Use Of Free Fetal DNA In Maternal Plasma To Noninvasive Prenatal Diagnosis β-thalassemia Methods Research

Background and objective:Prenatal diagnosis of genetic diseases commonly used techniques such as placentalvilli biopsy are invasive operation. Pregnant women and fetuses have a certain traumatic,resulting in practical and clinical application is limited. Rather than invasive prenatalscreening methods, such as serological (serum HCG, AFP quantitative) and ultrasound todetect more limited scope for genetic disease screening only as a way, not as a diagnostic tosolve the problems of patients. In recent years, studies of free fetal DNA with the deepeningof noninvasive prenatal diagnostic methods to make substantial progress, the detection offetal sex or RhD gene from maternal plasma, has been carried out in a number of prenataldiagnostic centers abroad; Using genome-wide high-throughput sequencing and digital PCRmethod has diagnosis part of chromosome abnormality and some single-gene disorders.However, the above techniques most prominent problem is the method of high technologyand cost requirements. Once the low cost and high yield program is established, prenataldiagnosis of fetal genetic disorders from maternal plasma will also be widely used inclinical practice.β-thalassemia is a mutation of β-globin gene lead to reduced β-globin chain synthesisor caused by a lack of monogenic autosomal recessive genetic disease (referred toβ-thalassemia), is a beta globin gene single nucleotide mutation and missing pieces as themain molecular basis. With the disease for the study, to build high specificity and highsensitivity can be used for low-abundance gene single nucleotide mutations and smalldeletions detection technology, is seeking a new simple low cost method for detect fetalmonogenic hereditary disease in maternal plasma.In2004，Lo in Lancet reported application of allelic specific fluorescence quantitativePCR technique to detect beta thalassaemia CD41-42fragment missing mutations(del-CTTT). But as a result of allelic specific PCR detection of low abundance mutations exist when sensitivity is not high, at present has not been applied to clinical. Professor Lifound that having3’�'5’ exonuclease activity of the polymerase fidelity identifiableprimer3’end of the upstream one to several mismatched bases, if the primer and templatematching completely, you can direct the synthesis. If the primer to the template matching isnot match,it will be send into the enzyme solution center correction, the corrected part maycontinue primers synthesized,portion can not be corrected in primer synthesis reaction isterminated. Binding to the3’end of the end of the sulfide modified base-specific primers,may constitute sensitive mutation "molecular switch." This research has been applied to thepart of the gene mutation and SNP detection. But it is still not used in the detection of lowabundance gene mutations.According to this research base, we will combine it withreal-time quantitative PCR, for the detection of trace deletion mutations, can be used todetect fetal DNA in maternal plasma.In our previous study, we used PCR/LDR combination of capillary electrophoresistechnology for fetal DNA in plasma experimental model for testing, found that thetechnology for low-abundance gene mutation detection sensitivity can be achieved1:10000,preliminary evidence that the technique can meet in maternal plasma fetal DNA pointmutation detection. But in this experiment, it is need to purified the PCR product, then，usethe purified product to the next Ligase detection reaction. It is easily contaminated duringthe purification process directly affects the accuracy of the experimental results. Therefore,a method for detecting point mutations in this study need to be simplified based onpreliminary experiments improved basis, intends PCR products were directly applied to theline after capillary electrophoresis LDR reaction to simplify procedures for the purpose ofreducing pollution.This study aims to use simple techniques to detect fetal maternal plasma paternalβ-thalassemia mutation and deletion mutation for noninvasive prenatal diagnosis of fetalmonogenic hereditary diseases to seek a new detection method.Materials and methods:1. The DNA polymerase has no3’-5’ circumscribed enzyme activity combine withcommon primers or the DNA polymerase has3’-5’ circumscribed enzyme activity combinewith Sulfide modified primers Amplified β-thalassemia CD41-42(del-CTTT) mutation inthe gene fragment Genomic extraction, primer annealing temperature test, the amplified fragment was cutplastic recycling sequencing.2. The DNA polymerase has3’-5’ circumscribed enzyme activity combine withSulfide modified primers constitute a "molecular switch". The "molecular switch" combinesreal-time quantitative PCR used to detectβ-thalassemia CD41-42(del-CTTT) mutation inthe gene fragment.The CD41-42(del-CTTT) heterozygous mutations in the genome DNA diluted intodifferent concentration gradient to amplify, then after amplification for detection. Normalgenomic DNA as a negative control.3. Combined with real-time quantitative PCR from maternal plasma to detectRASSF1A as targets to prove the existence of cell-free fetal DNA.Collected maternal peripheral blood5ml.Extracted DNA in maternal plasma afterplasma preparation,then use restriction enzyme Hinp1I and HhaI to digest unmethylatedsequences. Real-time quantitative PCR detect RASSF1A as targets to prove the existence ofcell-free fetal DNA.4. Improved methods of PCR/LDR/capillary electrophoresis to detect trace amountsof single nucleotide mutations4.1Extract human genome DNA for amplification, then send the products to companysequencing confirmed that the mutant gene types.With the different concentrations ofnormal peripheral blood DNA mixed with20pg mutant hybrid DNA as template for PCRamplification, normal peripheral blood DNA as the negative control. PCR/LDR/capillaryelectrophoresis to detect trace amounts of single nucleotide mutations and calculate thesensitivity.4.2Trace single nucleotide mutation detection from maternal plasmaThe20pg single nucleotide mutation heterozygous peripheral blood DNA was added innormal maternal plasma DNA for amplification,、electrophoresis of PCR products and LDRdetection reaction.Results:1. The DNA polymerase has no3’-5’ circumscribed enzyme activity combine withcommon primers visible PCR amplification can’t interpretation normal gene and CD41-42(del-CTTT) hybrid mutation; the DNA polymerase has3’-5’ circumscribed enzyme activity combine with Sulfide modified primers Amplified β-thalassemia CD41-42(del-CTTT) fragment that can interpretation normal gene and CD41-42(del-CTTT) hybridmutation until the annealing temperature to67.8℃.2. The "molecular switch" combines real-time quantitative PCR used to detectβ-thalassemia CD41-42(del-CTTT) mutation in the gene fragment. The beta thalassaemiaCD41-42(del-CTTT) heterozygous mutations in each genome DNA concentrationgradient can detect the fluorescence;300ng normal genomic DNA can detect thefluorescence, the rest of the concentration gradient has not been detected fluorescence.3. With genetic analyzer electrophoresis results of original，the detection sensitivitycan reach1:5000.Product peak area increased with the increase of normal peripheral bloodDNA concentration gradient decreases. To1:10000can not distinguish between peak andmiscellaneous,and no mutation in the amplified product was not detected in the negativecontrol LDR product.4. Normal maternal plasma DNA added20pg heterozygous mutation in peripheralblood DNA amplification products of LDR detection reaction results, product peaks can beclearly distinguished peaks and miscellaneous.Conclusions:1.The assay "molecular switch" combines real-time quantitative PCR will be expectedfor fetal DNA paternal deletion mutation detection in maternal plasma;2. New improved PCR/LDR/capillary electrophoresis technology is expected toprenatal diagnosis of fetal beta thalassaemia single nucleotide mutation.