Using SNPS Sites On The Non-Invasive Prenatal Diagnosis Of Down's Syndrome

In order to evaluate the feasibility by using the SNPS(single-nucleotidepolymorphism)sites on 21st chromosome on the Non-Invasive Prenatal Diagnosis ofDown's Syndrome. Establish a rapid, accurate, convenient diagnosis method for Down'sSyndrome, which is non-invasive on Prenatal and suitable for spread to clinical. we usedifferent experimental methods in multiple SNPS on 21st chromosome, in order to achievethe purpose of diagnostic down's syndrome. This study includes the following two parts:Part 1Choose DNA-SNPS sites using the technic based on heteroduplex dna generatedin single-tube PCR - DHPLC to detect down's syndromePurpose: To establish a technic based on heteroduplex dna generated in single- tubePCR - DHPLC(denaturing high performance liquid chromatography),For detecting thenumber of 21st chromosome.Method: According to the base sequencesof target SNPS up and down design PCRprimers.Optimizing the conditions of PCR and DHPLC. According to the peak of DHPLCto checkout the number of 21st chromosome. If the four DHPLC chromatogram peakheight ratio of 1:1:1:1 is the diploid,the ratio of 2:2:4:1 or 2:2:1:4is the diploid;Results: Using the technic of PCR-DHPLC test 27 cases of DNA samples of knowngenotype(14 cases of normal specimens, 13 cases of Down syndrome samples) to detectthe number of chromosome 21. Except 3 cases of Down syndrome in the samples can notmake a diagnosis,which target SNPs are homozygous .Others were consistent with theknown genotypes.Conclusion:Using the single-tube PCR-DHPLC technic based on heteroduplex dnagenerated to detect the number of chromosome 21.This method is accurate, simple, rapidand excellent repeatability, but for the samples which target SNPS site is homozygous can't make a diagnosis. It still exists limitations.ParPart 2Use the technic of MLPA—AB3130 test polymorphism of PLAC4-SNPs , evaluatethe feasibility by using the PLAC4-SNPS on the Non-Invasive Prenatal Diagnosis ofDown's Syndrome.Objective:Application of multiplex ligation dependent probe amplification (multiplexligation-dependent probe amplification, MLPA) United AB3130 (high-pressure capillaryelectrophoresis) detected multiple SNPs on the gene of PLAC4 on 21st chromosome ,. tostudy the polymorphism. Evaluate the feasibility by using the PLAC4-SNPS on theNon-Invasive Prenatal Diagnosis of Down's Syndrome.Method: Search the gene base sequence of 94 SNPs on the gene of PLAC4,select 13SNPs,and according to the base sequence synthesis probes. Using the technic ofMLPA-AB3130 test 100 case nomer DNA samples , with normal karyotype analysis, tostudy the polymorphism situation.For each target SNP site, the polyacrylamide gelelectrophoresis in the event of two peaks is the heterozygous, appear single peak is thehomozygous.Results: Using the technic test 100case samples of normal DNA, The geneRs4818219,Rs8130833,Rs59066201,Rs9977003 and Rs3804026 have higherheterozygosity in13 SNPs , 0.43; 0.41; 0.38; 0.29; 0.18. Can be further used the studyabout noninvasive prenatal detection of Down syndrome whith the technic of CffmRNA→cDNA→MLPA→AB3130.Conclusion: Although this study was selected in the presence of normal DNAsamples regional, and small sample size.However, the results of our study provides thebasis for the foundation of noninvasive prenatal diagnosis use CffmRNA-SNPs. Because,according to Lo and others theory,if there are 5 target SNPs,as long as there is oneheterozygote we can make a diagnosis of the trisomy 21sample.Therefore,make placentalexpressed mRNA in the maternal blood PLAC4 gene reverse transcription for cDNA, andthen applied to noninvasive prenatal diagnosis of Down syndrome use the technic of MLPA-AB3130 detect SNPs,with the feasibility and application prospect.