SOLID SUPPORT FOR HIGH-THROUGHPUT NUCLEIC ACID ANALYSIS

• US 20100248991A1
• Filed: 02/15/2010
• Published: 09/30/2010
• Est. Priority Date: 02/25/2009
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Heindl, Dieter, Froehlich, Thomas, Roesler, Angelika
• Granted Patent: US 9,347,092 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• 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 provides a solid support which is preferably a bead comprising at least two sequence specific amplification primers wherein at least one primer is bound to the support with an inducible cleavable linker. The present invention also provides various method for preparing a solid support comprising at least two sequence specific primers, further characterized in that at least one of the primers is cleavable.

Claims

1. A bead support comprising at least two sequence specific amplification primers wherein at least one primer is bound to said support with an inducible cleavable linker via a functional moiety selected from the group consisting of carboxy, aldehyde, azide, alkyne, amino, thiol, sulfonylalken, iodacetyl, amino hydrazine, hydroxylamino, and maleinimido.

2. The support according to claim 1, wherein said support is comprised of a material selected from the group consisting of silicon, titanium dioxide, aluminum oxide, lanthanide oxide, glass, silicates polystyrene, cellulose, Sepharose, and polyamide.

3. The support according to claim 2, wherein said cleavable linker is a photo-cleavable linker.

4. A library of bead supports according to claim 1.

5. A library according to claim 4 wherein each member of the plurality of primers which are bound to the support via a cleavable linker carries a different detectable tag.

6. A method for preparing a bead support comprising at least two sequence specific primers wherein at least one of said primers is cleavable, the method comprising:

providing a bead support carrying at least one functional group, and

reacting said functional group with a reactive group or groups of two sequence specific primers,

wherein a cleavable reactive moiety is present either within a spacer connecting said support with the functional group or within the functional groups or said cleavable moiety is present within the spacer connecting one of the sequence specific primers with the reactive group.

7. The method according to claim 6, wherein said first and second functional groups are connected to the support via a two-arm linker.

8. A method for preparing a bead support comprising at least two sequence specific primers wherein at least one of said primers is cleavable, the method comprising:

providing a bead support carrying a first and a second functional group, each functional group carrying a different protecting group,

deprotecting the first functional group and reacting said first functional group with a reactive group of a first primer,

deprotecting a second functional group and reacting said second functional group with a reactive group of a second primer.

9. A method for preparing a bead support comprising at least two sequence specific primers wherein at least one of said primers is cleavable, the method comprising:

providing a bead support carrying exactly one functional group, and

deprotecting said functional group and reacting said group with a mixture comprising a first and a second sequence specific primer, said first and second primers comprising identical reactive groups, wherein at least one of said primers is connected to its reactive group via a cleavable moiety.

10. A method for preparing a bead support comprising at least two sequence specific primers wherein at least one of said primers is cleavable, the method comprising:

providing a bead support carrying exactly one functional group, and

deprotecting said functional group and reacting said group with an oligonucleotide representing a first and a second amplification primer which are connected to said functional group by a cleavable moiety.