Restriction endonucleases and their uses
First Claim
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1. A method for determining the level of methylation in double stranded DNA, the method comprisingcontacting 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.
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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 recongition sequence 5′-TCGA-3′ are also disclosed.
20 Citations
13 Claims
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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. - View Dependent Claims (2, 3, 4)
- -TCGA-3′
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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). - View Dependent Claims (6, 7, 8, 9, 10, 11, 12)
- -TCGA-3′
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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.
- -TCGA-3′
Specification