Restriction endonucleases and their uses
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 recongition sequence 5′-TCGA-3′ are also disclosed.
1. A method for determining the level of methylation in double stranded DNA, the method comprising
contacting a first sample of the double stranded DNA with a first restriction endonuclease comprising SEQ ID NO. 4 or a sequence at least 85% identical to SEQ ID NO. 4, having a recognition sequence 5′-TCGA-3′, where a methylated cytosine residue in the recognition sequence impairs cleavage,
digesting the DNA comprising the recognition sequence 5′-TCGA-3′ in which the cytosine residue is unmethylated;
determining the amount of undigested DNA or digested DNA by the first restriction endonuclease,
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 sensitive to methylation of the cytosine residue.
2. The method of claim 1 wherein the double stranded DNA is genomic DNA.
3. The method of claim 1 wherein the amount of undigested DNA is determined using qPCR.
4. The method of claim 3 where the second restriction endonuclease is TaqI.
5. A kit for determining the modification status of a DNA duplex substrate, the kit comprising instructions for use and, in separate containers, (a) a restriction endonuclease comprising SEQ ID NO. 4 or a sequence at least 85% identical to SEQ ID NO. 4, having a recognition sequence 5′-TCGA-3′, where a modified cytosine residue in the recognition sequence impairs cleavage, (b) a second restriction endonuclease that has a recognition sequence 5′-TCGA-3′ and is not impaired by modification of the cytosine residue, and (c) a buffer comprising tris(hydroxymethyl)aminomethane) (Tris).
6. The kit of claim 5 wherein the second restriction endonuclease is TaqI.
7. The kit of claim 5 wherein the DNA duplex substrate is eukaryotic.
8. The kit of claim 5 wherein the buffer comprises Tris-HCl, Tris-acetate, MgCl 2 , Mg-acetate, K-acetate, NaCl, and/or bovine serum albumin (BSA).
9. The kit of claim 5 further comprising genomic DNA that is used for a control reaction involving the enzymes in the kit.
10. The kit of claim 5 wherein the modification status of a DNA duplex substrate comprises a modified cytosine residue.
11. The kit of claim 10 wherein the modified cytosine residue is a methylated cytosine.
12. The kit of claim 11 wherein the methylated cytosine is selected from the group consisting of 5-methylcytosine, 5-hydroxymethylcytosine, and glycosylated 5-hydroxymethylcytosine.
13. A composition comprising a first restriction endonuclease comprising SEQ ID NO. 4 or a sequence at least 85% identical to SEQ ID NO. 4, having a recognition sequence 5′-TCGA-3′, where a modified cytosine residue in the recognition sequence impairs cleavage, and (b) a second restriction endonuclease TaqI, that has a recognition sequence 5′-TCGA-3′ and is not impaired by modification of the cytosine residue.