REAL-TIME POLYMERASE CHAIN REACTION FOR GENOTYPING OF THE TRANSFORMING GROW FACTOR β1 POLYMORPHISM Thr263Ile IN PATIENTS WITH BALKAN ENDEMIC NEPHROPATHY AND IN A HEALTHY BULGARIAN POPULATION
Atanasova S1, von Ahsen N2, Dimitrov Tz3, Armstrong V2, Oellerich M2, Toncheva D1,*
*Corresponding Author: Professor Draga Toncheva, Ph.D., Department of Medical Genetics, Medical University Sofia, 2 Zdrave str., Sophia, Bulgaria; Tel/Fax: +359-2-9520-357; E-mail: draga@spnet.net
page: 37

MATERIALS AND METHODS

Subjects. A total of 207 unrelated individuals of Bul­garian origin were screened for presence of Thr263Ile in the TGFb1 gene. Ninety-five of these were diagnosed as having BEN at different stages, and fulfilled clinical and para-clinical criteria for BEN; place of birth and residence in an endemic village and familial occurrence of endemic nephritis. The cohort of BEN patients consisted of 21 men and 74 women (mean age 63  14 years). The patients were recruited from the Vratza endemic region. The con­trol group involved 112 healthy individuals from non endemic regions. A family history of renal and cancer diseases was an exclusion criteria. Controls were age and sex matched (58  10 years; 28 men and 84 women) with the patients. Written informed consent was obtained from all participants in this study.

DNA was extracted from 9 mL peripheral blood by a standard DNA isolation protocol that included an NaCl precipitation step [10].

Genotyping Procedure. We used a new, rapid and reliable method for high throughput analyses of Thr263Ile in TGFb1, that allows fast molecular epidemiological studies of large populations. A major advantage of the proposed method is the rapid thermal cycling for polymer­ase chain reaction (PCR) and the online, real-time detec­tion of the reaction kinetics. The method allows the geno­typing of 30 samples within 60 minutes.

Rapid cycle PCR and melting curve analysis on a LightCycler (Roche Biochemica, Mannheim, Germany) were used for genotyping. The primer pairs were designed with primer3 (http://www-genome.wi.mit.edu/cgi-bin/ primer/primer3_www.cgi). The variant allele (263Ile) was distinguished from the wild type allele (263Thr) by using detection and anchor probes binding to the target sequence. The hybridization probe had full match with the wild type allele and formed a mismatch with the polymor­phic allele. When the hybridization probe formed a perfect match with the target, a characteristic high melting tem­perature (Tm) was detected, while the low Tm was ob­served in cases where a mismatch was present. The charac­teristic melting temperatures were 63C for the wild type allele and 55C for the variant allele. Hybridization and anchor probes were designed with the MeltCalc software (http://meltcalc.com) [11].

PCR was performed in 10 mL reaction volume, con­taining 1 mL genomic DNA (500 ng/mL), 1 mL 10X PCR buffer (Invitrogen, Karlsruhe, Germany), 0.5 mmol/L for­ward primer (5-ACT GCA AGT GGA CAT CAA CG-3), 0.5 mmol/L reverse primer (5-CAA GGC TCA CCT GAA GCA AT-3), 0.2 mmol/l detection probe (5-CTT CTC ATG GCC ACC C-Fluoresceine-3) and 0.2 mmol/l anchor probe (5-Cy5.5-GCT GGA GAG GGC CCA GCA-Phosphate-3), 0.2 mmol/L of each dNTP, 2.5  mmol/L MgCl2, 500 mg/L bovine serum albumine (New England BioLabs, Schwalbach, Germany), 50 mL/L di­methylsulfoxide (Sigma, Steinheim, Germany) and 0.5 U/mL native Taq DNA polymerase (Invitrogen). The am­plification conditions were: one cycle at 95C for 2 min. and 45 cycles of of the following steps with the maximum ramp rate: 95C for 0 seconds; 55C for 10 seconds; and 72C for 20 seconds. The alleles were distinguished by denaturation at 95C for 20 seconds, holding the reaction at 60C for 20 seconds, 50C for 20 seconds, followed by melting with temperature increases from 28C to 80C at a ramp rate of 0.1C per second. The melting curve acqui­sition was done within channel three.




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