Method for performing multiple enzyme reactions in a single tube

• US 10,155,939 B1
• Filed: 06/15/2017
• Issued: 12/18/2018
• Est. Priority Date: 06/15/2017
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Vaisvila, Romualdas, Higgins, Lauren, Luck, Ashley
• Primary Examiner(s): Robinson, Hope
• Application Number:
  US15/623,756
• Publication Number:
  US 20180362960A1
• Time in Patent Office: 551 Days
• Field of Search: None
• CPC Class Codes: C07H 19/073 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C11D 3/122 Sulfur-containing, e.g. sulfates, sulfites or gypsum
• CPC Class Codes: C12N 15/09 Recombinant DNA-technology
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/2497 hydrolysing N- glycosyl compounds (3.2.2)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12Q 1/52 involving transaminase
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2521/537 Protease
• CPC Class Codes: C12Y 302/02015 DNA-deoxyinosine glycosylase (3.2.2.15)
• CPC Class Codes: C12Y 302/02027 Uracil-DNA glycosylase (3.2.2.27)
• CPC Class Codes: C12Y 402/99018 DNA-(apurinic or apyrimidinic site)lyase (4.2.99.18)

Abstract

Among other things, a method for performing multiple enzyme reactions in a single tube is provided. In some embodiments, the method may comprise producing a reaction mix comprising a thermolabile UDG, an AP lyase and DNA fragments that comprise one or more uracil residues, incubating the reaction mix at a relatively low temperature to cleave fragments at the one or more uracil residues, raising the temperature of the reaction mix to a relatively high temperature to inactivate the thermolabile UDG; and deaminating the fragments, thereby converting any cytosine in the fragments of DNA to uracil.

Claims

1. A method for performing multiple enzyme reactions in a single tube, comprising:

(a) producing a reaction mix comprising a thermolabile uracil-DNA glycosylase (UDG), a DNA-(apurinic or apyrimidinic site) lyase (AP-lyase) and fragments of genomic or plasmid DNA that comprise one or more uracil residues;

(b) incubating the reaction mix at a temperature of below 40°

C. to cleave the genomic or plasmid DNA fragments at the one or more uracil residues;

(c) raising the temperature of the reaction mix to at least 50°

C. for a period of time to inactivate the thermolabile UDG; and

(d) after step (c) deaminating the fragments of genomic or plasmid DNA, thereby converting any cytosine in the fragments of genomic or plasmid DNA to uracil.

2. The method of claim 1, wherein the fragments of genomic or plasmid DNA comprise an oligonucleotide, and wherein the oligonucleotide comprises the one or more uracil residues.

3. The method of claim 2, wherein the oligonucleotide is an adapter ligated to at least one end of the genomic or plasmid fragments.

4. The method of claim 3, wherein the adaptor is a loop adaptor.

5. The method of claim 1, wherein AP lyase is also thermolabile.

6. The method of claim 1, wherein AP lyase is Endonuclease III.

7. The method of claim 1, wherein the deaminating is performed using a deaminase enzyme.

8. The method of claim 7, wherein the deaminase enzyme has at least 90% sequence identity with SEQ ID NO:

1 (APOBEC).

9. The method of claim 1, wherein the deamination is performed by sodium bisulfite treatment.

10. The method of claim 1, further comprising (e) amplifying the fragments of genomic or plasmid DNA.

11. The method of claim 10, further comprising sequencing the amplification products.

12. The method of claim 11, further comprising identifying the sites of methylcytosines and/or hydroxymethylcytosine residues in the fragments of genomic or plasmid DNA.