**2.3 Polymerase chain reaction-RFLP**

442 Selected Topics in DNA Repair

Two millilitres of venous blood was stored in heparinised vacutainers for comet assay and assessment of DNA repair kinetics and stored at +4°C before further procedure. Detailed protocol is described before (Milić et al., 2010). Five millilitres of venous blood was stored in vacutainers with EDTA (ethylenediaminetetraacetic acid) at -20°C until further DNA isolation (Milić et al., 2010). Blood samples were irradiated with 60Co (Alcyon, CGR-MeV, France). The doses used were 2 and 4 Gy, with the same distance from the source (80 cm). Irradiation field was 15 x 15 cm2. After irradiation, samples were kept at + 4°C to prevent

Alkaline version of comet assay (Singh et al., 1988) with small modifications was used. Detailed procedure is described elsewhere (Milić, 2010; Milić et al., 2010). The slides were marked and stored at +4°C till the beginning of the irradiation. Control samples were put into cold lysis solution immediately after preparation and left there for 24 hours at +4°C (Milić, 2010). Irradiated blood gel samples were incubated at 37°C in serum free RPMI 1640 medium. Zero samples were immediately immersed into lysis solution. DNA repair kinetics was measured at 0, 15, 30, 60 and 120 minutes after exposure, and additional 24 hours for samples irradiated with the dose of 4 Gy. Specific measure points were based on the results of other researches (Singh et al., 1988; Price, 1993; Tice, 1995). The 2 Gy dose was a standard daily dose in radiotherapy, while the 4 Gy dose was chosen as a challenging dose for the exposed group. After the repair, slides were vertically placed into cold lysis solution at 4°C, overnight. Protein denaturation and DNA unwinding were done at 4°C in denaturation buffer (1 mM Na2EDTA and 300 mM NaOH) (pH 13.0) for 20 minutes. Horizontal electrophoresis in fresh cold denaturation buffer was done at 300 mA and 25 V for 20 minutes. The slides were washed in neutralisation buffer (0.4 M Tris-HCl, pH 7.5) three times. Slides were stained with 50 µL of ethidium bromide solution (20 μgmL-1, Sigma) (per slide), covered with cover slip and kept in container, in the dark

The procedure was done under dimmed light, in order to avoid additional DNA damage caused by the exposure to the normal light. Each slide was examined using a 250x magnification fluorescence microscope (Zeiss, Germany) equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm. A total of 200 comets per sample and per interval were scored (100 from each of two replicate slides). Comets were randomly captured at a constant depth of the gel, avoiding the edges of the gel, occasional dead cells and superimposed comets. Using a black and white camera, the microscope image was transferred to a computer-based image analysis system (Comet Assay II, Perceptive Instruments Ltd., U.K.). To avoid the variability, one well-trained scorer scored all comets. Three parameters of DNA damage were analysed: tail length (TL, presented in

Genomic DNA was isolated from peripheral lymphocytes according to modified protocol by Daly et al. (1996) or according to protocol for genomic DNA lymphocyte isolation from QUIAGEN (mini KIT). DNA was purified with two times centrifugation at 4°C, 500 μL of 70 percent ethanol added every time. The pellet was dried overnight at room temperature and

the repair of the damage. Details are also described before (Milić et al., 2010).

**2.1 Comet assay** 

conditions at 4°C.

**2.2 DNA isolation** 

micrometres), tail DNA (TI, %) and tail moment (TM).

In a total volume of 10 ml, 10 ng of genomic DNA was amplified for each sample. Compounds for the reaction mixture are given in Table 2.

PCR reaction was completed in six steps: (I) incubation at 94 °C (2 min) for Taq DNA polymerase activation; (II) incubation at 94 °C (30 s) for denaturation of double stranded DNA; (III) incubation at specific temperature that depended on the specific gene (30 s), for hybridisation of primers (Table 3); (IV) incubation at 72 °C (30 s) for DNA synthesis. Steps No.2 to No. 4 were repeated for 34 times. After that there were steps: (V) incubation at 72 °C (5 min) and (VI) incubation at 10 °C.


