Research On Methods For The Detection And Analysis Of Fetal Nucleic Acids

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...