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
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
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.
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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.
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Specifications
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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.
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