Whole blood genomic dna extraction kit

Genomic DNA was extracted using a whole blood genomic DNA extraction kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s instructions, and then analyzed with 0.8% agarose gel electrophoresis. Amplification primers for the promoter region of the 5-HTT gene were as follows: Forward primer, 5′-GCGCTCCTGCATCCCCCATTA-3′; and reverse primer, 5′-GGGATGCGGGGGAATACTGGT-3′. 5-HTTLPR polymorphism in the 5-HTT promoter region was assessed using PCR amplification. A total of 25 μl PCR reaction solution was prepared by mixing 10X PCR reaction buffer [containing 2.0 mmol/l (NH₄)₂SO₄ and MgCl₂; Invitrogen Life Technologies, Carlsbad, CA, USA], 0.16 μmol/l each primer, 1.5 units Taq enzymes and 160 μmol/l deoxyribonucleotide triphosphates. The PCR conditions were as follows: Denaturation at 95°C for 2 min, followed by 35 cycles of 95°C for 60 sec, 62°C for 60 sec and 72°C for 60 sec, and then extension at 72°C for 10 min. The PCR products were separated by 2% agarose gel electrophoresis, and the gel was visualized and analyzed by a Bio-Rad image analysis system (Bio-Rad, Hercules, CA, USA). Two different allelic fragments with the lengths of 297 bp (L) and 253 bp (S) were detected among the PCR products, and the genotype was determined accordingly (Fig. 1) (14 (link)).

A clinically tested blood sample was drawn and anticoagulated using EDTA-2Na-(Solarbio, China, Batch number: E8030). The DNA was extracted using the Qiagen Whole Blood Genomic DNA Extraction Kit (Qiagen, China, Batch number: 20150114). Thirty DNA standard samples of 30 PAH genotypes identified by DNA sequencing were 27 in 9 mutants and 3 in wild-type. The R243Q (G → A) genotype was an artificial plasmid used for the establishment of this method. (This study was submitted to and approved by Guangzhou University of Chinese medicine vhospital institutional ethics committee, and all participants were from Guangzhou University of Chinese medicine hospital).