本文選題：深度測序 + 單細(xì)胞��； 參考：《中國人民解放軍醫(yī)學(xué)院》2013年碩士論文

【摘要】：【背景】非整倍體是最常見的染色體異常，可導(dǎo)致胚胎停育與自發(fā)流產(chǎn)，是限制試管嬰兒成功率的重要原因之一。胚胎植入前遺傳學(xué)篩查（preimplatationgenetic screening，，PGS）是篩選染色體數(shù)目和結(jié)構(gòu)正常的胚胎進(jìn)行移植，以此期望提高移植成功率與妊娠率。目前PGS技術(shù)主要包括:原位熒光雜交技術(shù)、比較基因組雜交以及微陣列比較基因組雜交和單核苷酸多態(tài)性芯片技術(shù)等，但各項技術(shù)均存在缺點。為了尋找一種更快速、全面檢測非整倍體的方法，我們以二代測序技術(shù)為基礎(chǔ)，嘗試一種新方法完成對單細(xì)胞21-三體的診斷，從而建立該技術(shù)平臺為后續(xù)研究奠定基礎(chǔ)。 【目的】初步建立深度測序技術(shù)在單細(xì)胞水平診斷21三體的技術(shù)平臺 【方法】采用深度測序技術(shù)對正常女性外周血DNA進(jìn)行檢測，評估該方法檢測的穩(wěn)定性。對不同濃度DNA的21-三體樣本應(yīng)用多重置換擴(kuò)增方法進(jìn)行全基因組擴(kuò)增，對其產(chǎn)物進(jìn)行深度測序，評估該方法對21-三體診斷的可行性。利用深度測序技術(shù)對單細(xì)胞21-三體樣本進(jìn)行檢測，初步建立深度測序方法檢測單細(xì)胞-21三體的技術(shù)平臺。 【結(jié)果】1.采用深度測序方法對14例樣本進(jìn)行檢測，各染色體標(biāo)準(zhǔn)差均處于低水平，其中最大標(biāo)準(zhǔn)差為0.078。21-三體樣本21號染色體拷貝數(shù)是正常樣本21號染色體拷貝數(shù)的1.5倍，其余染色體與正常樣本相比未發(fā)現(xiàn)拷貝數(shù)的明顯異常。3. cut off值0.5、1、1.5分別代表單倍體、二倍體和三倍體，21-三體樣本測序結(jié)果顯示21號染色體cut off均值為1.586，其99%可信區(qū)間為[1.537,1.635]。采用深度測序方法檢測單細(xì)胞21-三體具有可行性。 【結(jié)論】1.采用深度測序方法檢測正常女性DNA具有穩(wěn)定性。2.通過對不同濃度DNA模板擴(kuò)增產(chǎn)物進(jìn)行深度測序，證實該檢測方法對診斷21-三體具有可行性。3.深度測序方法檢測單細(xì)胞-21三體具有可行性。
[Abstract]:Background: aneuploidy is one of the most common chromosomal abnormalities, which can lead to aborted embryo and spontaneous abortion, which is one of the important reasons to limit the success rate of IVF.Preimplantation genetic screening (PGSs) is a method of screening embryos with normal chromosome number and structure in order to improve the success rate and pregnancy rate.At present, PGS technology mainly includes: in situ fluorescence hybridization, comparative genomic hybridization, microarray comparative genomic hybridization and single nucleotide polymorphism chip technology.In order to find a more rapid and comprehensive method for the detection of aneuploidy, we try a new method to complete the diagnosis of single-cell 21-trisomy based on the second-generation sequencing technique, and establish the platform for further research.[objective] to establish a technique platform for the diagnosis of trisomy 21 at single cell level by deep sequencing.[methods] the DNA in peripheral blood of normal women was detected by deep sequencing and the stability of this method was evaluated.The whole genome of 21- trisomy samples with different concentrations of DNA was amplified by multiplex replacement amplification method, and its products were deeply sequenced to evaluate the feasibility of this method in the diagnosis of 21-trisomy.The technique platform of detecting single cell trisomy 21 was established by using the deep sequencing technique to detect the single cell 21 trisomy.[result] 1.14 samples were detected by deep sequencing. The standard deviation of each chromosome was low, and the maximum standard deviation was 0.078.21- chromosome 21 copy number of trisomy sample was 1.5 times that of normal sample.There was no obvious abnormality of copy number in the other chromosomes. The cut off value of 0.5 ~ 1. 5 represented haploid, diploid and triploid trisomy 21, respectively. The results of sequencing showed that the mean value of cut off on chromosome 21 was 1.586, and its 99% confidence interval was [1.537 鹵1.635].It is feasible to detect single cell 21-trisomy by deep sequencing.[conclusion] 1.The method of deep sequencing was used to detect the stability of DNA in normal women. 2. 2.By deep sequencing of DNA template amplification products with different concentrations, it is proved that this method is feasible for the diagnosis of 21 trisomy. 3.It is feasible to detect trisomy-21 by deep sequencing.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R394;R3416

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