Selective 5′ ligation tagging of RNA

• US 8,309,335 B2
• Filed: 02/29/2012
• Issued: 11/13/2012
• Est. Priority Date: 08/17/2007
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Epicentre Technologies Corporation (Illumina Incorporated)
• Inventors: Jendrisak, Jerome, Vaidyanathan, Ramesh, Dahl, Gary
• Primary Examiner(s): Woolwine, Samuel
• Assistant Examiner(s): Pande, Suchira
• Application Number:
  US13/408,851
• Publication Number:
  US 20120156753A1
• Time in Patent Office: 258 Days
• Field of Search: None
• US Class Current: 435/194
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/525 Phosphatase
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA

Abstract

The present invention provides novel compositions, kits and methods employing RNA 5′ polyphosphatases, RNA 5′ monophosphatases, capping enzymes, decapping enzymes, nucleic acid pyrophosphatases and RNA ligases, as well as other enzymes, for selective 5′ ligation tagging of desired classes of RNA molecules that differ with respect to particular chemical moieties on their 5′ ends. The 5′ tagged RNA molecules can be used for synthesis of tagged first-stand cDNA, double-stranded cDNA, and sense or antisense RNA for a variety of uses.

Claims

1. A kit for generating 5′

ligation-tagged RNA from RNA in a sample, wherein the kit comprises an RNA 5′

polyphosphatase (RPP) comprising the sequence of SEQ ID NO 2 and at least one other component selected from the group consisting of;

an RNA 5′

monophosphatase (RMP);

an alkaline phosphatase (AP);

a nucleic acid pyrophosphatase;

a decapping enzyme;

a capping enzyme;

an RNA ligase;

an RNA acceptor oligonucleotide;

a poly(A) polymerase;

a poly(U) polymerase;

an RNA-dependent DNA polymerase (RT);

a first-strand cDNA synthesis primer;

an RNase H;

a second-strand cDNA synthesis primer;

an RNA polymerase (RNAP);

5′

exoribonuclease (Xrn);

a polynucleotide kinase (PNK); and

an RNA molecule that has a 5′

triphosphate or diphosphate group wherein the beta or gamma phosphate of said group is labeled.

2. The kit of claim 1, wherein the kit comprises the RNA 5′

polyphosphatase, an RNA ligase and an RNA acceptor oligonucleotide.

3. The kit of claim 2, wherein the kit further comprises a first-strand cDNA synthesis primer.

4. The kit of claim 3, wherein the kit further comprises a second strand cDNA synthesis primer.

5. The kit of claim 2, wherein the kit further comprises a poly(A) polymerase.

6. The kit of claim 2, wherein the kit further comprises RNA.

7. The kit of claim 2, wherein the kit further comprises an RNA 5′

monophosphatase.

8. The kit of claim 2, wherein the kit further comprises a nucleic acid pyrophosphatase.

9. The kit of claim 2, wherein the kit further comprises a decapping enzyme.

10. The kit of claim 2, wherein the kit further comprises a capping enzyme.

11. The kit of claim 2, wherein the kit further comprises an RNA polymerase.

12. The kit of claim 2, wherein the kit further comprises a polynucleotide kinase.

13. The kit of claim 2, wherein the kit further comprises an RNA molecule that has a 5′

triphosphate or diphosphate group wherein the beta or gamma phosphate of said group is labeled.