本文選題：液滴數(shù)字PCR + TaqMan探針��； 參考：《大連醫(yī)科大學》2016年碩士論文

【摘要】：目的:針對目前實時定量PCR(real-time quantitative PCR,qPCR)和液滴數(shù)字PCR(droplet digital PCR,ddPCR)只能檢測已知突變位點的不足,嘗試建立一種基于Taqman探針和染料相結合的ddPCR方法用于檢測未知突變序列,并應用該方法檢測PIK3CA基因E545K突變位點的三種基因型(野生型、雜合突變型和純合突變型)。方法:本研究屬于方法學研究,即先建立方法,然后與已有方法進行統(tǒng)計學分析比較,確定方法的可靠性。1.選擇包含PIK3CA基因的野生型(MCF-7細胞)和雜合突變型(H460細胞)細胞系以及合成包含PIK3CA基因的純合突變型雙鏈DNA,針對PIK3CA基因E545K突變位點設計合成特異性引物及一對野生型(FAM標記)和突變型(VIC標記)探針。2.提取各細胞系的核酸,PCR擴增后測序擴增產物,確定各細胞系包含的PIK3CA基因E545K位點的基因型。3.實時定量PCR檢測引物和探針的特異性,初步建立基于Taqman探針和DNA結合染料(EvaGreen)聯(lián)合使用的qPCR反應體系,并優(yōu)化反應體系,使三種基因型純樣本之間的qPCR終熒光信號值差別最大,從而便于對三種基因型純樣本進行鑒別區(qū)分。4.理論上qPCR反應體系可以直接應用于ddPCR,但在實際實驗中需要對體系的某些條件進行微調,然后以qPCR反應體系為基礎,應用ddPCR建立和優(yōu)化雙探針法(野生型+突變型)與野生型探針聯(lián)合EvaGreen染料的方法,用于PIK3CA基因E545K突變位點三種基因型的檢測,確保能夠從ddPCR結果圖中清晰的區(qū)分不同基因型的液滴團。5.應用上述兩種ddPCR方法對同時含有三種基因型的混合樣本進行定量檢測,分別得到三種基因型的拷貝數(shù),對三組數(shù)據(jù)分別做配對樣本均數(shù)的t檢驗,通過P值判斷兩種檢測方法所得到的結果的差異是否具有統(tǒng)計學差異,驗證方法的可靠性。結果:1.由染料法qPCR優(yōu)化體系及PCR循環(huán)條件,得到最佳退火溫度為62℃,此時體系擴增效率(amplification efficiency,E)E=0.918l。良好的擴增效率可降低ddPCR中各液滴之間因擴增效率不同導致的熒光信號差異和結果的不準確。2.采用qPCR雙探針體系分別檢測三種基因型純樣本,由擴增曲線可知探針特異性良好。3.初步建立了聯(lián)合使用野生型探針和EvaGreen染料的qPCR體系,分別對等拷貝數(shù)的三種基因型純樣本進行擴增檢測,優(yōu)化后各樣本間野生型探針(VIC)信號差值最大。4.基于ddPCR建立的雙探針法體系,經(jīng)優(yōu)化可用于檢測同時含有三種基因型的混合樣本,包含不同基因型的液滴團可清晰區(qū)分。5.基于ddPCR建立的聯(lián)合使用野生型探針和EvaGreen染料的體系,優(yōu)化后也可檢測同時包含三種基因型的混合樣本,各基因型對應的液滴團清晰可分。6.應用ddPCR的雙探針法和聯(lián)合使用野生型探針和EvaGreen染料兩種方法分別對同一組含有三種基因型的混合樣本進行檢測,分別得到三種基因型的拷貝數(shù),分別對三組樣本數(shù)據(jù)進行配對樣本均數(shù)的t檢驗。野生型組:P=0.9540.05;雜合突變型組:P=0.4510.05;純合突變型組:P=0.3080.05,說明兩種檢測方法差異無統(tǒng)計學意義。結論:成功建立了基于ddPCR聯(lián)合使用TaqMan探針和染料檢測PIK3CA基因E545K突變位點三種基因型的方法。該方法只使用野生型探針,可檢測與此野生型探針序列不匹配的任何SNP或其他類型的突變,打破了普通雙探針法只能對突變型探針所設計的已知突變位點進行檢測的局限,有望降低多突變位點檢測的成本,并用于突變的篩查。
[Abstract]:Objective: in order to detect the deficiency of the known mutation site in real-time quantitative PCR (real-time quantitative PCR, qPCR) and droplet digital PCR (droplet digital PCR, ddPCR), a ddPCR method based on the combination of Taqman probe and dye is tried to detect the unknown mutation sequence, and the method is used to detect the mutation of the mutation. Three genotypes of the loci (wild, heterozygous and homozygous mutant). Methods: This study belongs to methodological study, first to establish a method, and then to compare with the existing methods for statistical analysis, and to determine the reliability of the method.1. selection of the wild type (MCF-7 cell) and heterozygous mutant (H460 cell) cell lines containing the PIK3CA gene and the cell lines, as well as the cell lines of the heterozygous mutant (H460 cell). The homozygous mutant double stranded DNA containing the PIK3CA gene was synthesized and the specific primers were designed for the E545K mutation site of the PIK3CA gene, and a pair of wild type (FAM markers) and the mutant (VIC marker) probe.2. were used to extract nucleic acid from each cell line. PCR amplified the product after amplification and determined the genotype.3. of the PIK3CA gene E545K loci of each cell line. The specificity of the primers and probes was detected by real-time quantitative PCR. The qPCR reaction system based on the combination of Taqman probe and DNA binding dye (EvaGreen) was initially established, and the reaction system was optimized to make the maximum difference between the qPCR terminal fluorescence signal values between the three genotypes of pure samples. Thus, the.4. theory was differentiated