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 ATC TTT 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 ´ Protein Asn 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 of family 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