ЭФФЕКТИВНОСТЬ ОПРЕДЕЛЕНИЯ 5-МЕТИЛЦИТОЗИНА В ДНК КЛЕТОК ESCHERICHIA COLI, НЕСУЩИХ ГЕНЫ БАКТЕРИАЛЬНЫХ ДНК- МЕТИЛТРАНСФЕРАЗ, С ПОМОЩЬЮ УСТАНОВКИ OXFORD NANOPORE

Abstract

The MinION system (Oxford Nanopore Technologies Limited) was used for direct sequencing of genomic DNA of two recombinant E. coli strains. In one case, the cells contained a plasmid with the M.HpaII gene of DNA methyltransferase, which methylates the second cytosine in CCGG site; in the second case, the E. coli strain contained M.HspAI DNA methyltransferase, which modifies the central cytosine in the GCGC sequence. In both cases, DNA methyltransferases methylate cytosine to 5-methylcytosine. It has been shown that when DNA is sequenced at high coverage, the presence of 5-methylcytosine in DNA can be detected with high accuracy. In particular, in E. coli DNA containing the cloned gene of DNA methyltransferase M.HspAI, at 1300coverage, 98.9% of the GCGC sites are identified as methylated at the first cytosine. At the same time, only 0.09% of the remaining tetranucleotides, which have the CpG dinucleotide in the middle, give a false positive result being identified as methylated at the central cytosine...