| BackgroundCNV is the submicroscopic mutation caused by a particular DNA segment range from Kb to Mb,including deletion and duplication.The formation mechanism of CNV is primarily divided into two parts:recombination and mis-copy,therefore,recombination includes non-allelic homologous recombination and non-homologous end joining.Non-allelic homologous recombination usually occurs undergoing meiotic,which is caused by the recombination of homogenous DNA sequences in different gene sites.What the difference from non-allelic homologous recombination is that non-homologous end joining can generate without homogenous DNA sequences.However,mis-copy is the DNA strand transposition by homogenous DNA sequences during DNA replication.CNV is the important part of structural variation,which not only gives rise to genetic polymorphism and genetic differences,but also causes serious diseases.According to study,CNV is linked with single gene disorders,rare diseases and complex diseases.It probably participates in the occurrence and development of diseases by the change of gene expression,protein struction and locality effect,so it may cause diseases.The common diseases caused by CNV are α-thalassaemia Down Syndrome,infantile autism,SMA and so on.CNV is one of structural variation participating in the occurrence and development of diseases,so the rapid detection of CNV is quite important.At present,the methods for CNV detection are array comparative genomic hybridization,aCGH;customized fluorescence in situ hybridization,FISH;multiplex ligation-dependent probe amplification,MLPA;next-generation sequencing,NGS and so on.However,there are both advantages and disadvantages for any method,and the most obvious disadvantage is high cost and fussy operation.The detection of CNV is the copy number detection of targeted gene,and any detection method is in order to determine the deletion or duplication of targeted gene.The objective of study is to establish a rapid,accurate,convenient,high throughout and low-cost method for CNV detection.MethodsBased on the pathogenic mechanism of CNV,we can diagnose diseases accurately by CNV determination.In resent years,qPCR has been widely used in clinical tests for its high throughout amd no-subsequent processing,which has been for CNV detection as well.According to Xn=X0(1+Ex)n,suppose the amplification efficiency reachs 100%.If the original copy nunber(X0)is 1,in order to get 8(Xn)copy number,it needs 3(8=1*2n,n=3)cycles.If the original copy number(X0)is 2,it needs 2(8=2*2n,n=2)cycles.If the original copy number(X0)is 3,it needs 1.42(8=3*2n,n= 1.42)cycles.In theory,that means the △Ct between 3 copies and lcopy is 1.58;△Ct between 2 copies and 1copy number is 1;△Ct between 3 copies and 2copies is 0.58.So,it may get false negative result for low-ratio copy number variation such as 1.5-fold copy number variation.In order to solve this problem,we try to establish a new method-a template block qPCR for rapid detection of copy number variations.Based on the research strategy,we carry out programs and methods as follows.1.Block templates.We suppose that one targeted sequence is 4 copies in normal samples;6 copies in positive samples,therefore,the ratio of copy number between them is 1.5-fold.It can just get 0.585 △Ct by qPCR so that it may get ambiguous result.If there are respectively 2 copies been blocked,not being templates for PCR amplification,the ratio of copy number between positive samples(6-2)and normal samples(4-2)will turn to be 2.0-fold.It can get 1.0△Ct by qPCR,which can response the difference clearly.Based on the assumption,we try to block templates.The principle of it is that design a special oligonucleotide coverred with primer binding site,insert 1 to 3 uncomplementary bases in 3,end and modify with spacer C3.We define such a modified oligonucleotide as blocked probe.During PCR,blocked probes combine with templates by complementary sequences without extension,for uncomplementary bases in 3’end and modified spacer C3 preventing from 5’-3’polymerase activity.2.Establish tripartite qPCR.We select Down Syndrome as a disease model in our research.Choose DSCR3 and DSCR4 as targeted genes,GAPDH as a reference gene,analyzing the relative copy numbers of targeted genes by ACt.It calculates relative copy numbers by Ct value during index growth and the more copy numbers,the low Ct value.For normal samples,the copy number of DSCR3,DSCR4 and GAPDH arc identical,so the △ct are constant.However,for trisomic samples,the Ct value will reduce for increasing copy number of targeted genes,and the △Ct will increase.In theory,we can analyse the situation of targeted genes by relative quantitative to diagnose diseases.The way of △Ct relative quantitative is based on fixed volume and fixed gDNA dose to measure the ratio of targeted