Restriction endonucleases and their uses

• US 9,650,675 B2
• Filed: 12/17/2015
• Issued: 05/16/2017
• Est. Priority Date: 07/05/2012
• Status: Active Grant

• Current Assignee: Thermo Fisher Scientific Baltics, UAB (Thermo Fisher Scientific Incorporated)
• Original Assignee: Thermo Fisher Scientific Baltics, UAB (Thermo Fisher Scientific Incorporated)
• Inventors: Rimseliene, Renata, Lubys, Arvydas, Skirgalia, Remigijus
• Primary Examiner(s): Mondesi, Robert
• Assistant Examiner(s): EKSTROM, RICHARD C
• Application Number:
  US14/972,566
• Publication Number:
  US 20160194706A1
• Time in Patent Office: 516 Days
• Field of Search: None
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• 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 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

A restriction endonuclease with a recognition sequence 5′-TCGA-3′. The restriction endonuclease is sensitive to the presence of a modified cytosine residue in the recognition sequence. Methods and kits using the restriction endonuclease with a recognition sequence 5′-TCGA-3′ are also disclosed.

Claims

1. A method for determining the level of methylation in eukaryotic double stranded DNA, the method comprising

contacting a first sample of the eukaryotic double stranded DNA with a restriction endonuclease comprising SEQ ID NO. 4 or a sequence at least 70% identical to SEQ ID NO. 4, having a recognition sequence 5′

-TCGA-3′

where a modified cytosine residue in the recognition sequence impairs cleavage,

digesting the DNA comprising the recognition sequence 5′

-TCGA-3′

in which the cytosine residue is unmodified; and

determining the amount of undigested DNA or digested DNA.

2. The method of claim 1 further comprising

contacting a second sample of the double stranded DNA with a second restriction endonuclease;

and

determining the amount of DNA digestion,

where the second restriction endonuclease has a recognition sequence 5′

-TCGA-3′ and

is not impaired by modification of the cytosine residue.

3. The method of claim 1 wherein the eukaryotic double stranded DNA is genomic DNA.

4. The method of claim 1 wherein the amount of undigested DNA is determined using qPCR.

5. The method of claim 2 where the second restriction endonuclease is TaqI.

6. The method of claim 1 wherein the impaired ability to cleave the recognition sequence means that under conditions at which the restriction endonuclease is most active, optimal reaction conditions, the presence of the modified cytosine residue in the recognition sequence reduces the endonuclease'"'"'s ability to cleave the sequence by more than 80%.

7. The method of claim 6 wherein the presence of the modified cytosine residue in the recognition sequence reduces the endonuclease'"'"'s ability to cleave the sequence by more than 90%.

8. The method of claim 6 wherein the percent of DNA cleavage is determined by qPCR analysis.

9. The method of claim 1 wherein the modified cytosine residue is a methylated cytosine.

10. The method of claim 9 wherein the methylated cytosine is selected from the group consisting of 5-methylcytosine, 5-hydroxymethylcytosine, and glycosylated 5-hydroxymethylcytosine.