Product Class: Other

APOBEC3A
Gray property icon for cloned at NEB Gray property icon for recombinant Gray property icon for engineered for performance Gray property icon for incubation temperature 37° 70 Heat Epi


Catalog #M0648

Product Introduction

Mechanism of APOBEC3A, a cytidine deaminase

 

Deaminase enzyme that deaminates cytosine to uracil, or 5-methylcytosine to thymine.

APOBEC3A is ideal for:

  • Cytosine Deamination
  • Base editing

 

Product Information

Description

APOBEC3A (Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A) is a cytidine deaminase enzyme that catalyzes the deamination of cytosine or 5-methylcytosine in single-stranded DNA to produce uracil or thymine.

  • Product Source: An E. coli strain that carries a cloned APOBEC3A gene.

 

Figure 1: APOBEC3A deaminates cytosine to uracil or 5-methylcytosine to thymine

Conversion of cytosine to uracil and 5-methylcytosine to thymine by APOBEC3A

APOBEC3A catalyzes the deamination of cytosine or 5-methylcytosine in single-stranded DNA to produce ammonia and either uracil (cytosine) or thymine (5-methylcytosine).

 

Figure 2: APOBEC3A substrate specificity

Line graph depicting substrate specificity of APOBEC3A

Activity on ssDNA substrates containing various cytosine modifications was determined by incubating 16 units of APOBEC3A with 1 µg (73 pmol) of a 44-nucleotide single-stranded DNA oligo containing a single dC (or modified cytosine) at 37°C for the indicated time in a total reaction volume of 40 µl in 1X Deamination Reaction Buffer. Following heat inactivation of the APOBEC3A and Thermolabile Proteinase K (NEB #P8111) treatment, the ssDNA oligos were cleaned up using the Monarch Spin PCR & DNA Cleanup Kit (NEB #T1130) Oligonucleotide Cleanup Protocol, eluted in water and then digested to nucleosides using the Nucleoside Digestion Mix (NEB #M0649). The extent of deamination was then determined by liquid chromatography.
This product is related to the following categories:
Base Modifying Enzymes,
DNA Methylation Analysis,
DNA Modifying Enzymes & Cloning Technologies,

Properties & Usage

Materials Required but not Supplied

  • dC- or 5mC-containing ssDNA
  • Formamide (for dsDNA denaturation if needed)
  • Microcentrifuge tubes
  • Thermocycler or heat block

Unit Definition

One unit is defined as the amount of enzyme required to deaminate 5 pmol of a fluorescently labeled 44-mer oligonucleotide containing a single cytosine base in 30 minutes at 37°C in a total reaction volume of 20 µl in 1X Deamination Reaction buffer.

Storage Temperature
-20°C

Reaction Conditions

1X Deamination Reaction Buffer
Incubate at 37°C

Heat Inactivation

70°C for 10 minutes

Unit Assay Conditions

5 pmol of fluorescently labeled 44-mer oligonucleotide containing a single cytosine is incubated with 1 unit of APOBEC3A for 30 minutes at 37°C in a total reaction volume of 20 µl in 1X Deamination Reaction Buffer. The enzyme generated uracil-containing ssDNA is then incubated with USER enzyme in 1X rCutSmart Buffer to cleave the phosphodiester backbone at the uracil site. The resulting product is measured by capillary electrophoresis to determine APOBEC3A deamination activity.

Features

Deamination of cytosine by APOBEC3A generates a uracil, while deamination of 5-methylcytosine by APOBEC3A generates a thymine.

Protocols, Manuals & Usage

Protocols

  1. Deamination of Single Stranded DNA (ssDNA) Using APOBEC3A (NEB #M0648)

FAQs & Troubleshooting

FAQs

  1. What is the activity of APOBEC3A (NEB #M0648) in different NEBuffers?
  2. Can APOBEC3A (NEB #M0648) be heat inactivated?
  3. What is the molecular weight of APOBEC3A (NEB #M0648)?
  4. Is APOBEC3A (NEB #M0648) a tagged protein?
  5. What is the relative activity of APOBEC3A (NEB #M0648) on different modified dC-containing ssDNA substrates?
  6. Which DNA modifications protect cytosine from deamination by APOBEC3A (NEB #M0648)?
  7. Is APOBEC3A (NEB #M0648) active on double stranded DNA?
  8. Is APOBEC3A (NEB #M0648) active on RNA?
  9. Does APOBEC3A (NEB #M0648) exhibit any sequence preference?
  10. What temperatures is APOBEC3A (NEB #M0648) active?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

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]

Certificate Of Analysis

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

Safety DataSheets

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

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

Notice to Purchaser: No rights are conveyed for the use of this product in NEB’s EM-seq technology, which is covered by patents on compositions and/or methods including US Patent Nos.10,227,646, 10,260,088, 10,619,200, 11,001,876, and 11,124,825; EP3368688; JP6224689; and CN108699598, and corresponding pending patent applications. Use of this product for NEB’s EM-seq technology requires a license from NEB. For more information, please email busdev@neb.com.