Background: Single nucleotide polymorphism association studies among cases and controls have been widely used for genetic analysis. The pyrosequencing method is based on indirect luminometric quantification of the pyrophosphate that is released as a result of nucleotide incorporation onto an amplified template. It has the advantages of accuracy, flexibility, automatization and speed when compared with PCR-RFLP method.
Aim: To develop a protocol for allele frequency determination using pyrosequencing technology in the detection of the polymorphism at position -31 of the interleukin-1beta (IL-1beta) gene.
Methods: 162 patients (F/M = 0.93) who were enrolled at the Hospital Universitario Dr "José Eleuterio Gonzalez" were studied. 123 patients had non-ulcer dyspepsia and 39 had histologically confirmed gastric cancer (GC). The polymorphism of IL-1beta -31 was determined by both RFLP and pyrosequencing methods. PCR-RFLP method used Alul restriction endonuclease. The same specific primers for PCR-RFLP and pyrosequencing were used for initial amplification and an additional biotinylated specific primer was designed for sequencing.
Results: 157 (96.9%) samples were clearly typed by the pyrosequencing method and the results were in accordance with the results of the PCR-RFLP method. The results of 5 samples (3.1%) were not in accordance between both methods. Two of them were T/T and 2 were C/T by sequencing method and all four were C/C by RFLP. Another sample was C/ C by sequencing and T/T by RFLP.
Conclusion: The pyrosequencing method is not only suitable for the IL-1beta -31 genotyping but is a fast and unexpensive way of genotyping since requires smaller amounts of DNA, and required significantly less time in the generation of results than the RFLP technique. The protocol developed is useful for the typing of the IL-1beta -31 polymorphism.