**4. Genetic analysis: Detection of mutations in gene of alpha-synuclein (SNCA)**

*DNA Isolation:* under aseptic conditions, from each one of the patients, blood sample was taken, with the function of a vein. Each of those blood samples are kept invaccum pots (Vaccutainer), and anticoagulant EDTA Na2 is added in each of the pots.

*Amplification:* amplification of regions of SNCA gene was made with polymerase chain reaction.

*Detection of mutations:* to detect the mutations in selected regions of SNCA gene, polymerase chain reaction- Restriction Fragment Length Polymorphism, was used.

Amplification of the region of egzon 3 of SNCA gene was made, using the primers 5'- GTC TCA CAC TTT GGA GGG TTT C-3' and 5' CAC CTA CCT ACA CAT ACC TCT GAC and TC-3'.

PCR amplification of region of egzon 4 of SNCA gene was made by using the primers 5' GCT AAT CAG CAA TTT AAG GCT AG-3' and 5' GAT ATG TTC TTA GAT GCT and CAG-3'. All of these amplifications have length of 215 bp.

Amplificated products were digested with appropriate restrictive endonuclease under the optimal conditions.

G88C mutation in egzon 3 of SNCA gene was detected with restricted digestion with enzyme Mval.

G209A mutation in egzon 4 of SNCA gene was detected with enzyme Mael.
