METHOD OF DETERMINING THE METHYLATION SITES PuCGPy REGULATORY REGIONS OF GENES-MARKERS OF COLORECTAL CANCER BY GLAD-PCR-ANALYSIS AND OLIGONUCLEOTIDE PRIMERS AND FLUORESCENT-LABELLED PROBES FOR REALISING SAID METHOD Russian patent published in 2016 - IPC C12Q1/68 

FIELD: biology.

SUBSTANCE: group of inventions refers to molecular biology and can be used in molecular-genetic diagnosis of oncological diseases. Method involves bio information analysis of previous publications on genes-tumor markers of colorectal cancer and extraction of genes, methylation of PuCGPy sites at regulatory regions of which occurs with high frequency in DNA cells of colorectal cancer, taking into account possibility of detecting gene methylation at early stages of disease and formation of panel of following genes-tumor markers of colorectal cancer: CNRIP1, ELMO1, ESR1, FBN1, RXRg, RYR2, SEPT9b, SOCS3 and UCHL1. It is followed by hydrolysis of highly pure genomic DNA of the analysed biomaterial methyl dependent site-specific DNA-endonuclease GlaI, ligation of universal oligonucleotide adapter with further amplification in real time using genomic primers and probes, complementary sequences of regulatory regions of genes CNRIP1, ELMO1, ESR1, FBN1, RXRg, RYR2, SEPT9b, SOCS3 and UCHL1 in investigated DNA, and hybrid primers, 3'-ends of which is complementary 5'-ends of DNA in specified places hydrolysis GlaI, and remaining part is complementary to adapter sequence. Conclusion on presence of sequence of Pu(5mC)GPy (where 5mC is 5-methylcytosine, Pu - A or G, Py - T or C) in regulatory regions of genes CNRIP1, ELMO1, ESR1, FBN1, RXRg, RYR2, SEPT9b, SOCS3 and UCHL1 is made at onset of fluorescent signal. Adapter sequence is universal oligonucleotide adapter 5'-CCTGCTCTTTCATCG-3'/3'-p-GGACGAGAAAGTAGC-p-5'.

EFFECT: using the inventions enables simplifying and improving accuracy of determining of PuCGPy sites methylation.

3 cl, 1 dwg, 4 tbl, 3 ex