METHOD OF DETERMINING NUCLEOTIDE SEQUENCE R(5mC)GY IN PRESET POSITION OF EXTENDED DNA Russian patent published in 2016 - IPC C12Q1/68 

FIELD: biotechnologies; molecular genetics.

SUBSTANCE: described is method of determining nucleotide sequence (site) 5'-R(5mC)GY-3'/3'-YG(5mC)R-5' in preset position of extended DNA. Method involves hydrolysis of highly pure methyl dependent genomic DNA by site-specific DNA-endonuclease GlaI, ligation of universal oligonucleotide adapter with further amplification in real time using genome primer and probe, complementary tested DNA and hybrid primer, 3' end of which is complimentary to 3' end of DNA from specified point of GlaI hydrolysis, and remaining part of adapter is complementary to adapter sequence and making conclusion on availability of R(5mC)GY sequence (where 5mC is 5-methylcytosine, R is A or G, Y is Τ or C) at onset of fluorescent signal. Adapter sequence - 5'-CCTGCTCTTTCATCG-3'/3'-p-GGACGAGAAAGTAGC-p-5'. Method is carried out in single reaction mixture with addition of composition of components ensuring restoring S-S bonds in proteins, stabilizing enzymes and are donor of methyl groups.

EFFECT: simplified method of determining R(5mC)GY nucleotide sequence in preset position of extended DNA, expands its functional capabilities and reduces analysis time.

3 cl, 2 dwg, 2 ex