Single-tube-genotyping of gastric cancer related SNPs by directly using whole blood and paper-dried blood as starting material.

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 0.5-1.0 microL 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.