Table 2. Compounds for PCR-RFLP reaction (10 μl of reaction mixture (9 μl of Master Mix and 1 μl of DNA sample).


Table 3. Gene, forward and reverse primers, method used for determination of polymorphism, hybridisation temperature, restriction enzymes and the DNA fragments.

The Influence of Individual Genome Sensitivity in DNA Damage Repair

Rad, Hercules, USA).

**3. Results** 

same in both groups.

equilibrium.

Assessment in Chronic Professional Exposure to Low Doses of Ionizing Radiation 445

After PCR reaction, DNA products were cut with specific restriction enzymes (Fermentas, Vilnius, Litva) that have cut DNA samples on specific places and have given different DNA fragments in order to recognize which samples were homozygote wild type, heterozygote and polymorphic homozygotes. Treatment with restriction enzymes was performed at 37 °C in a period of 3 hours or overnight (due to the specification of specific restriction enzyme used in the reaction). Enzymes, time intervals of cutting, the temperature for those enzymes and the resulting DNA fragments after the cutting are given in Table 3. After electrophoresis that lasted 30 minutes at 200 V, PCR products were analysed on 10%-polyacrilamid gel (Bio-

PCR products were also amplified with Real-Time PCR (Real-Time PCR ABI Prism 7300 thermocycler, Applied Biosystems, Foster City, USA) with TaqMan allelic discrimination assay (Applera, Foster City, USA). Allelic determination was done by their software. Compounds for the Real-Time PCR mixture is shown in Table 4. Forty cycluses were performed for division between VIC and FAM fluorescence stain. The intensity of those

Ingredients Volume/μl

reH2O 5.33

DNA sample 0.5

TaqMan Master Mix 6.5

Specific primers for every gene 0.65

Table 4. Compounds for Real Time PCR reaction. Steps in Real Time PCR-a were: (I) 95 °C

DNA repair kinetics after the exposure to gamma radiation of 2 and 4 Gy was measured on

The groups differed in average age, gender, smoking status and alcohol consumption. The mean values for both groups did not significantly differ, although inter-individual differences were notable. In control group higher level of DNA damage compared to exposed group was observed, but without statistical difference. The repair dynamic was the

After genotyping, heterozygotes and polymorphic homozygotes were grouped together to evaluate polymorphic allele appearance. Number of individuals carrying particular gene is given in Table 5. Frequency of genotyping did not differ from expected Hardy-Weinberg

a group of 126 subjects, 70 medical workers and 56 controls (Figure 1).

Genotype results were regularly confirmed by random repetition of the samples.

stains is selecting the samples into three categories (Angelini et al., 2005).

(10 min); (II) 92 °C (15 s); and (III) 60 °C (10 min).

After PCR reaction, DNA products were cut with specific restriction enzymes (Fermentas, Vilnius, Litva) that have cut DNA samples on specific places and have given different DNA fragments in order to recognize which samples were homozygote wild type, heterozygote and polymorphic homozygotes. Treatment with restriction enzymes was performed at 37 °C in a period of 3 hours or overnight (due to the specification of specific restriction enzyme used in the reaction). Enzymes, time intervals of cutting, the temperature for those enzymes and the resulting DNA fragments after the cutting are given in Table 3. After electrophoresis that lasted 30 minutes at 200 V, PCR products were analysed on 10%-polyacrilamid gel (Bio-Rad, Hercules, USA).

Genotype results were regularly confirmed by random repetition of the samples.

PCR products were also amplified with Real-Time PCR (Real-Time PCR ABI Prism 7300 thermocycler, Applied Biosystems, Foster City, USA) with TaqMan allelic discrimination assay (Applera, Foster City, USA). Allelic determination was done by their software. Compounds for the Real-Time PCR mixture is shown in Table 4. Forty cycluses were performed for division between VIC and FAM fluorescence stain. The intensity of those stains is selecting the samples into three categories (Angelini et al., 2005).


Table 4. Compounds for Real Time PCR reaction. Steps in Real Time PCR-a were: (I) 95 °C (10 min); (II) 92 °C (15 s); and (III) 60 °C (10 min).
