The Study Of Detection On β-thalassemia By Chip-based Capillary Electrophoresis And Noninvasive Prenatal Diagnosis

Thalassemia is the commonest genetic disorder worldwide.There is high incidence rate in southern china.The homozygous state is associated with high morbidity and mortality,thus screening of at-risk pregnancies and prenatal testing are stongly advocated. Fetal cells and cell free fetal DNA in the maternal circulation are found before 10 years which can be used for noninvasive prenatal diagnosis on fetal sex,21 trimsome and RhD, but it was not applied widely,partly because of less sample and complex technology.The diagnostic reliability of circulating DNA analysis depends on the fractional concentration of the targeted DNA,the analytical sensitivity,and the specificity.The discrimination of single-nucleotide difference between circulating DNA samples is technically challenging and demands the adoption of highly sensitive and specific analytical systems.Chip-based capillary electrophoresis can detect a little sample speedily and conveniently.This technique may be useed for prenatal diagnosis,preimplantation genetic diagnosis which sample is precious and less.It could resolve many problems which the gene detection was slow,tardiness,much sample to need which is not fit for prenatal diagnosis.Objectives:We construct the technique platform of detection the point mutation by the chip-based capillary electrophoresis.It can be used to detect other genetic diseases in clinic as a celerity and sensitive means.In our study we should do as follows:1) For sample preparation we should construct the clones of the mutation genes ofβ-thalassemia which is important for the experiment.The steady detection system of primer extension is to be accomplished.By the gel to be detected the wildness and mutation genotype of sample it was be proved that the primers can discriminate the different of mutation types sensitively and specially.2) Multiplex primer entension reaction:we amplify six loci through multiplex primer entension reaction and detect the six genotypes simultaneity by sensitive HPLC. 3) Detection theβ-thalassemia by chip-based capillary electrophoresis:we construct the technical platform to detect mutation by chip-based capillary electrophoresis and progress the ability to differentiate.In the end we should accomplish the rapid prenatal diagnosis ofβ-thalassemia by chip-based capillary electrophoresis.Mehods:our experiment involves three parts as follows:1) The construction of gene mutation clone and primer extension reactionWe construct the clone of nomalβ-globin gene and six clones ofβ-thalassemia mutation genes:CD28,CD17,CD26,CD41,CD71,IVS654 by site-directed mutagenesis technique which were be proved by sequencing.The primers were designed whose anneal temperature and length is close each other and primer dimmers don't been produced so that the multiplex can be successful.We study the conditions which may influence the detecting system.Many factors have been studied include reaction of procedure,the concentration of primers,the sequence of primers,the conctration of Mg ion,artificial mismatch primer and so on.Detected by gel the primers were proved to be special and sensitive.In order to differentiate the wildness and mutation genotype we added ten bases of T at the location of primer 5' end so that they can be differentiated by chip-based capillary electrophoresis.2) In the second part multiplex primer extention reaction were operated to amplify the six point mutation simultaneously.The products of primer extention action were detected by HPLC.3) The third parts were to construct the technical platform to detect the different length PCR product by chip-based capillary electrophoresis.The condition of multiplex primer entension experiments were designed to detect sixβ-globin gene mutation loci simultaneously.We study the raw materials made of capillary electrophoresis chips.To improve the ability to differentiate we studied the concentration of denaturalization gel and the procedure.Through detection of STR standard sample we preliminarily identified the capility of chip-based capillary electrophoresis.We can detect the ladder and samples ofβ-thalassemia simultaneously by the two channels labeled with different fluorescence. Through comparing the ladder with sample we could discern the genotype of the detected sample.All sample including the clone and patient's blood were detected by this system. The detection systems were validated by the previous methods.At last we processed prenatal dianosis for three families in which parent wereβ-thalassemia patients or carriers. The fetal samples were collected from the amniotic fluid,cord blood and maternal plasma fetal DNA.We compared the kit detection method and chip-based capillary electrophoresis.Results:We constructed the clones ofβ-thalassemia mutation gene by site-directed mutagenesis technique.The sequences of clones were accoding with publicized date completely at NCBI.The primers designed can work and no primer dimmer produced.In the procedure of multiplex primer extension action the three temperature cycle was better than single temperature cycle which primers can work well.In the second