MOLECULAR ANALYSIS OF FRIEDREICH’S ATAXIA IN MACEDONIAN PATIENTS
Kocheva S1,2, Trivodalieva S1, Vlaski-Jekic S3, Kuturec M2, Efremov GD1,*
*Corresponding Author: Professor Dr. Georgi D. Efremov, Macedonian Academy of Sciences and Arts, Research Center for Genetic Engineering and Biotechnology, Aven Krste Misirkov 2, POB 428, 1000 Skopje, Republic of Macedonia; Tel: +3892-120253; Fax: +3892-115434; E-mail:gde@manu.edu
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MATERIALS AND METHOD

 

Patients and Controls. Forty patients from the Republic of Macedonia with spinocerebellar ataxia referred to our institution for molecular diagnosis of FRDA, were studied. Fifteen had an early onset of progressive ataxia (before 25 years), while 25 were older than 25 years at the time of diagnosis. Fifty healthy individuals were also studied. GAA Repeat Analysis. Genomic DNA was extracted from venous blood leukocytes using a standard phenol/ chloroform extraction procedure [17]. Amplification of normal and expanded alleles was obtained by long range polymerase chain reaction (PCR) procedures using primers that flank the GAA repeat in intron 1 of the frataxin gene (GAAF: 5’- GGG ATT GGT TGC CAG TGC TTA AAA GTT AG-3’ and GAAR: 5’-GAT CTA AGG ACC ATC ATG GCC ACA CTT GCC-3’) and generate a PCR product of 457 + 3n bp (n = number of GAA repeats). Long-range PCR conditions, which gave efficient amplification, consisted of: 20 cycles at 94°C’s for 20 seconds, 68°C for 2 min. and 72°C for 2 min., followed by 17 cycles in which the length of the 68°C steps was increased by 15 seconds per cycle. The PCR products were analyzed by 1% agarose gel electrophoresis, visualized by ethidium bromide staining and recorded on a digital camera. The size of the PCR products was determined by comparing their migration rate with a molecular weight size standard [DNA molecular weight marker IX, 72- 1353 bp (GE Healthcare Bio-Sciences AB, Uppsala,Sweden)].




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