AbaSI

Description

Recognition sequence: x is ghmC, hmC, mC or C

AbaSI is a DNA modification-dependent endonuclease and it is an EpiMark™ validated product. AbaSI recognizes 5-glucosylhydroxymethylcytosine (5ghmC) in double stranded DNA. It also recognizes 5-hydroxymethylcytocine (5hmC) but at a lower efficiency. It does not recognize DNA with 5-methylcytosin or un-modified cytosine. AbaSI selectively cleaves DNA that contains the modified bases, 5ghmC or 5hmC on one or both strands and introduces a double-stranded DNA break on the 3´ side away from the modified cytosine producing a 2-base or 3-base 3´-overhang. Sites with two 5ghmC on opposite strands are cleaved most efficiently; sites with one 5ghmC and another C or 5mC are cleaved less efficiently.

Product Source

An E. coli strain that carries the synthetic AbaSI gene from Acinetobacter baumannii SDF species.

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
CutSmart® Buffer-2010X

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to digest 1 μg of T4 wild-type phage DNA (fully ghmC-modified) in 1 hour at 25°C in a total reaction volume of 50 μl.

Reaction Conditions

1X CutSmart® Buffer
Incubate at 25°C

1X CutSmart® Buffer:
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 μg/ml BSA
pH 7.9 @ 25°C

Activity in NEBuffers

NEBuffer 1.1: 25%
NEBuffer 2.1: 50%
NEBuffer 3.1: 50%
CutSmart® Buffer: 100%

Diluent Compatibility

Storage Temperature

-20°C

Storage Conditions

100 mM KCl
10 mM Tris-HCl
1 mM DTT
0.1 mM EDTA
0.5% Tween® 20
0.5% IGEPAL® CA-630
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

65°C for 20 min

Methylation Sensitivity

dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Not Sensitive

Notes

  1. Optimization of the amount of enzyme for complete digestion of genomic DNA may be required.

FAQs

  1. Does AbaSI cut hemi-glucosylated CpG site?
  2. How do I know my DNA is cut?
  3. How much enzyme should be used in digesting genomic DNA?
  4. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?
  5. Why do I see additional DNA bands on my gel after a restriction digest?
  6. Why do I see a DNA smear on an agarose gel after a restriction digest?
  7. Why is my Restriction Enzyme not cutting DNA?

Protocols

  1. Protocol for Glucosylation and digestion of Genomic DNA using AbaSI (#R0665)

Selection Charts

Usage Guidelines & Tips

Interactive Tools

NEB Publications

  • Horton JR, Borgaro JG, Griggs RM, Quimby A, Guan S, Zhang X, Wilson GG, Zheng Y, Zhu Z, Cheng X (2014). Structure of 5-hydroxymethylcytosine-specific restriction enzyme AbaSI in complex with DNA Nucleic Acids Res. 42(12), 7947-59. PubMedID: 24895434, DOI: 10.1093/nar/gku497
  • Sun Z, Terragni J, Borgaro JG, Liu Y, Yu L, Guan S, Wang H, Sun D, Cheng X, Zhu Z, Pradhan S, Zheng Y (2013). High-resolution enzymatic mapping of genomic 5-hydroxymethylcytosine in mouse embryonic stem cells Cell Rep. 3(2), 567-576. PubMedID: 23352666, DOI: 10.1016/j.celrep.2013.01.001
  • Wang H, Guan S, Quimby A, Cohen-Karni D, Pradhan S, Wilson G, Roberts RJ, Zhu Z, Zheng Y (2011). Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine Nucleic Acids Res. 39(21), 9294-305. PubMedID: 21813453, DOI: 10.1093/nar/gkr607

Citations

  • Mooijman D, Dey S S, Boisset JC, Crosetto N, van Oudenaarden A (2016). Single-cell 5hmC sequencing reveals chromosome-wide cell-to-cell variability and enables lineage reconstruction Nature Biotechnology. 34, 852-857. PubMedID: 27347753, DOI: 10.1038/nbt.3598
  • McKernan KJ, Spangler J, Zhang L, Tadigotla V, McLaughlin S, Warner J, Zare A, Boles RG (2014). Expanded genetic codes in next generation sequencing enable decontamination and mitochondrial enrichment PLoS One. 9(5), e96492. PubMedID: 24788618, DOI: 10.1371/journal.pone.0096492

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Endonuclease Activity (Nicking):
    The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
  • Exonuclease Activity (Radioactivity Release):
    The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
  • Non-Specific DNase Activity (16 hour):
    The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
  • Protein Purity (SDS-PAGE):
    The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
  • RNase Activity (16 Hour Digestion):

    The product is tested in a reaction containing a RNA substrate.  After incubation for 16 hours there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.