from the pure samples of the three genotypes. The upper qPCR reaction system can be directly applied to ddPCR, but in actual experiments, some conditions of the system need to be adjusted. Then, based on the qPCR reaction system, the method of combining the double probe (wild type + mutant) and wild type probe combined with EvaGreen dye is established and optimized by ddPCR, which is used for the three genes of the E545K mutation site of the PIK3CA gene. To ensure that the two ddPCR methods were used to detect the mixed samples containing three genotypes at the same time, and the number of copies of the three genotypes was obtained, and the three sets of data were tested by the t test of the average number of paired samples, and two kinds of P values were used to judge two kinds of ddPCR. Whether the difference between the results obtained by the detection method has statistical difference, and the reliability of the method is verified. 1. the optimum annealing temperature is 62 C by the dye method qPCR optimization system and the PCR cycle condition. The good amplification efficiency of the system amplification efficiency (amplification efficiency, E) E= 0.918l. can reduce the cause of each droplet in ddPCR. The difference of the fluorescence signal and the inaccuracy of the results were detected by the different amplification efficiency. The qPCR dual probe system was used to detect three pure samples of the genotypes. The amplification curve showed that the probe specificity was good.3., and the qPCR system of the combined use of wild type probe and EvaGreen dye was preliminarily established, and the pure samples of the three genotypes of the peer-to-peer number were identified respectively. The double probe system based on ddPCR based on the maximum.4. signal difference of the wild type probe (VIC) between the samples was optimized. The optimization could be used to detect the mixed samples containing three genotypes. The droplets containing different genotypes could clearly distinguish.5. based on ddPCR and EvaGreen dyeing. The system of material can also be optimized to detect the mixed samples containing three genotypes at the same time. The corresponding droplets of each genotype can be clearly divided into two kinds of mixed samples containing three genotypes, including three kinds of genes, respectively, using the double probe method of.6. using ddPCR and the combined use of wild type probe and EvaGreen dye. The copy number of the type is t test for the average number of paired samples of three groups of samples. The wild type group: P=0.9540.05; the heterozygous mutant group: P=0.4510.05; the homozygous mutant group: P=0.3080.05, indicating that there is no statistical difference between the two detection methods. Conclusion: a successful establishment of a ddPCR combined with a TaqMan probe and a dye for the detection of the PIK3CA gene E. The method of three genotypes of the 545K mutation site. This method only uses a wild type probe to detect any SNP or other types of mutations that do not match the sequence of the wild type probe. It breaks the limitation that the common double probe can only detect the known mutation sites designed by the mutant probe, and it is expected to reduce the detection of multiple mutation sites. Ben, and for mutation screening.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R440

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