AIM: To demonstrate an inexpensive method for typing gastric cancer related single nucleotide polymorphisms (SNPs) using whole blood or paper-dried blood as starting materials. METHODS: PCR amplification is directly c...AIM: To demonstrate an inexpensive method for typing gastric cancer related single nucleotide polymorphisms (SNPs) using whole blood or paper-dried blood as starting materials. METHODS: PCR amplification is directly carried out from the whole blood or paper-dried blood sample without any DNA extraction step. Before PCR, a blood sample, four primers, and all of biological reagents necessary for PCR were added at a time; After PCR, the amplified products were directly separated by slab gel electrophoresis or microchip CE without any purification. SNP typing was performed by tetra-primer PCR with two inner primers specific to each allele and two outer primers defining the length of allele-specific amplicons. Genotypes were directly discriminated by the size of amplicons specific to each allele, thereby avoiding any post-PCR process. RESULTS: Using a special PCR buffer, inhibitory substances in blood (including the anticoagulant in blood) and filter paper were effectively suppressed; a 'true' single-tube-genotyping is thus realized. We successfully determined genotypes IL-1B-511 and IL-1B-31 polymorphisms at the gene IL-1B by using whole-blood and paper-dried blood samples as starting materials respectively. The method is so sensitive that 0.5-1.0μL of blood sample is enough to give a satisfactory typing results. The genotyping results were confirmed by RFLP-PCR using purified genome DNA, indicating that amplification specificity was not affected by inhibitory components (including coagulants) in blood or filter paper. CONCLUSION: Compared with SNP typing methods based on purified DNA, the proposed method is labor saving, simple, inexpensive, and less cross-contaminated. It is promising to use this method to type other SNPs.展开更多
基金Supported by National Natural Science Foundation of China,No. 30270368
文摘AIM: To demonstrate an inexpensive method for typing gastric cancer related single nucleotide polymorphisms (SNPs) using whole blood or paper-dried blood as starting materials. METHODS: PCR amplification is directly carried out from the whole blood or paper-dried blood sample without any DNA extraction step. Before PCR, a blood sample, four primers, and all of biological reagents necessary for PCR were added at a time; After PCR, the amplified products were directly separated by slab gel electrophoresis or microchip CE without any purification. SNP typing was performed by tetra-primer PCR with two inner primers specific to each allele and two outer primers defining the length of allele-specific amplicons. Genotypes were directly discriminated by the size of amplicons specific to each allele, thereby avoiding any post-PCR process. RESULTS: Using a special PCR buffer, inhibitory substances in blood (including the anticoagulant in blood) and filter paper were effectively suppressed; a 'true' single-tube-genotyping is thus realized. We successfully determined genotypes IL-1B-511 and IL-1B-31 polymorphisms at the gene IL-1B by using whole-blood and paper-dried blood samples as starting materials respectively. The method is so sensitive that 0.5-1.0μL of blood sample is enough to give a satisfactory typing results. The genotyping results were confirmed by RFLP-PCR using purified genome DNA, indicating that amplification specificity was not affected by inhibitory components (including coagulants) in blood or filter paper. CONCLUSION: Compared with SNP typing methods based on purified DNA, the proposed method is labor saving, simple, inexpensive, and less cross-contaminated. It is promising to use this method to type other SNPs.
