5-Carboxylcytosine DNA Standard

by Active Motif
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5-Carboxylcytosine (5-caC) is a recently discovered modification of DNA. Just as 5-hydroxymethylcytosine (5-hmC) is generated when the Tet family of cytosine oxygenase enzymes converts 5-methylcytosine (5-mC), it has been shown that continued activity of the Tet proteins on hydroxymethylcytosine will generate 5-formylcytosine (5-fC) and 5-carboxylcytosine. It is believed that this conversion process may represent a pathway of DNA demethylation.

To better understand the role of 5-carboxylcytosine in genomic DNA, Active Motif has created the 5-Carboxylcytosine DNA Standard Kit. This kit includes two double-stranded DNA oligonucleotides, one oligonucleotide contains a total of 12 carboxylcytosine modifications and the other oligonucleotide contains only unmodified cytosine residues. These oligonucleotides can be used as positive and negative controls in the interrogation of 5-carboxylcytosine. To learn more about the 5-Carboxylcytosine DNA Standard Kit, click on the Documents tab below to download our data sheet.

Name Format Cat No. Price  
5-Carboxylcytosine DNA Standard 0.5 µg 55014 $230 Buy Now

5-Carboxylcytosine DNA Standard Kit

The 5-Carboxylcytosine DNA Standard Kit consists of two oligonucleotides, each containing 0.5 µg double-stranded DNA. The Carboxylcytosine Standard consists of a 38-mer with 8 carboxylcytosine modifications on the forward strand and 4 carboxylcytosine modifications on the reverse strand. The annealed dsDNA, therefore, contains a total of 12 carboxylcytosine residues. The Unmodified Standard consists of the same 38-mer DNA sequence without any modifications to the cytosine residues such that it can be used as a negative control.

5-caC Specificity

The 5-Carboxylcytosine DNA Standard Kit is quality control tested by dot blot using Active Motif's 5-Carboxylcytosine antibody (Catalog No. 61225). The antibody specifically recognizes the 5-Carboxylcytosine Standard and does not detect DNA that is unmodified or contains 5-methylcytosine or 5-hydroxymethylcytosine methylation (Figure 1).

Figure 1: Dot blot analysis of 5-Carboxylcytosine specificity.

Varying amounts of double-stranded DNA from the Methylated DNA Standard Kit (Catalog No. 55008) or 5-Carboxylcytosine DNA Standard Kit were spotted onto a nylon membrane and probed with a 1:2000 dilution of Active Motif's 5-Carboxylcytosine antibody (Catalog No. 61225).

5-caC Sensitivity

To determine the ability of the 5-Carboxylcytosine antibody to recognize both single-stranded (ss) and double-stranded (ds) DNA, another dot blot was run using both the Carboxylcytosine DNA Standard and the Unmodified DNA Standard. For single-stranded DNA, the standards were heat denatured for 10 minutes at 95°C prior to spotting onto a nylon membrane. Double-stranded DNA was spotted without denaturation. Active Motif's 5-Carboxylcytosine antibody (Catalog No. 61225) was used overnight at a 1:2000 dilution. Following incubation with an anti-rabbit HRP antibody and ECL reagents, the results show that the antibody is capable of recognizing the 5-Carboxylcytosine DNA in either a ssDNA or dsDNA configuration.

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