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 -

presymtomatic

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

Source of DNA

DNA of certain gene – all nuclear cells

RNA of certain gene - only these cells, where this gene is

expressed

Principle of testing

DNA isolation

PCR (amplification of a DNA region)

+ other analyses

visualization of result in gel

Contents of 3-weeks practice

Practice 1: Own DNA isolationPractice 2: PCR - amplifying CFTR and HFE genesPractice 3: RFLP-restriction digestion of HFE gene

The most frequent mutations

Cystic fibrosis :• CFTR gene, on chromosome 7

delta F508 mutationHemochromatosis :• HFE gene, on chromosome 6

C282Y mutation

Delta F508 mutationNormal sequenceDNA 5 ´ … AAT ATC ATC TTT GGT GIT … 3´

Protein Asn Ile Ile Phe Gly ValPosition 505 506 507 508 509 510

Mutated DNADNA 5 ´ … AAT ATC AT - - - T GGT GIT …

3´

Protein Asn Ile Ile - Gly ValPosition 505 506 507 508 509 510

C282Y mutationNormal DNA5 ´......G / TGC….. 3´

3´…. C / ACG….. 5´

C – Cys – Cysteine (TGC) at position 282 of protein

Mutated DNA5 ´......G / TAC….. 3´

3´…. C / ATG….. 5´

Y – Tyr – Tyrosine (TAC) at position 282 of protein

Characteristics of DNA diagnostics

Detection of certain polymorphism of the predisposition gene

• TARGET ANALYSES• COMPLETE ANALYSES

Target analyses

• The localization and the whole sequence of a gene is known

• The mutation of the gene is known

An examination of family membersis not needed

Complete analyses

• The localization and the whole sequence of a gene is known

• Mutations of the gene are unknown

An examination of family membersis necessary

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