A Universal Gene Chip Based On Multiplex Invasive Reaction For SNP Detection And Its Application Evaluation In Personalized Medicine

Multiplex detection of drug-related SNPs is required in gene-oriented personalized medicine.However,conventional methods can detect only one SNP locus once.On the contrary,gene chip enables high-throughput detection,whereas it is expensive and easy to be cross-contaminated due to manipulating of PCR.Dye labels and locus-specific probe hybridization also limit gene chip's application in field of personalized medicine.To address problems above,a novel universal gene chip based on multiplex invasive reaction combined with zipcode technique was proposed for SNPs detection.By multiplex invasive reaction generating specific zipcodes corresponding to different SNPs respectively,SNPs loci were identified.Zipcodes were amplified and then captured by oligonucleotides on chip.They were hybrided with universal fluorescent probes to detect all SNPs loci.This universal SNP on-chip-detection method,with a low risk of cross-contamination,low cost,and high throughput,will be a useful tool for personalized medication.Firstly,with optimization of modification on chip,spotting solution,probes concentration and hybridization temperature,we detected zipcodes with specificity on chip based on base-stacking and universal fluorescent probes.Due to the weakness of base-stacking,it was proved to be hard to generate signal when plenty of downstream probes exist.Secondly,we established the detection based on ligation instead of base-stacking,with method optimization of ligase,mixture concentration,hybridization temperature and time.We achieved to distinguish one base-mismatch within 5 bases near ligation site.The least amount of zipcode fragments could be detected with high specificity was 2 pM.Thirdly,to combine multiplex invasive reaction with ligation reaction,we designed reasonable zipcodes,and optimizated mixture components,reaction time,probes concentration and Afu FEN1 amount.The sensitivity of this chimeric method increased to 105 copies of target templates.Nevertheless,one-step invasive reaction based on ligation could not detect genomic DNA target directly and trial of two-step invasive reaction based on ligation was failure.We finally hit the target through PCR pre-amplification followed by the universal SNP on-chip-detection based on multiplex invasive reaction and ligation.This method can detect 14 SNPs loci in single-tube at the same time.We used this method to detect 14 SNPs loci of 20 clinical samples.Results shown the correspondence with Sanger sequencing,which reflected the exactly accuracy and clinical-usefulness of our new SNP detection method.In summary,we successfully established a universal SNP on-chip-detection method based on multiplex invasive reaction and ligation,with PCR pre-amplification.This new method had advantages of single-tube,inability to be cross-contaminated and universality.It solved the problems of existing SNP detection methods.We decreased the cost as much as we can and simplified the procedure to meet the needs of multiplex drug-related SNPs loci detection,which provided a useful tool in clinical gene-oriented personalized medicine.