胎儿核酸检测分析方法的研究
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摘要
产前诊断一直人类遗传学领域的研究重点,目前临床上常用的诊断方法主要有羊水穿刺和绒毛膜诊断等,这些方法对产妇和胎儿均有较大的危害,而且操作相对繁琐。近年来胎儿DNA的发现为这一问题的解决提供了可能,使得无创性产前诊断不再是梦想。随后的研究证实胎儿RNA也存在于孕妇的外周血液中,这为该研究的进一步发展提供了新的途径。尽管无创性产前诊断在近年来取得了较大的进展,但仍然与临床实际应用存在着很大的差距,特别是在胎儿核酸的高灵敏度和高通量检测方面。为此,我们以不同怀孕时期的孕妇外周血液为研究对象,结合基因芯片技术,发展了胎儿DNA和RNA的高通量检测技术并且应用于胎儿相关基因的检测分析中。本论文的主要研究内容有:胎儿核酸分离纯化方法的研究;胎儿DNA的扩增检测方法的研究;胎儿RNA定量检测分析方法的研究;孕妇血浆DNA甲基化的初步研究。具体内容如下:
1.孕妇血浆核酸提取方法的研究我们以现有的核酸提取分离方法为基础,结合孕妇外周血液中胎儿核酸的特性,发展了改良的胎儿核酸提取纯化方法。我们首先利用甲醛溶液来处理孕妇的外周血液,在新鲜的孕妇血液中加入1%的甲醛溶液,处理30分钟后,离心分离孕妇血浆,然后用常规的树脂吸附法提取血浆DNA,以Y染色体特异性序列作为男性胎儿DNA的标志物,定量检测胎儿DNA的浓度。结果发现采用甲醛处理的实验组提取获得的胎儿DNA占血浆总DNA的比例有了较大的提高。我们还改良了RNA的提取方法,结果发现利用改良的“Trizol”提取法获得的胎儿RNA浓度比普通方法有了较大的提高。这些胎儿核酸的分离纯化方法为无创性产前诊断方法的发展打下了基础。
2.微乳液多重PCR技术聚合酶链式反应(PCR)主要用于DNA片断的扩增分析,是生物医学领域一项重要的核酸分析技术。尽管如此,PCR在检测灵敏度、特异性方面仍存在一些问题,多重PCR扩增也因引物等成分之间的干扰而受到了较大的限制。微乳液(油包水)是一种水相均匀的分散到油相中形成的稳定均一的分散系,近年来有人利用微乳液的特殊的结构进行生物化学反应获得了一定的成功。我们通过优化微乳液制备方法和PCR反应体系,成功地在微乳液中进行了PCR扩增。实验结果表明,微乳液体系中的微小液滴结构不但能够提高PCR扩增的效率,而且能够有效的降低多重PCR体系中各个成分特别是引物之间的干扰,从而使多重PCR能够更加有效的进行。我们利用该技术进行了Y染色体微缺失的检测,在不育的男性样品和怀有男性胎儿的孕妇外周血液中我们均检测到了微缺失位点的存在。结果显示微乳液多重PCR技术不但具有很高的灵敏度和特异性,而且能够用于相关疾病的分析检测,可以检测孕妇外周血液中胎儿DNA用于无创性产前诊断,大大推动了产前诊断技术的进一步发展。
Noninvasive prenatal diagnosis is a long-sought goal in human genetics. Recent interest in cell-free DNA in plasma and serum has led to the discovery of fetal DNA in maternal plasma. The presence of fetal DNA in maternal plasma made noninvasive prenatal diagnosis possible, and this approach avoids the risks associated with conventional invasive techniques, such as amniocentesis and chorionic-villus sampling. Fetal RNAs were also found in maternal plasma and serum in subsequently researches, this provided new approach for non invasive prenatal diagnosis. Many progresses have been made in the research of fetal nucleotide acids in maternal plasma and serum. However, most techniques are expensive and time-consuming or technically demanding. An inexpensive technique which is sensitive and high throughput is needed for noninvasive prenatal diagnosis. In this thesis, we developed several microarray based methods which could be used for the detection of fetal nucleotide acids in maternal plasma and serum. It has been showed great potential for clinical researches and diagnosis. The main works during my Ph.D. study are as follows:
1. Modified methods for extraction and purification of fetal nucleic acids in maternal plasma In order to obtain high quantity and quality fetal nucleic acids from plasma of pregnant women, we modify the methods for extraction and purification of fetal nucleotide acids. 1% formaldehyde solution was added in peripheral blood of pregnant women for about 30 min, then the plasma was separated and DNA was extracted, and real-time PCR was carried out to measure it. Fetal RNA in maternal plasma was also extracted with modified“TRIZOL”method and measured with real-time PCR. Results showed that treatment with formaldehyde could increase the proportion of fetal DNAs in total plasma DNA. Fetal RNA extracted from maternal plasma was also increased greatly with our modified“TRIZOL”method. It will benefit the future noninvasive prenatal diagnosis.
2. Microemulsions Multiplex Polymerase Chain Reaction PCR is a very important tool in molecular biology researches. However, it has less the ability of parallel detection. We developed a method to carry out PCR and multiplex PCR in microemulsions. Microemulsion is used as the reaction media to avoid the interference of primers and templates. Y chromosome micro-deletions were detected. 13 STS sites in Y chromosome related with micro-deletions and SRY were selected for simultaneous amplification. All sites were successfully amplified by microemulsion multiplex PCR. Results showed that the PCR in micoemulsion obtained higher sensitivity and specificity; and multiplex PCR is sensitive, efficient and reliable for multiplex gene amplification, which has great potentials in practical applications of both clinical researches and diagnosis.
