules.

11. The method of claim 10 wherein said oligonucleotide tags are single stranded oligonucleotides.

12. The method of claim 11 wherein said step of determining said nucleotide sequence of said cDNA molecules is carried out simultaneously for said population of cDNA molecules by a single base sequencing method.

13. The method of claim 12 wherein each said portion of each of said sorted cDNA molecules is from 12 to 50 nucleotides in length.

14. The method of claim 13 wherein each said portion of each of said sorted cDNA molecules is from 12 to 25 nucleotides in length.