Materials and sequence analysis
The 88 recruiting samples all came from a coke oven plant. The male and female workers are 54 and 34 respectively, and the ages of male and female workers are 22–55 years old and 25–50 years old respectively.
The POT1 gene information and polymorphism site rs10250202 for POT1 were obtained from NCBI website (http://www.ncbi.nlm.nih.gov). Sequence analysis shows that the sequence GATC which consist of polymorphism site rs10250202 can be cleaved by BfuCI, Sau3AI, DpnI, DpnII and MboI. The cost of above mentioned enzymes is much higher. To find a lower cost PCR–RFLP method, a new BclI enzyme site was created to detect the POT1 gene polymorphism.
The prices of above mentioned enzymes from New England BioLabs website (NEB) are as Table 1. From Table 1 we can see that compared to using DpnI enzyme, $70 would be saved when 1000 U enzyme was used to detect 200 samples.
Table 1 Identification of the sequence and prices of some kinds of endonucleases in NEB Co
The primers were designed by primer premier 5.0 software. The utilization of the NEB cutter program for restriction enzyme site mapping (http://nc2.neb.com/NEBcutter2/, http://www.neb-china.com) showed that the BclI restriction enzyme would be useful to distinguish the different alleles of the SNP of rs10250202 in POT1 gene. Peripheral blood leukocytes of 88 Chinese Han individuals were used as a source of genomic DNA for PCR amplifications by genomic DNA isolation kit (Bio Teke Corporation, Beijing, China). The protocols were approved by Life Sciences of Institutional Review Board of Zhengzhou University Ethics, China (IRB 00006861, FWA00014064); all participants provided written informed consent.
PCR primers and PCR conditions
The primers designed by primer premier 5.0 software are as follows: the forward primer: 5′-AATTTACTTGATTACGTGTATCTCAAAGC-3′, the reverse primer: 5′-CATATTTCAATATTGTCCAATTAAATGAT-3′.
The PCR was performed in a final volume of 15 μl containing the following: 100 ng of genomic DNA, 0.3 μM each primer(Sangon Biotech, Shanghai, China), 2 × PCR Mix (Lifefeng Biotech, Shanghai, China) and double-distilled water. The cycling conditions are as follows: 95 °C for 5 min; 35 cycles of 95–60–72 °C for 20 s each; and 72 °C for 5 min. Electrophoresis were performed on 2 % agarose gel dyed ethidium bromide for 20 min.
Genotype of DNA
Enzyme digestion was conducted in a 20 μl final volume consists of 5U of BclI enzyme (New England Biolab) and 10 μl of PCR products. The reaction was conducted at 50 °C overnight and the digested products were electrophoresed on 3 % agarose gel containing ethidium bromide (0.5 μg/ml) for 40 min, the agarose gel was observed under UV transilluminator.
PCR products sequencing
PCR products of the sample were Sanger sequenced with Reverse primer by Sangon Biotech Company in Shanghai, China.
Quality control
We follow in strict accordance with the instructions of DNA extraction kit to extract the genomic DNA. The concentration and purity of DNA sample must meet the PCR requirements. The DNA concentration must be above 50 ng/μl. A260/A280 is in the scope of 1.8–2.0.
PCR lab was divided into four areas, namely, reagent preparation area, sample preparation area, PCR area and product analysis area. The lab must be sterilized by ultraviolet radiation method to avoid cross contamination. Manual Pipettes and PCR instruments must be periodically calibrated to ensure the accuracy.
The positive control and negative control were used in CRS-PCR–RFLP progress. Ten DNA samples were randomly selected to be amplified by PCR and genotyped by enough restriction enzyme digestion. PCR and enzyme-digested products were confirmed by electrophoresis. In PCR progress, one of the above DNA samples of successful PCR was used to be positive control, and water as negative control. The wild and mutant homozygous genotypes of DNA samples were acted as the quality control samples in each enzyme digestion reaction.