Methods of Modifying Support Surfaces for the Immobilization of Particles and the Use of the Immobilized Particles for Analyzing Nucleic Acids

• US 20090099027A1
• Filed: 08/22/2008
• Published: 04/16/2009
• Est. Priority Date: 08/23/2007
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: STUART, Jeremy, TAING, Meng C., MENCHEN, Steven M., GREINER, Douglas P.
• Application Number:
  US12/197,132
• Publication Number:
  US 20090099027A1
• Time in Patent Office: Days
• US Class Current: 506/4
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07C 335/20 being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• 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 40/04 Libraries containing only organic compounds
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/18 using a particular method of attachment to the solid support
• 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

Methods of modifying a nucleophilic surface of a support are described. The methods involve reacting a multifunctional electrophilic reagent with nucleophilic groups on the surface of the support. The resulting electrophilic surface can be used for the covalent attachment of particles (e.g. beads) having nucleophilic functional groups. For example, nucleic acid templates with nucleophilic (e.g., amine) groups can be attached to a surface of the particles. The nucleophilic groups on the nucleic acid templates can then be used to attach the particles to the modified surface of the support. The resulting support-bound particles can be used to analyze (e.g., sequence) the nucleic acid templates on the particles.

Claims

1. A method comprising:

reacting a nucleophilic group on the surface of a substrate with a molecule comprising a plurality of electrophilic groups thereby providing one or more free electrophilic groups on the surface of the substrate; and

reacting nucleophilic groups on a surface of a particulate material with the one or more free Electrophilic groups on the surface of the substrate to covalently attach the particulate material to the substrate.

2. The method of claim 1, wherein the molecule comprising a plurality of electrophilic groups is represented by the following structure:

E₁-(L-E₂-)_nL-E₃

wherein E₁, E₂ and E₃ are electrophilic groups, L is a linker and n is 0 or a positive integer.

3. The method of claim 2, wherein the molecule comprising a plurality of electrophilic groups is:

4. The method of claim 2, wherein the molecule comprising a plurality of electrophilic groups is a polymer having a repeat unit represented by the formula:

5. The method of claim 1, wherein a nucleic acid comprising nucleophilic groups and having a 5′

end and a 3′

end is attached to the surface of the particulate material.

6. The method of claim 5, wherein the nucleic acid comprises a nucleophilic group of the formula:

7. The method of claim 6, wherein the nucleic acid is attached to the particulate material via the 5′

end of the nucleic acid and wherein the nucleophilic group is at the 3′

end of the nucleic acid.

8. The method of claim 1, wherein the particulate material comprises cross-linked polystyrene.

9. An article of manufacture made by the method of claim 1.

10. An article of manufacture comprising:

a particulate material comprising surface functional groups;

a support comprising surface functional groups;

wherein surface functional groups of the particulate material are covalently attached to surface functional groups on the support via a linker group comprising the moiety;

11. A method comprising:

reacting a nucleophilic group on the surface of a substrate with the compound represented by the formula;

12. The method of claim 11, wherein the nucleophilic group on the surface of the substrate is an amino group.

13. An article of manufacture made by the method of claim 11.

14. An article of manufacture comprising a moiety covalently attached to a support surface, wherein the moiety is represented by the formula.

15. A method comprising:

(a) hybridizing an initializing oligonucleotide probe to a target polynucleotide to form a probe-target duplex, wherein the oligonucleotide probe has an extendable probe terminus, wherein the target polynucleotide is attached to a particulate material and wherein the particulate material is covalently attached to the surface of a support;

(b) ligating a first end of an extension oligonucleotide probe to the extendable probe terminus thereby forming an extended duplex containing an extended oligonucleotide probe, wherein the extension oligonucleotide probe comprises a cleavage site and a detectable label;

(c) identifying one or more nucleotides in the target polynucleotide by detecting the label attached to the just-ligated extension oligonucleotide probe;

(d) cleaving the just-ligated extension oligonucleotide probe at the cleavage site to generate the extendable probe terminus, wherein cleavage removes a portion of the just-ligated extension oligonucleotide probe that comprises the label from the probe-target duplex; and

(e) repeating steps (b), (c) and (d).

16. The method of claim 15, wherein the cleavage site can be cleaved under conditions that will not cleave phosphodiester bonds and wherein cleavage occurs under conditions that will not cleave phosphodiester bonds.

17. The method of claim 15, wherein the extension oligonucleotide probe is an octamer.

18. The method of claim 15, wherein the detectable label is a fluorescent moiety.

19. The method of claim 15, wherein a second end of the extension oligonucleotide probe opposite the first end comprises a non-extendable probe terminus.

20. The method of claim 15, wherein the extension oligonucleotide probe has a sequence as set forth below:

*NNNNN-zzz

wherein each “

N”

represents, independently, A, C, T or C, “

z”

represents a universal base, “

*”

represents the first end of the probe and “

-”

represents the cleavage site.

21. The method of claim 20, wherein the detectable label is attached to one of the universal bases.

22. The method of claim 15, wherein surface functional groups of the particulate material are covalently attached to functional groups on the support via a linker group comprising the moiety.

23. The method of claim 15, wherein the target nucleic acid comprises a nucleophilic group comprising one or more amino groups of the formula:

24. A method of sequencing a nucleic acid comprising:

(a) hybridizing a primer to a target polynucleotide to form a primer-target duplex, wherein the target polynucleotide is attached to a particulate material at a 5′

end and wherein the particulate material is covalently attached to the surface of a support;

(b) contacting the primer-target duplex with a polymerase and one or more different nucleotide analogs to incorporate a nucleotide analog onto the 3′

end of the primer thereby forming an extended primer strand, wherein the incorporated nucleotide analog terminates the polymerase reaction and wherein each of the one or more nucleotide analogs comprises (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine and uracil and their analogs (ii) a unique label attached to the base or analog thereof via a cleavable linker;

(iii) a deoxyribose; and

(iv) a cleavable chemical group which caps an —

OH group at a 3′

-position of the deoxyribose;

(c) washing the surface of the support to remove any unincorporated nucleotide analogs;

(d) detecting the unique label attached to the just-incorporated nucleotide analog to thereby identify the just-incorporated nucleotide analog;

(e) optionally, permanently capping any unreacted —

OH group on the extended primer strand;

(f) cleaving the cleavable linker between the just incorporated nucleotide analog and the unique label;

(g) cleaving the chemical group capping the —

OH group at the 3′

-position of the deoxyribose of the just incorporated nucleotide analog to uncap the —

OH group;

(h) washing the surface of the support to remove cleaved compounds;

(i) repeating steps (b)-(h).