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Description
Prokaryotic Argonaute (pAGO) is the pivotal enzyme in the host defense system by mediating nucleic acid molecules. As the DNA-guided endonuclease from eubacterium Thermus thermophilus, TtAGO protein targets cognate DNA without the restriction of a PAM (protospacer adjacent motif) or PFS (protospacer flanking site) on the target sequence, expanding the selection range of available target DNA sequence. Besides, compared with CRISPR/Cas systems, TtAGO have remarkable advantages as TtAGO endonuclease utilizes guide DNA rather than guide RNA in directing target DNA cleavage, which is more convenient for in vitro applications as guide DNA is more stable in ambient temperature, easier and cost-saving to synthesize than guide RNA.
These capacities enable TtAGO as a powerful programmable DNA endonuclease directed by short guide DNAs in efficient Nucleic Acid Detection systems as well as in versatile Synthetic Biology platforms.
Features
High Biological Activity
Highly Specific
Low Endotoxin Level
Application
Nucleic Acid Detection, Cancer Diagnostics, Genetic Screening, Synthetic Biology
Source
TtAgo gene from Thermus thermophilus (NCBI Reference Sequence: WP_011174533.1) expressed in E. coli
Purity
Greater than 95% as determined by SDS-PAGE and FPLC
Activity
Purified 370 bp PCR product dsDNA digested after 1 hour reaction at 83°C.
The reaction is performed as follow:
Reaction system total 10 µl, including 50 nM TtAGO Enzyme, 0.5 µM each of guide DNA, 100 ng purified 370 bp PCR product dsDNA as target DNA and 2ul 5x reaction buffer.
Reaction at 83°C for 1 hour, then incubate at 95°C for 10 min to terminate the reaction.
Storage Condition
-20 °C
5X Reaction Buffer
100 mM Tris-HCI, pH 8.8,
50 mM (NH4)2SO4,
50 mM KCl,
40 mM MgSO4,
0.5% Tween-20,
4 M Betaine
Storage Buffer
10 mM Tris-HCl
300 mM NaCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.4 at 25°C
Experimental Data
References
1. Gang Sheng et. al. (2014). PNAS. 111 (2) 652-657.
2. Daan C. Swarts et. al. (2014). Nature. 507(7491):258-261.
3. Yuqing Qin et. al. (2022). Trends Biotechnol. 40(8):910-914.
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