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Analysis of C282Y mutation in hemochromatosis gene

Analysis of C282Y mutation in hemochromatosis gene. PCR – muta tion C282Y (hemochromatosis). MIX – per 1 sample 15,8 ul H 2 O 2,5 ul buffer 1,5 ul primer 1 (P1) 1,5 ul primer 2 (P2) 2,0 ul MgCl 2 0,5 ul dNTP 1,0 ul DNA 0,2 ul Taq polymer asis (add on ice).

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Analysis of C282Y mutation in hemochromatosis gene

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  1. Analysis of C282Y mutation in hemochromatosis gene

  2. PCR – mutation C282Y(hemochromatosis) • MIX – per 1 sample • 15,8 ul H2O • 2,5 ul buffer • 1,5 ul primer 1 (P1) • 1,5 ul primer 2 (P2) • 2,0 ul MgCl2 • 0,5 ul dNTP • 1,0 ul DNA • 0,2 ul Taq polymerasis (add on ice)

  3. PCR – mutation C282Y - ELFO

  4. RFLP –Restriction Fragment Length Polymorphism restriction analysis of DNA by its digestion with restrictionendonucleases (RE)in specific restriction sites in the case the sequence difference (polymorphism) creates or disturbs a specific site for RE,after restriction, fragments withdifferent sizes are formed

  5. RFLP –Restriction Fragment Length Polymorphism restriction analysis of DNA by its digestion with restrictionendonucleases (RE)in specific restriction sites in the case the sequence difference (polymorphism) creates or disturbs a specific site for RE,after restriction, fragments withdifferent sizes are formed

  6. RFLP –Restriction Fragment Length Polymorphism • Restriction endonucleases (RE) • known about 2100 bacterial RE • RE recognize variously short nucleotides sequences (4,6,8), in which then they digest covalent phosphodiester bonds • Principle of the analysis • starting DNA(genomic DNA, PCR product) • digestion with a restriction enzyme into fragments with different sizes • fragments electrophoresis separation

  7. Sequence of C282Y mutation(hemochromatosis) Primersrestriction endonucleasis RsaI restriction site (GTAC) mutation C282Y (GTGC → GTAC)

  8. RFLP – mutation C282Y(hemochromatosis) • MIX – per 1 sample • 7,0 ul H2O • 2,0 ul buffer • 10 ul PCR product • 1,0 restriction endonukleasis RsaI (add on ice) • Incubation 37°C 2 h

  9. RFLP – mutation C282Y - ELFO

  10. Hemochromatosis • autosomal recessive disease affecting iron metabolism • excessive iron absorption, its deposition in organs (mainly parenchymal) and subsequent damage of the organism • hepatopathy (cirrhosis, hepatocellular carcinoma) • diabetes mellitus, arthropathy, hypogonadism, kardiomyopathy, amenorhea • serum iron, transferrin saturation, ferritin, liver biopsy • repeated phlebotomy

  11. HFE gene mutations

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