Enzymatic ligation of nucleic acids

• US 10,494,671 B2
• Filed: 01/17/2012
• Issued: 12/03/2019
• Est. Priority Date: 01/17/2011
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Hendricks, Stephen, King, David
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US13/980,280
• Publication Number:
  US 20140378315A1
• Time in Patent Office: 2,877 Days
• CPC Class Codes: A61J 1/062 Carpules
• CPC Class Codes: A61J 1/14 Details; Accessories therefor A61J7/00 takes precedence; openers B65D, B67B7/00
• CPC Class Codes: A61J 2200/76 for fluid level
• CPC Class Codes: A61J 2205/30 Printed labels
• CPC Class Codes: A61M 5/008 Racks for supporting syringes or needles A61M5/001 takes precedence
• CPC Class Codes: A61M 5/5086 for indicating if defective, used, tampered with or unsterile
• CPC Class Codes: B65B 3/003 Filling medical containers such as ampoules, vials, syringes or the like
• CPC Class Codes: B65B 3/006 Related operations, e.g. scoring ampoules
• CPC Class Codes: B65B 31/046 the nozzles co-operating, or being combined, with a device for opening or closing the container or wrapper B65B31/06 takes precedence
• CPC Class Codes: B65B 55/08 by irradiation
• CPC Class Codes: B65B 55/10 by liquids or gases
• CPC Class Codes: B65D 15/02 of curved, or partially curved, cross-section, e.g. cans, drums
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2531/137 Ligase Chain Reaction [LCR]
• CPC Class Codes: C12Q 2533/107 : Probe or oligonucleotide ligation
• 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 2537/149 : Sequential reactions
• CPC Class Codes: C12Q 2563/107 : fluorescence
• CPC Class Codes: C12Q 2563/149 : Particles, e.g. beads
• CPC Class Codes: C12Q 2565/102 : Multiple non-interacting labels
• CPC Class Codes: C12Q 2600/16 : Primer sets for multiplex assays
• CPC Class Codes: C12Y 605/01001 : DNA ligase (ATP) (6.5.1.1)
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Methods, assays, compositions and kits for the ligation of short polynucleotides are presented herein. The short polynucleotides are optionally no more than 7 nucleotides in length, and can be as short as 3 or 4 nucleotides in length. The ligation is optionally performed by CV ligase.

Claims

1. A method of template-dependent ligation, said method comprising:

a. enzymatically ligating a proximal terminus of a first oligonucleotide and a proximal terminus of a second oligonucleotide, wherein the proximal termini of the first and the second oligonucleotides are adjacently hybridized to a template to form a ligation product, wherein the ligation is performed by a Chlorella Virus ligase (CV ligase), wherein the first oligonucleotide and/or second oligonucleotide is less than 8 nucleotides in length, wherein the first oligonucleotide hybridizes to a region on the template adjacent to the proximal terminus of the second oligonucleotide, and wherein the first oligonucleotide is fluorescently labeled, wherein the first oligonucleotide comprises a proximal portion that is (i) perfectly hybridized to the template and is (ii) L nucleotides long, wherein the first oligonucleotide'"'"'s L+1th nucleotide is mismatched with the template; and

b. detecting the fluorescent label of the first oligonucleotide that has ligated to the second oligonucleotide.

2. The method of claim 1, wherein the effiency of the ligation is at least 80%.

3. The method of claim 1, wherein the proximal terminus of the first oligonucleotide is its 3′

terminus and the proximal terminus of the second oligonucleotide is its 5′

terminus.

4. The method of claim 1, wherein the proximal terminus of the first oligonucleotide is its 5′

terminus and the proximal terminus of the second oligonucleotide is its 3′

terminus.

5. The method of claim 1, wherein the second oligonucleotide and/or template is fluorescently labeled.

6. A method of claim 1, wherein at least one of the first oligonucleotide, the template, and/or the second oligonucleotide is immobilized.

7. The method of claim 1, wherein ligation is performed in the presence of a multiplicity of additional oligonucleotides that are at least partially complementary to the same region on the templae as the first oligonucleotide.

8. The method of claim 7, wherein at least one of the multiplicity of additional oligonucleotides is labeled distinguishably from the first oligonucleotide.

9. The method of claim 7, wherein the portion of the second oligonucleotide that is hybridized to the template is 2 to 20 nucleotides in length.

10. The method of claim 1, wherein the ligation is repeated at least once.

11. The method of claim 10, any ligation product of a previous ligation reaction is used as the second oligonucleotide of a next ligation.

12. The method of claim 10, further comprising detecting whether the first oligonucleotide has ligated to the second oligonucleotide before repeating the ligation.

13. The method of claim 10, wherein any ligation product of the previous ligation reaction is used as the template of the next ligation reaction.

14. The method of claim 1, wherein the method comprises a ligase chain reaction.

15. The method of claim 1, wherein the first oligonucleotide serces a probe function.

16. The method of claim 1, wherein the second oligonucleotide serves a primer function.

17. The method of claim 1, wherein the second oligonucleotide is extendable at its 3′

terminus.

18. A method of template-dependent ligation, said method comprising:

a. enzymatically ligating a first oligonucleotide and a second oligonucleotide, wherein said first oligonucleotide and said second oligonucleotide are hybridized to an RNA template, to form a ligation product, wherein the ligation is performed by a Chlorella Virus ligase (CV ligase), wherein the portion of the first oligonucleotide that is hybridized to the RNA template is 2 to 8 nucleotides in length and the portion of the second oligonucleotide that is hybridized to the RNA template is 2 to 20 nucleotides in length, wherein the first oligonucleotide is at least partially complementary to a region on the RNA template adjacent to the proximal terminus of the second oligonucleotide, and wherein the first oligonucleotide is labeled;

b. amplifying the ligation product; and

c. detecting the label of the amplified ligation product.

19. The method of claim 18, wherein the efficiency of the ligation is atleast 80%.

20. The method of claim 18, wherein the label is a fluorescent label.

21. The method of claim 18, wherein the RNA template is an miRNA template.

22. The method of claim 18, wherein the amplifying is performed by a polymerase chain reaction (PCR).