3. Detection of fetal DNA in maternal plasma by microarray based method Recently, fetal DNA has been used in several genetic disease diagnoses, many challenges still remained in the detection methods. In order to set up a sensitive and reliable method for fetal DNA
1.孕妇血浆核酸提取方法的研究我们以现有的核酸提取分离方法为基础,结合孕妇外周血液中胎儿核酸的特性,发展了改良的胎儿核酸提取纯化方法。我们首先利用甲醛溶液来处理孕妇的外周血液,在新鲜的孕妇血液中加入1%的甲醛溶液,处理30分钟后,离心分离孕妇血浆,然后用常规的树脂吸附法提取血浆DNA,以Y染色体特异性序列作为男性胎儿DNA的标志物,定量检测胎儿DNA的浓度。结果发现采用甲醛处理的实验组提取获得的胎儿DNA占血浆总DNA的比例有了较大的提高。我们还改良了RNA的提取方法,结果发现利用改良的“Trizol”提取法获得的胎儿RNA浓度比普通方法有了较大的提高。这些胎儿核酸的分离纯化方法为无创性产前诊断方法的发展打下了基础。
2.微乳液多重PCR技术聚合酶链式反应(PCR)主要用于DNA片断的扩增分析,是生物医学领域一项重要的核酸分析技术。尽管如此,PCR在检测灵敏度、特异性方面仍存在一些问题,多重PCR扩增也因引物等成分之间的干扰而受到了较大的限制。微乳液(油包水)是一种水相均匀的分散到油相中形成的稳定均一的分散系,近年来有人利用微乳液的特殊的结构进行生物化学反应获得了一定的成功。我们通过优化微乳液制备方法和PCR反应体系,成功地在微乳液中进行了PCR扩增。实验结果表明,微乳液体系中的微小液滴结构不但能够提高PCR扩增的效率,而且能够有效的降低多重PCR体系中各个成分特别是引物之间的干扰,从而使多重PCR能够更加有效的进行。我们利用该技术进行了Y染色体微缺失的检测,在不育的男性样品和怀有男性胎儿的孕妇外周血液中我们均检测到了微缺失位点的存在。结果显示微乳液多重PCR技术不但具有很高的灵敏度和特异性,而且能够用于相关疾病的分析检测,可以检测孕妇外周血液中胎儿DNA用于无创性产前诊断,大大推动了产前诊断技术的进一步发展。
Noninvasive prenatal diagnosis is a long-sought goal in human genetics. Recent interest in cell-free DNA in plasma and serum has led to the discovery of fetal DNA in maternal plasma. The presence of fetal DNA in maternal plasma made noninvasive prenatal diagnosis possible, and this approach avoids the risks associated with conventional invasive techniques, such as amniocentesis and chorionic-villus sampling. Fetal RNAs were also found in maternal plasma and serum in subsequently researches, this provided new approach for non invasive prenatal diagnosis. Many progresses have been made in the research of fetal nucleotide acids in maternal plasma and serum. However, most techniques are expensive and time-consuming or technically demanding. An inexpensive technique which is sensitive and high throughput is needed for noninvasive prenatal diagnosis. In this thesis, we developed several microarray based methods which could be used for the detection of fetal nucleotide acids in maternal plasma and serum. It has been showed great potential for clinical researches and diagnosis. The main works during my Ph.D. study are as follows:
1. Modified methods for extraction and purification of fetal nucleic acids in maternal plasma In order to obtain high quantity and quality fetal nucleic acids from plasma of pregnant women, we modify the methods for extraction and purification of fetal nucleotide acids. 1% formaldehyde solution was added in peripheral blood of pregnant women for about 30 min, then the plasma was separated and DNA was extracted, and real-time PCR was carried out to measure it. Fetal RNA in maternal plasma was also extracted with modified“TRIZOL”method and measured with real-time PCR. Results showed that treatment with formaldehyde could increase the proportion of fetal DNAs in total plasma DNA. Fetal RNA extracted from maternal plasma was also increased greatly with our modified“TRIZOL”method. It will benefit the future noninvasive prenatal diagnosis.
2. Microemulsions Multiplex Polymerase Chain Reaction PCR is a very important tool in molecular biology researches. However, it has less the ability of parallel detection. We developed a method to carry out PCR and multiplex PCR in microemulsions. Microemulsion is used as the reaction media to avoid the interference of primers and templates. Y chromosome micro-deletions were detected. 13 STS sites in Y chromosome related with micro-deletions and SRY were selected for simultaneous amplification. All sites were successfully amplified by microemulsion multiplex PCR. Results showed that the PCR in micoemulsion obtained higher sensitivity and specificity; and multiplex PCR is sensitive, efficient and reliable for multiplex gene amplification, which has great potentials in practical applications of both clinical researches and diagnosis.
3. Detection of fetal DNA in maternal plasma by microarray based method Recently, fetal DNA has been used in several genetic disease diagnoses, many challenges still remained in the detection methods. In order to set up a sensitive and reliable method for fetal DNA
引文
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