| Objective1.Single nucleotide polymorphism(SNP)typing technologies including first-generation sequencing,Taq Man probe qPCR,DNA microarray,etc.at present,which have the limitations of high detection cost,long cycle,and the detection results are easy to appear the false positive.Li et al.created a universal probe which based intermediate prime-initiated qPCR(upip-qPCR)technology.The purpose of this study was to develop a deafness-related SNP detection kit based on UPIP-qPCR technology.2.Differences between genomes may be caused by SNP or copy number variation(CNV).The mutation rate of CNV is much higher than that of SNP,and the quantitative detection of CNV in genomes by nucleic acid is an important indicator for clinical detection.UPIP-qPCR was used to detect CNV of metabolic genes related to tumors(breast cancer and lung cancer),such as glutathione S-transferase(GSTs)isozymes M1 and T1(GSTM1 and GSTT1),we will be analyzing the feasibility,sensitivity and resolution of this technology.To explore the versatility of this technique in different tissue samples(tumor cells,tumor tissues)and large samples.Methods1.In this study,the peripheral blood DNA of healthy people was used as the template,and we were selected the common deafness SNP mutation sites in Chinese as candidate SNPs.We designed the specific primers of UPIP-qPCR and the primers of gold standard Sanger sequencing,and compared the UPIP-qPCR with Sanger sequencing to prove the feasibility of the technology;Then,we constructed standard samples(wild homozygous,mutant heterozygous and Mutant homozygous)and used it as templates.We used this technology to type and verify the deafness-related SNP sites to prove the effectiveness of the primers in the kit.2.We were designed UPIP-qPCR specific primers for target genes(GSTM1,GSTT1)and reference genes(GAPDH)and qPCR primers for Taq Man probe(gold standard for CNV detection).Using normal peripheral blood DNA as template,we obtained the gene copy number in DNA samples by absolute quantitative method.We used the UPIP-qPCR technique to detect the sensitivity of GSTM1 at different concentrations,the resolution of GSTM1 in different copies,and the generality of CNV in large samples and samples from different sources(tumor cells and tumor tissues).Result 1.We designed primers based on UPIP-qPCR technology to obtain specific amplification signals and typical amplification curves in single and multiple reactions.indicating that the deafness gene of healthy people was wild pure type,and the typing results were consistent with the results of Sanger sequencing.It was confirmed that the primers and the technology could specifically distinguish the wild pure sum,wild heterozygous and complete mutant SNP sites which are associated with deafness bythe testing of standard products.2.The same results were obtained by UPIP-qPCR and Taq Man probe qPCR(the gold standard)in the copy number detection of target genes(GSTM1 and GSTT1)in healthy people,which proved that UPIP-qPCR could detect CNV in human genome and was feasible.The UPIP-qPCR technique can identify the approximate copy of the most accurate multiple is 1.1 times CNV,and the highest sensitivity is 0.01ng/10μL.The positive detection rate of CNV in large sample is 100%,which meets the need of clinical mass detection.We used UPIP-qPCR to perform a general experiment,and the results showed that the copy number of GSTM1 could be detected in samples from different sources.Conlusion1.We used UPIP-qPCR technology to complete SNP typing detection for deafness-related gene loci,and developed a deafness-related SNP detection kit based on UPIP-qPCR technology.2.The UPIP-qPCR technology was used to develop a new method for CNV detection,which has high accuracy,high resolution and high sensitivity,and can be used to detect CNV in large quantities of tumor samples from different sources,meeting the requirements of application in the detection of tumor mutants CNV. |
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