Oligonucleotide-linked magnetic particles and uses thereof

• US 5,512,439 A
• Filed: 07/06/1994
• Issued: 04/30/1996
• Est. Priority Date: 11/21/1988
• Status: Expired due to Term

• Current Assignee: Life Technologies AS (Perspecta, Inc.)
• Original Assignee: Dynel Comapny
• Inventors: Hornes, Erik, Korsnes, Lars
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): TRAN, PAUL
• Application Number:
  US08/272,372
• Time in Patent Office: 664 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.5, 514/44, 436/526
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: G01N 33/54326 Magnetic particles

Abstract

Monodisperse, superparamagnetic particles carrying a plurality of molecules of an oligonucleotide are disclosed and may be used inter alia for sequencing single-stranded nucleic acids. The oligonucleotide may be covalently attached or affinity bonded to the particles either by their 3'"'"' or 5'"'"' termini. The particles have a specific gravity in the range 1.1-1.8 and are 1-10 microns in diameter. A kit for the isolation or processing of target nucleic acid is also disclosed.

Claims

1. A plurality of monodisperse, superparamagnetic particles, wherein:

each particle comprises (i) superparamagnetic iron oxide dispersed within a polymer particle, (ii) a coating which reduces non specific binding, and (iii) a functional group carried by said coating for bonding a nucleic acid

the particles are monodisperse and have a diameter standard deviation of less than 5%, and

particles of said plurality carry a plurality of molecules of an oligonucleotide.

2. Particles as claimed in claim 1, wherein a 5'"'"'-amino group on said oligonucleotide is covalently bonded to the particle surface via a 5'"'"'-amido group formed with a carboxyl group on the particle.

3. Particles as claimed in claim 2 in which the particles have a coating of a polyacrylic or polymethacrylic acid to provide carboxyl groups which form said 5'"'"'-amido group by reaction with a 5'"'"'-amino group on the oligonucleotide.

4. Particles as claimed in claim 1 in which the oligonucleotide is covalently bonded directly to the particle surface via a 5'"'"'-amino group on said oligonucleotide.

5. Particles as claimed in claim 1 in which the oligonucleotide is bonded to the particle surface by a 5'"'"'-biotinyl group on said oligonucleotide which bonds to avidin or streptavidin on the particle.

6. Particles as claimed in claim 1 in which the oligonucleotide is covalently bonded to the particle by a 3'"'"'-hydroxy group on said oligonucleotide which bonds to hydroxyl on the particle surface.

7. Particles as claimed in claim 1 having a specific gravity in the range 1.1 to 1.8.

8. Particles as claimed in claim 1 in the size range 1 to 10 microns.

9. Particles as claimed in claim 1 in which the oligonucleotide has a chain length in the range 12 to 200 bases.

10. Particles as claimed in claim 1 in which the oligonucleotide is polydT.

11. Particles as claimed in claim 1 wherein said oligonucleotide hybridizes specifically to a DNA or RNA target.

12. Particles as claimed in claim 11 in which the oligonucleotide hybridizes specifically to a conserved region of a family of target nucleic acids.

13. Particles as claimed in claim 1 in which the oligonucleotide comprises a region hybridizing specifically to a target nucleic acid and, joined to the particle, a linker sequence containing a restriction endonuclease restriction site.

14. A method of preparing magnetic particles as claimed in claim 1, wherein monodisperse, superparamagnetic particles carrying functional groups on the surface thereof are reacted with an oligonucleotide carrying one or more functional groups reactive with the functional groups on said particles, whereby said oligonucleotide is bonded to said particles.

15. A process as claimed in claim 14 in which the oligonucleotide is bonded by a reaction or synthesis selected from the group consisting of:

(a) reaction of biotin on the oligonucleotide with avidin or streptavidin on the particle;

(b) reaction of a 5'"'"'-amino group on the oligonucleotide with a carboxyl or tosyloxy group on the particle; and

(c) direct chemical synthesis of the oligonucleotide on particles carrying hydroxyl or protected hydroxyl groups.

16. A method of immobilizing a target nucleic acid whereby said nucleic acid is contacted in solution with particles as claimed in claim 1 whereby the oligonucleotide thereon hybridizes to a nucleotide sequence on said target nucleic acids, thereby immobilizing said target nucleic acid.

17. A method as claimed in claim 16 in which the target nucleic acid is mRNA and the particles are subsequently magnetically aggregated to a surface and separated from said solution.

18. A method as claimed in claim 16 in which the target nucleic acid when immobilized on said particles is subjected to amplification by the polymerase chain reaction using the oligonucleotide attached to said particles as one of the two primers, further particles carrying said oligonucleotide being present or added subsequently to provide a sufficient amount of the said primer to permit amplification.

20. A kit for the detection or isolation of target nucleic acids, comprising:

(a) magnetic particles as claimed in claim 1, and at least one of the following;

(b) a polymerase

(c) a reverse transcriptase

(d) a restriction endonuclease

(e) appropriate buffers

(f) dideoxynucleotides some of which may carry, or be adapted to carry a label

(g) deoxynucleotides, some of which may carry, or be adapted to carry, a label

(h) an oligonucleotide carrying, or adapted to carry, a label

(i) a 5'"'"'-primer or a 3'"'"'-primer, or both, which may be labelled.

19. A method of sequencing single stranded nucleic acids which comprises the steps of:

(a) preparing a plurality of superparamagnetic monodisperse particles wherein each particle comprises (i) superparamagnetic iron oxide dispersed within a polymer particle and (ii) a coating which reduces non-specific binding; and

which carries a functional group for bonding a nucleic acid and the particles being monodisperse and having a diameter standard deviation of less that 5%, and particles of said plurality carrying a plurality of moleceles of an oligonucleotide to be sequenced;

(b) either

(i) dividing the particles into four aliquots and adding to each aliquot a polymerase, mixed nucleoside triphosphates, a single dideoxynucleoside triphosphate, the latter being different for each aliquot and, a primer, if required, wherein at least one of the primer or nucleoside or dideoxynucleoside is labelled;

or

(ii) adding to all the particles a polymerase, mixed nucleoside triphosphates, four different dideoxy nucleoside triphosphates each carrying a different label and, a primer, whereby, a series of labelled DNA strands are synthesized each having different chain lengths and ending with a particular dideoxy base,

(c) liberating the labelled DNA strands and size fractionating these; and

(d) determining the sequence of the nucleic acids.