A new single nucleotide polymorphism genotyping method based on gap ligase chain reaction and a microsphere detection assay

Abstract

The establishment of new methods to detect human genetic variations, such as single nucleotide polymorphisms, is of importance in hereditary disease diagnosis and pharmacogenomics. Several single nucleotide polymorphism genotyping technologies have been presented in recent years. However, techniques which would allow accurate, fast and cheap allelic determination and multiple single nucleotide polymorphism detection in parallel are still in great need. Here, we present a new genotyping technique based on gap ligase chain reaction and a fluorescent polystyrene microsphere measurement platform. We chose the human polymorphism rs3730386 as our candidate to establish this method. Probes for gap ligase chain reaction were designed to recognize the target alleles sensitively and specifically and to produce templates for amplifying the correspondent target fragments used in the following hybridization. The genotypes were finally determined by a hybridization process based on the Luminex fluorescent polystyrene microspheres measurement platform. This method was successfully applied for the detection of selected single nucleotide polymorphisms with high sensitivity and specificity. The genotypes were validated by DNA sequencing. The new genotyping method benefited from the high sensitivity and specificity of gap ligase chain reaction and the detection platform of Luminex. The method allows multiplex analysis of single nucleotide polymorphism, because 100 types of microspheres are available from Luminex.