temperature cycle 67℃was fit to detect without any false positive results,moreover the primer extionsion were influenced by the concentration of iron and primer and the sequences of primers. Artifical mismatch primers were used on experiment to differentiate the wild and mutation primers and cut down the nonspecia produce.The ten bases T were added to 5' end of the reverse loci specific primers to lengthen PCR product of wild primer and don't influence anneal temperature and other primers.Through the preliminary detection of clinical sample the primers were be proved to be special and sensitive.We establish the steady system to detectβ-thalassemia by gel.The result of detection was according with sequencing of the patients' sample.This method is easy,convenient,common material used,less cost to operate.So it may be useful for the convenient detection ofβ-thalassemia and reducing the cost of gene detection.The result was according with the sequencing and previous result.Through the multiplex primer extension six loci were amplificated and detected.The PCR products were detected by HPLC which can differentiate these six loci.We constructed the technique platform of chip-based capillary electrophoresis to detect the point mutation.We utilized the polymer substrates PMMA which is less expensive and easy to make.An improvement of this chip-based electrophoresis detector from conventional confocal setup was to use a hole reflecting mirror instead of a dichroic mirror, which could pass through both the exciting and reflected lasers while reflect most of the emitted fluorescences to the collecting pathway.This setup woμld significantly eliminate the impact of reflected lasers on the fluorescence detection.Another feature of this detector was that lasers were focused onto a very small spot(diameter of 5μm) by the objective. Two advantages could be resulted from this feature.One is that with the smaller focused spot,higher intensity of laser could be obtained,causing higher exciting efficiency of fluorescence.The other advantage is that the focused spot is much smaller than the width of separation channel(60μm),thus avoiding the illumination of rough side walls,which would cause scattering of lasers.Both of the two advantages would increase the sensitivity. The microvolumetr characteristics of chip-based capillary electrophoresis provide obvious advantages over slab-gel electrophoresis and capillary electrophoresis for biomedical and clinical applications.We sudied and optimizied the concentrations of denaturalization gels. The 3%LPA gel was used to detect with the best results which could differentiate the discrepancy of 4bp between the 100bp to 200 and 300-400bp and 10bp between 100-600bp. We detected size ladders and samples simultaneously by a dual-channel detection system which labeled respectively with Cy3 and Cy5 fluorescence so as to improve the detection precision.The work exploited the efficient separation of amplified DNA and differentiated the wide and mutation genotype by PCR products length.The genotype including homozygous wild type,heterozygous variant,and homozygous variant can be known.Sensitivity study show the detection system of the chip-based capillary electrophoresis could detect 0.5ng genomic DNA sample(0.5ng/μl).Chip-based capillary electrophoresis allowed the analysis time for the genotypes to be decreased to 200 sec.The high sensitivity is important for prenatal diagnosis in which the samples are always precious and a little, especially in early pregnancies.We utilized the chip-based capillary electrophoresis and developed rapid assays for prenatal diagnosis ofβ-thalassemia.We processed the prenatal diagnosis on three cases by the amniotic or a umbilical blood sample and maternal plasma fetal DNA.The results of chip-based capillary electrophoresis are according with that of the previous method and sequencing.Fetal genotypes were predicted correctly in all cases studied.The microvolume characteristics of CE provide obvious advantages for biomedical and clinical applications.Conclusion:1) we finished the experiments form the construction,primer extention reaction and detection.The clones of mutation gene were a suitable method for preparation the mutation samples.The study of factors inflenced the primer extension reacton would be based for quickly constrcting the reaction condition.We construct a suit of method to study and detect the point mutation which can be used to other diseases related to gene mutation. moreover we constructed the convenient method to detectβ-thalassemia by gel which can be used at common hospital.2) We constructed the platform of chip-based capillary electrophoresis to detect the point mutation and processed prenatal diagnosis ofβ-thalassemia quickly by maternal plasma fetal DNA.This detection system of chip-based capillary electrophoresis could be used to noninvasive prenatal diagnosis ofβ-thalassemia. It woμld facilitate prenatal diagnosis of genetic disorder rapidly and sensitively.3) we compared the three methods including agarose gel,HPLC and chip-based capillary electrophoresis.The results show chip-based capillary electrophoresis is a quick,sensitive detection instrument with stronger discrimination power.