Miniaturized, high-throughput nucleic acid analysis

• US 8,822,158 B2
• Filed: 01/22/2013
• Issued: 09/02/2014
• Est. Priority Date: 02/25/2009
• Status: Active Grant

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: Froehlich, Thomas, Heindl, Dieter, Roesler, Angelika, Heckel, Tobias
• Primary Examiner(s): HORLICK, KENNETH R
• Application Number:
  US13/746,812
• Publication Number:
  US 20130190191A1
• Time in Patent Office: 588 Days
• Field of Search: 435/6.12, 435/91.2
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2547/101 by confinement to a single tube/container
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The present invention is directed to method for analyzing multiple nucleic acid molecules of interest comprising in the steps of (i) providing a plurality of beads, characterized in that each bead comprises at least two sequence specific amplification primers, further characterized in that at least one of the primers is bound to the bead via a cleavable linker, (ii) capturing the nucleic acid molecules of interest from a sample, (iii) clonally isolating the plurality of beads, (iv) cleaving the at least one primer, (v) clonally amplifying the nucleic acid thereby creating multiple amplification products, and (vi) analyzing the amplification products.

Claims

1. A method for analyzing a sample comprising nucleic acid molecules of interest, the method comprising the steps of:

(a) providing a plurality of beads and a plurality of sequence specific amplification primer pairs, wherein each bead comprises one pair of sequence specific amplification primers comprising a first primer and a second primer, and wherein the first primer is bound to the bead via photo-cleavable linker,

(b) capturing onto the beads the nucleic acid molecules of interest from the sample by hybridization of the nucleic acid molecules to the second primer,

(c) clonally isolating said plurality of beads with captured nucleic acid molecules,

(d) cleaving the first primer by photo-induction, while the second primer remains covalently attached to the bead,

(e) clonally amplifying said nucleic acid, thereby generating multiple amplification products, and

(f) analyzing said amplification products.

2. The method according to claim 1, wherein step (c) comprises the generation of an emulsion wherein each bead is encapsulated in a single micelle.

3. The method according to claim 1, wherein step (f) further comprises sequencing said amplification products.

4. The method according to claim 3, wherein said sequencing is performed by a sequencing by synthesis reaction.

5. The method according to claim 1, wherein the generation of amplification products is monitored.

6. The method according to claim 5, wherein said amplification products are monitored by means of a specifically double-stranded DNA binding fluorescent entity or a sequence specific hybridization probe.

7. The method according to claim 5, wherein said amplification products are monitored by means of a thermal gradient.

8. The method according to claim 1, wherein step (c) comprises the distribution of said plurality of beads into the cavities of a micro- or picotiter plate.

9. The method according to claim 1, wherein steps (e) and (f) are performed simultaneously by means of real time PCR.

10. The method according to claim 1, wherein the first primer carries a detectable label.

11. The method according to claim 10, wherein said detectable label is selected from the group consisting of a mass-tag, a color label, an e-tag, and a hapten which is detectable by an antibody.

12. The method according to claim 10, wherein said detectable label is a fluorescent label which is quenched as long as said labeled primer has not been elongated.

13. The method according to claim 1 wherein each member of the plurality of primer pairs which are bound to a bead via a cleavable linker carries a different detectable label.

14. The method according to claim 1 wherein clonal amplification comprises physical separation of amplification products originating from different target molecules by immobilization on a bead enclosed in a micelle.

15. The method according to claim 1 wherein clonal amplification is performed by emulsion PCR.

16. The method according to claim 1 wherein step (c) comprises the generation of a water-in-oil emulsion wherein each bead is encapsulated in a single micelle containing an aqueous solution and clonal amplification is performed by emulsion PCR.

17. The method according to claim 16, wherein bead density in a 1:

2 water to oil emulsion is about 3000 beads per 1 microliter emulsion.

18. The method according to claim 8 wherein a size of the cavities corresponds to a diameter of the beads in such a way that one cavity can only be filled with one bead.

19. A method according to claim 9 wherein a hot-start PCR protocol is applied.

20. The method according to claim 4 wherein the sequence by synthesis reaction comprises a pyrophosphate sequencing reaction.