Catalyzes the cleavage of a phosphodiester bond 5′ from deoxyxanthosine (dX), deoxyinosine (dI), deoxyuracil (dU), and an abasic (AP) site, creating a nick with 3′-OH and a 5′-phosphate
Thermostable Endonuclease Q (EndoQ) is an archaeal DNA endonuclease that cleaves the phosphate backbone of a DNA substrate 5′ to the position of the modified nucleobases of deoxyxanthosine (dX), deoxyinosine (dI), deoxyuracil (dU), and an abasic (AP) site. The result is a 3′-OH and a 5′-phosphate. Thermostable Endonuclease Q generally prefers single-stranded DNA over double-stranded DNA, and exhibits higher activity on dU over dI and dX.
Product Source: An E. coli strain that carries a cloned archaeal Endonuclease Q gene with a C-terminal his-tag.
Figure 1: Relative Activity of Thermostable Endonuclease Q on Various DNA Substrates
Thermostable Endonuclease Q has different specificities for modified nucleotides in ssDNA and dsDNA. Thermostable Endonuclease Q (#M0701) activity was tested on 1 pmol of ssDNA or dsDNA substrates with fluorescently-labeled oligonucleotides containing modified nucleotides of interest (red) for 1 hour in 1x NEBuffer r2.1 at 65°C. Cleaved products were detected by capillary electrophoresis. Thermostable Endonuclease Q prefers ssDNA, and exhibits higher activity on dU over dI and dX. Figure 2: Temperature Profile of Thermostable Endonuclease Q
Thermostable Endonuclease Q is active over a wide range of temperatures. Thermostable Endonuclease Q (#M0701, 0.2 U) activity was tested on 1 pmol of a fluorescently-labeled single-stranded oligonucleotide containing a single uracil for 1 hour in 1x NEBuffer r2.1 at various temperatures. Cleaved products were detected by capillary electrophoresis. Thermostable Endonuclease Q is active over a wide range of temperatures (37°C – 90°C), but displays optimal activity between 65°C and 85°C. Figure 3: Thermostable Endonuclease Q is active in some DNA:RNA contexts
Thermostable Endonuclease Q is active on some DNA:RNA contexts. Thermostable Endonuclease Q (#M0701, 0.2 U) activity was tested on 1 pmol of substrate containing a single deoxyuracil (dU) or ribouracil (rU) within a DNA oligonucleotide (gray) or RNA oligonucleotide (blue) for 1 hour in 1x ThermoPol® Reaction Buffer. Activity was compared with Thermostable USER® III Enzyme (#M5509) for comparison. Cleaved products were detected by capillary electrophoresis. Thermostable Endonuclease Q can cleave at dU and rU located within DNA oligonucleotide strands regardless of presence or type (DNA or RNA) of complement.
This product is related to the following categories:
One unit is defined as the amount of enzyme required to cleave 1 pmol of a 60-mer oligonucleotide ssDNA containing a single deoxyxanthosine site* in a total reaction volume of 20 µl after 1 hour at 65°C.
*A deoxyxanthosine site is synthetically prepared with dX at the 24th position of a 5′ FAM-labelled 60-mer ssDNA oligo.
Reaction Conditions
1X NEBuffer™ r2.1
Incubate at 65°C
1X NEBuffer™ r2.1
50 mM NaCl
10 mM Tris-HCl
10 mM MgCl2
100 µg/ml Recombinant Albumin
(pH 7.9 @ 25°C)
Storage Buffer
10 mM Tris-HCl
500 mM NaCl
0.1 mM EDTA
1 mM DTT
50% Glycerol
pH 7.4 @ 25°C
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Specifications & Change Notifications
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