Applied biosystems 3730 l automated sequencer

The genotypes of SNPs in the CXCR2 gene were examined using the polymerase chain reaction method (PCR). PCR was performed in a volume of 25 μL, including 100 ng DNA, 0.5 μM primers, 4 U of Taq DNA polymerase, 2mM buffer, and 0.5 mM deoxyribonucleoside triphosphate mix. The reaction condition was as follows: denaturation for 5 min at 95°C followed by 35 cycles of 95°C for 1 min, annealing at 58°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 7 min. The primer sequences of + 785C/T (rs2230054) and + 1440G/A (rs1126580) were summarized in Table 3.Table 3

Primer Sequences of CXCR2 Gene rs2230054 and rs1126580 Polymorphisms

Variations	Primer Sequences
rs2230054	Forward 5ʹ-TCGTCCTCATCTTCCCGCT-3’
	Reverse 5ʹ-GGAGTCCATGGCGAAACTTC-3’
rs1126580	Forward 5ʹ-AGGCTGGCCAACGGGG/A-3’
	Reverse 5ʹ-TCATAGCAGCTTATTCACAAGAC-3’

The purification of PCR products was conducted with ExoSAP-IT (USB Corp) and sequenced with an Applied Biosystems 3730×l automated sequencer (Applied Biosystems, Foster City, CA, USA), and all sequences were analyzed by Vector NTI software.

The target fragments for NAT2 gene 2 polymorphisms rs1799930 and rs1799931 were directly amplified by multiple polymerase chain reaction method (PCR). The primer sequences for the 2 SNPs were designed by Primer Premier 5.0, and synthesized by Sangon Biotech (Shanghai, China) (Table 1). The designed primer sequences were verified by nucleotide-nucleotide BLAST (blastn). The results demonstrated that the primers used in this study was specific to NAT2 sequences, and could be used for the following analysis. The PCR procedures consisted of an initial degeneration at 94°C for 5 minutes, followed by 11 cycles of 94°C degeneration for 20 seconds, annealing at 65°C for 40 seconds, and extension at 72°C for 1.5 minutes, then 24 cycles of 94°C degeneration for 20 seconds, annealing at 59°C for 30 seconds, and extension at 72°C for 1.5 minutes were followed, and a final extension at 72°C for 2 minutes and saved at 4°C.
Then the PCR products of the 2 polymorphisms were first purified by ExoSAP-IT (USB Corp) and directly sequenced by automated DNA sequencing with an Applied Biosystems 3730 × l automated sequencer (Applied Biosystems, Foster City, CA), and sequence analysis was performed using Vector NTI software.