A Method Using One Fluorophore Signal in Sanger Read to Determine CpG Methylation in Bisulfite Converted DNA

• Published in: Russian journal of genetics Vol. 59; no. 11; pp. 1255 - 1262
• Main Author: Karpenko, D. V.
• Format: Journal Article
• Language: English
• Published: Moscow Pleiades Publishing 01-11-2023
• Subjects: Animal Genetics and Genomics; Biomedical and Life Sciences; Biomedicine; Human Genetics; Mathematical Models and Methods; Microbial Genetics and Genomics; Sanger sequencing of multiclonal PCR product; asymmetric PCR; CpG; 5mC; bisulfite sequencing
• ISSN: 1022-7954; 1608-3369
• DOI: 10.1134/S1022795423110066

Epigenetics is a growing section of biologic studies. One of the most studied epigenetic marks is 5mC modification in CpG context. Modern technologies provide solutions to proceed multiple samples and to acquire big massive data for 5mC. Technologies demonstrate high efficiency relative to a single piece of information, but such solutions could be cost ineffective for studies focused on a local DNA region. We previously presented an original method, and here I present improvements to make it faster and more reproducible. The method uses Sanger sequencing of total PCR product from bisulfite converted DNA. The key feature of the method is a utilization of only one fluorophore channel to determine a methylation ratio in a CpG. Stability of a fluorophore signal profile determined by a sequence context is enough to measure methylation of a CpG and a complicated establishing of a rarely presented counter signal from other fluorophore in a read is not required.