genes and reference gene.We establish tripartite qPCR,with identical component determining targeted genes and reference gene,which eliminate difference due to single tube.In addition,we adopted template pre-amplification,compounding detection templates in low temperature and detecting in high temperature,to guarantee the specificity and efficiency.3.Optimize the template block qPCR.According to some relative articles,it has to be high and consistent efficiency for every PCR to apply 2’△△Ct method.After the specificity of primers and TaqMan probes of DSCR3,DSCR4 and GAPDH had been verified,components and reactive condition,including primers concentration,TaqMan probes concentration,PCR Buffer,Mg2+ concentration,gDNA dosage,blocked probes concentration and cycle numbers,annealing temperature,extension time,was optimized and adjusted separately.At last,we comfirmed the components and reactive condition by analyzing the comprehensive efficiency and consistency efficiency.4.Analyze the template block qPCR.The establishment of one method is to diagnose diseases clinically.So,for the establishment of one method,optimization is premise,accuracy is critical,sensitiveness and repeatability are guaranteed.Whether the method we established,blocked templates with tripartite qPCR,can achieve the goal for screening and routine detection,it needs future evaluation,including accuracy,sensitiveness and repeatability.5.Double-blinded detection with the established method.We prepared 139 gDNA clinical samples,which were concentration greater than 100ng/ul,OD260/280 between 1.7 and 1.9,comfired genetype by karyotype analysis.After numbering them blindly,we tested them with the established method.Finally,compared the results of this method with the karyotype analysis.6.Explore the resolving power of the template block qPCR.To explore the resolving power of the method we established,we tested different ratios of trisomy samples and normal samples,including 0%,10%,20%,30%,40%,50%and 60%.In addition,we prepared 18 gDNA clinical samples,which were concentration greater than 100ng/ul,OD260/280 between 1.7 and 1.9,comfired genetype by karyotype analysis.After numbering them blindly,we tested them with the established method.Finally,compared the results of this method with the karyotype analysis.ResultsBased on the objective and strategy,implementing the program and method,we get the results as follows.1.Establish tripartite qPCR.The selection of genes and their sequences were characteristic and reliable.The amplification of tripartite qPCR was parallel without interference with each other.The pre-amplification of templates,compounding detection templates in low temperature and detecting in high temperature,was feasible.And the assumpition to alter the copy number ratio of samples by blocking templates was achievable as well.2.Comfirm the template block qPCR.After the optimization and adjustion of components and reactive condition,the analysis of comprehensive efficiency and consistency efficiency,we comfirmed the system.It was 20ul,including 1.5x PCR Buffer,5.Ommol/L Mg2+,125nmol/L primer,125nmol/L TaqMan probe,lOnmol/L blocked probe,0.2mmol/L dNTPs,1U Ex Taq and +200ng gDNA.The reactive condition was 95 ℃ 5min→2cycles(95 ℃ 3 0sec+65 ℃ 1 min+52 ℃ 30sec)→35cycles(94℃30sec+62℃ 1min).In such an optimized system,the efficiency of DSCR3,DSCR4 and GAPDH were high and consistant,106.0%,107.5%,100.9%respectively.And the slopes of their analytical curves for consistency efficiency were both 0.0002.3.Evaluate the template block qPCR.The accuracy of this system for Down Syndrome can reach 100%.In such an optimized system,even thought the gDNA as low as 7.25ng,it can get the perfect detection result.In addition,it can achieve the ideal results for intra and inter assay evaluation.4.Detecte CNV with the template block qPCR.We applied the template block qPCR to detect 139 gDNA blindly,and obtained 39 trisomic samples,which was identical with the result of karyotype analysis.In addition,the template block qPCR can not only determine trisomic samples but also containing 40%trisomic samples.We obtained 8 positive samples,which was identical with the result of karyotype analysis.ConclusionVia the study,we established a novel approach-block templates,to alter the ratio of copy number variations in theory.And the template block qPCR was confirmed to determine CNV in methodology.The novel method we established-template block qPCR,solved the false negative problem for CNV detection with traditional qPCR,especially for less than 2.0-fold CNVs.In addition,it can realize multiple samples detection simultaneously,which can reduce work,cost and time,thus increase throught and efficience.The establishment of the template block qPCR can provide reference for other research of genetic diseases. |
