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DNA diagnostics

DNA diagnostics. What can we detect ?. Monogenic and polygenic inherited diseases Some types of tumors Disease progress during the therapy Identification of people in forensic medicine HLA-typization in cases of transplantation Prevention - examination: - preimplantation - prenatal

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DNA diagnostics

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  1. DNA diagnostics

  2. What can we detect ? • Monogenic and polygenic inherited diseases • Some types of tumors • Disease progress during the therapy • Identification of people in forensic medicine • HLA-typization in cases of transplantation • Prevention - examination: - preimplantation - prenatal - presymtomatic

  3. DNA polymorphisms Variability in DNA sequence between different individuals of the same species • Allele polymorphisms physiological function, with frequency > 1% predisposition to polygenic diseases • Mutations pathological function, with frequency < 1% cause of monogenic diseases

  4. Source of DNA DNA of certain gene – all nuclear cells RNA of certain gene - only these cells, where this gene is expressed

  5. Principle of testing DNA isolation PCR (amplification of a DNA region) + other analyses visualization of result in gel

  6. Contents of 3-weeks practice Practice 1: Own DNA isolation Practice 2: PCR - amplifying CFTR and HFE genes Practice 3: RFLP-restriction digestion of HFE gene

  7. The most frequent mutations Cystic fibrosis : • CFTR gene, on chromosome 7 delta F508 mutation Hemochromatosis : • HFE gene, on chromosome 6 C282Y mutation

  8. Delta F508 mutation Normal sequence DNA 5 ´ … AAT ATC ATCTTT GGT GIT … 3´ Protein Asn Ile Ile Phe Gly Val Position 505 506 507 508 509 510 Mutated DNA DNA 5 ´ … AAT ATC AT - - - T GGT GIT … 3´ ProteinAsn Ile Ile - Gly Val Position 505 506 507 508 509 510

  9. C282Y mutation Normal DNA 5 ´......G / TGC….. 3´ 3´…. C / ACG….. 5´ C – Cys – Cysteine (TGC) at position 282 of protein Mutated DNA 5 ´......G / TAC….. 3´ 3´…. C / ATG….. 5´ Y – Tyr – Tyrosine (TAC) at position 282 of protein

  10. Characteristics of DNA diagnostics Detection of certain polymorphism of the predisposition gene • TARGET ANALYSES • COMPLETE ANALYSES

  11. Target analyses • The localization and the whole sequence of a gene is known • The mutation of the gene is known An examination of family members is not needed

  12. Complete analyses • The localization and the whole sequence of a gene is known • Mutations of the gene are unknown An examination offamily members is necessary

  13. Polymorphism detection of certain predisposition gene Target analyses: • PCR with sequence specific primers (CFTR delta F508) • PCR with general primers + 1) RFLP with restriction enzymes (Restriction Fragment Length Polymorphism) (HFE C282Y) + 2) hybridization with DNA probes Complete analyses: PCR with general primers DNA sequencing

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