Methods of making oligonucleotide probes

• US 10,370,702 B2
• Filed: 09/22/2016
• Issued: 08/06/2019
• Est. Priority Date: 10/18/2012
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Wu, Chao-ting, Beliveau, Brian
• Primary Examiner(s): Priest, Aaron A
• Application Number:
  US15/273,041
• Publication Number:
  US 20170002402A1
• Time in Patent Office: 1,048 Days
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

The present invention relates to methods of making linear nucleic acid probes using rolling circle amplification methods.

Claims

1. A plurality of nucleic acid probes in the form of a single stranded nucleic acid concatamer of repeating nucleic acid probe sequences wherein each nucleic acid probe sequence comprises

a polymerase recognition site at one end, a first nucleic acid sequence complementary to a target sequence, and a label addition site

at the other end,

and further comprising a hybridized oligonucleotide at the polymerase recognition site to form a hybrid that is recognizable by an endonuclease.

2. The nucleic acid probes of claim 1 wherein the single stranded nucleic acid concatamer is DNA.

3. The nucleic acid probes of claim 1 wherein the single stranded nucleic acid concatamer is RNA.

4. The nucleic acid probes of claim 1 wherein the polymerase recognition site is an RNA polymerase recognition site.

5. The nucleic acid probes of claim 1 wherein the polymerase recognition site is a DNA polymerase recognition site.

6. The nucleic acid probes of claim 1 wherein the label addition site comprises a sequence complementary to a secondary probe including a label.

7. The method of claim 1 wherein the hybridized oligonucleotide is DNA and the polymerase recognition site is DNA to produce a DNA/DNA hybrid recognizable by the endonuclease.

8. The method of claim 1 wherein the hybridized oligonucleotide is DNA and the polymerase recognition site is RNA to produce a DNA/RNA hybrid recognizable by the endonuclease.

9. The method of claim 1 wherein the hybridized oligonucleotide is RNA and the polymerase recognition site is DNA to produce a RNA/DNA hybrid recognizable by the endonuclease.

10. The method of claim 1 wherein the hybridized oligonucleotide is RNA and the polymerase recognition site is RNA to produce a RNA/RNA hybrid recognizable by the endonuclease.