PstI (10 U/µL)

Protocol tips

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	The XPD genotypes (Lys751Gln/A to C) were determined by the PCR amplification and restriction digestion of the products with PstI (Thermo Fisher Scientific Inc. (EU), Lithuania). The PCR amplicon (436 bp) was digested overnight at 37°C with PstI.	The restriction/digestion products were resolved on 3% agarose gel containing 0.5 μg/ml ethidium bromide using a gel electrophoresis system at 100 V for 30–40 min and visualized under UV light. The PstI digestion resulted in two fragments of 290 bp and 146 bp for homozygous wild genotype (Lys/Lys), three fragments of 227 bp, 146 bp, and 63 bp for homozygous variant genotype (Gln/Gln), and four fragments of 290 bp, 227 bp, 146 bp, and 63 bp for heterozygous genotype (Lys/Gln) (Figure 2).

Protocol tips
The XPD genotypes (Lys751Gln/A to C) were determined by the PCR amplification and restriction digestion of the products with PstI (Thermo Fisher Scientific Inc. (EU), Lithuania). The PCR amplicon (436 bp) was digested overnight at 37°C with PstI.
Downstream tips
The restriction/digestion products were resolved on 3% agarose gel containing 0.5 μg/ml ethidium bromide using a gel electrophoresis system at 100 V for 30–40 min and visualized under UV light. The PstI digestion resulted in two fragments of 290 bp and 146 bp for homozygous wild genotype (Lys/Lys), three fragments of 227 bp, 146 bp, and 63 bp for homozygous variant genotype (Gln/Gln), and four fragments of 290 bp, 227 bp, 146 bp, and 63 bp for heterozygous genotype (Lys/Gln) (Figure 2).

Publication protocol

The XPD genotypes (Lys751Gln/A to C) were determined by the PCR amplification and restriction digestion of the products with PstI (Thermo Fisher Scientific Inc. (EU), Lithuania). A region of 436 bp carrying the restriction site for PstI was amplified by PCR using the following set of primers: forward primer (5- GCC CGC TCT GGA TTA TAC G-3) and reverse primer (5- CTA TCA TCT CCT GGC CCC C-3) (Integrated DNA Technologies, Coralville, Iowa). All the reactions were carried out in a total reaction volume of 25 μl containing 50–75 ng of genomic DNA template and 1 U of Taq polymerase (Thermo Fisher Scientific Inc. (EU), Lithuania). The PCR conditions were initial denaturation for 10 minutes at 94°C, followed by 30 cycles each of 1-minute denaturation at 94°C, 30-second annealing phase at 65°C, and 45-second extension at 72°C, followed by a final extension at 72°C for 7 minutes. The amplified product was resolved on 2% agarose gel containing 0.5 μg/ml ethidium bromide and visualized under UV light using a transilluminator system. The PCR amplicon (436 bp) was digested overnight at 37°C with PstI. The restriction/digestion products were resolved on 3% agarose gel containing 0.5 μg/ml ethidium bromide using a gel electrophoresis system at 100 V for 30–40 min and visualized under UV light. The PstI digestion resulted in two fragments of 290 bp and 146 bp for homozygous wild genotype (Lys/Lys), three fragments of 227 bp, 146 bp, and 63 bp for homozygous variant genotype (Gln/Gln), and four fragments of 290 bp, 227 bp, 146 bp, and 63 bp for heterozygous genotype (Lys/Gln) (Figure 2)

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