Utilizing Duplex Nested Polymerase Chain Reaction Assay And Multiple Displacement Amplification For Sex-linked Hereditary Desease On Single Cell

Objectives: 1.To develop a simple, quick and highly accurate method to determine sex on single cell level in order to provide a theory or method basis for noninvasive prenatal diagnosis of single gene inheritance disease and preimplantation genetic diagnosis(PGD),and to prepare to build single cell PCR technique platform in our centre. 2.To develop one of whole genome amplification(WGA)--multiple displacement amplification(MDA)on single cell and single blastomere retrieve the shortage of template .This study is to set up a technique of single blastomere PCR, and to p rovide a basis for preimplantation genetic diagnosis.METHODS: 1.We extracted a total of 150 single lymphocytes from peripheral blood of male and female donors respectively, which were randomly divided into three groups including 50 male and female lymphocytes. Then we developed a duplex nested polymerase chain reaction assay to co-amplify Steroid sulfatase gene/Y-Pseudogene and Amelogenin homologous sequences on X and Y chromosome, the amplification efficiency and accuracy was then compared to that of the single nested polymerase chain reaction assay.2.We developed multiple displacement amplification to amplify whole genome of 5 single lymphocytes and 10 single blasomeres.1% agarose electrophoresis detect amplification efficiency of MDA.Regular PCR for STS and Alphoid detect homogenicity of MDA product. Results :1.By the aid of simultaneous amplification of STS and AMEL homologous sequences, the amplification efficiency and accuracy of sex determination was 98% and 96% respectively. Compared to that of the only amplification of steroid sulfatase gene/Y-Pseudogene(84%,76%) or Amelogenin (84%,84%) homologous sequences respectively, a significantly increase in the amplification efficiency and accuracy was found, the differentiation was statistically significant(P<0.05,P<0.01).2.All 5 single lymphocytes (including three male cells and two femal cells)are amplified successfully,the rate of amplification is 100%;the rate of accuracy is 100% by detecting STS second round sequence.The four out of ten blastomeres are successfully amplified,the rate of amplification is 40%;the product of all amplified successfully blastomere are of homogenicity because two male and two female blastomeres have been detected with STS second round primer.unfortunately,the four blastomeres are from four defferent embryos,they can not check each other.But we can judge that MDA product is homogenicity on the basis of detecting STS.CONCLUSION:1.For the application of sex determination on the single cell level,the duplex nested polymerase chain reaction might possess higher amplification efficiency and accuracy than that of single nested polymerase chain reaction. Besides,it is also helpful to find the phenomenon of allele drop-out(ADO) and amplification failure.Duplex nested polymerase chain reaction assay has considerable application value in noninvasive prenatal diagnosis of single gene inheritance disease and preimplantation genetic diagnosis (PGD).It deserves to be further popularized clinically. 2.Preliminary experiment tell us MDA can make up the shortage of small template of single cell and only being used once.But its stability and confidence need be sovled before it is used in noninvasive prenatal diagnosis of single gene inheritance disease and pre-implantation genetic diagnosis (PGD).