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US 11,530,456 B2Granted
Detection of endonuclease activity
AssigneesAarhus Universitet, The Chinese University of Hong Kong
InventorsMarianne Smedegaard Hede, Birgitta Ruth Knudsen, Magnus Stougaard, Yi-Ping Ho
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Abstract
The present invention relates to methods for determining endonuclease activity in a sample. In particular, the present invention relates to a method for determining viable pathogenic bacteria in a sample based on patterns of endonuclease activity.
Claims23
A method for screening for Type II restriction endonuclease activity in a sample, the method comprising:
- a) adding to a provided sample to be analyzed at least one first oligonucleotide, to obtain a first reaction composition, the at least one first oligonucleotide comprising a hairpin structure, wherein the stem of said hairpin structure comprises a first cleavage site for a first Type II restriction endonuclease;
- b) adding a ligase to the first reaction composition obtained in a), to circularize at least two of the first oligonucleotides if a Type II restriction endonuclease present in the sample has cleaved the cleavage site in the first oligonucleotide, wherein the produced circularized molecules comprise part of stem-loop structures of the first oligonucleotide after Type II restriction endonuclease cleavage;
- c performing an amplification reaction using the at least first circularized oligonucleotides as template;
- and
- d detecting the amplified product from step c), thereby determining Type II restriction endonuclease activity in said sample.
2 more elements
A method for screening for Type II restriction endonuclease activity in a sample, the method comprising:
- a) adding to a provided sample to be analyzed at least one first oligonucleotide, to obtain a first reaction composition, the at least one first oligonucleotide comprising a hairpin structure, wherein the stem of said hairpin structure comprises a first cleavage site for a first Type II restriction endonuclease;
- b) adding a ligase and a support oligonucleotide to the first reaction composition obtained in a), the support oligonucleotide facilitating hybridization by bridging the 5′-end and the 3′-end of said first oligonucleotide after endonuclease cleavage, to circularize the first oligonucleotides if a Type II restriction endonuclease present in the sample has cleaved the cleavage site in the first oligonucleotide, wherein the produced circularized molecules comprise part of the stem-loop structures of the first oligonucleotides after Type II restriction endonuclease cleavage;
- c) performing an amplification reaction using the at least first circularized oligonucleotides as template;
- and
- d) detecting the amplified product from step c), thereby determining Type II restriction endonuclease activity in said sample.
2 more elements
A method for screening for Type II restriction endonuclease activity in a sample, the method comprising:
- a) adding to a provided sample to be analyzed at least one first oligonucleotide and at least one second oligonucleotide, to obtain a first reaction composition, the at least one first oligonucleotide and the at least one second oligonucleotide comprising a hairpin structure, wherein the stem of said hairpin structure comprises a first cleavage site for a first Type II restriction endonuclease;
- b) adding a ligase to the first reaction composition obtained in a), to circularize the at least first oligonucleotide and the at least one second oligonucleotide if a Type II restriction endonuclease present in the sample has cleaved the cleavage site in the first oligonucleotide, wherein the produced circularized molecules comprise part of stem-loop structures of the first oligonucleotide and part of stem-loop structures of the second oligonucleotide after Type II restriction endonuclease cleavage;
- c) performing an amplification reaction using the circularized oligonucleotides as template;
- and
- d) detecting the amplified product from step c), thereby determining Type II restriction endonuclease activity in said sample, wherein the first oligonucleotide and the second oligonucleotide are different in sequence.
2 more elements
Dependencies are inferred from the claim text.