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
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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 63°C for the wild type allele and 55°C 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 95°C for 2 min. and 45 cycles of of the following steps with the maximum ramp rate: 95°C for 0 seconds; 55°C for 10 seconds; and 72°C for 20 seconds. The alleles were distinguished by denaturation at 95°C for 20 seconds, holding the reaction at 60°C for 20 seconds, 50°C for 20 seconds, followed by melting with temperature increases from 28°C to 80°C at a ramp rate of 0.1°C per second. The melting curve acquisition was done within channel three.
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