A New Method For Detection Of Single Nucleotide Polymorphisms Combined With The Fill In-ligation Strategy And Magnetic Separation System

With the discovery of large number of single nucleotide polymorphisms related to human diseases and drug therapy, many approaches for the genotyping of SNP have be developed. However, most of methods can not be performed in local clinical laboratories readily due to the low sensitivity, expensive instrumentation and regents and the limitation of professional manipulation.A new flexible genotyping method for the SNP and insertion/deletion of short fragment was developed with designing specific detection probes. The detection process include following steps:(1) The amplification of DNA sequence containing SNP sites; (2) The allele of different SNP locus were discriminated based on the dual specificity of DNA polymerase and ligase; (3) Magnetic particles coupled with streptavidin were used to purify the detection fragments to eliminate the redundant components out in the reaction system to further enhance the specificity of this method; (4) The allele specificity fragments after purification were amplified to increase the detection sensitivity; (5) The genotype was detected by agarose gel electrophoresis, ELISA detection and common oligonucleotide microarray technology.The genotyping with agarose gel electrophoresis and ELISA detection can be achieved with high specificity and sensitivity, without the requirement of expensive instrument and reagents, and this method can be used for the detection of heterogeneous samples in the conventional laboratory. The genotyping with common oligonucleotide microarray technology can be performed for the high throughput detection with simple fluorescence scanner and data analysis method in single fluorescence labeled system.The homobifunctional crosslinker 1,4-phenylene diisothiocyanate (PDC) was coupled to amine-terminated magnetic particle surface to prepare the activated magnetic particles, and the streptavidin was immobilized on the activated magnetic particles covanlently for the SNP genotyping.Eight of SNPs and one of ins/del locus in cyp3A4, cyp2B6 and EGFR were detected by developed method with agarose gel electrophoresis. The results show that the method could detect the minor mutant from the mixture DNA sample even if the proportion of variant allele is as lower as 0.5%. Eight of samples in 35 non-small cell lung cancer tissues were identified as heterozygote with the detection of agarose gel electrophoresis. However, only two samples were identified with the sequencing method. It indicates that the new method had high sensitivity and was adopted to the genotyping of SNP for the heterogeneous samples. The result of ELISA detection for eight heterozygote sample had more obvious comparision of positive and negative signal compared to the result of agarose gel electrophoresis. The results show that ELISA detection had more sensitivity and could be performed with more samples.The genotyping with common oligonucleotide microarray was studied by detecting the SNP sites in CYPs. The results showed that seven of SNP loci in CYP2D6 all had significant positive fluorescence signal value and obvious ratio of positive and negative signal. The result show that the method could be used for the medium or high throughput SNP genotyping in the near future.