The Application Of MDA In Sex Diagnosis Of Preimplanted Embryos

The object of our study is to develop multiple displacement amplification (MDA) protocol of single cell, and then analyse 16 STR loci of the MDA products. In the second part of the study, we proceeded sex diagnosis of preimplanted embryos using MDA protocol of single blastomere.Our study is made of two parts. In the first part,we developed isothermal multiple displacement amplification protocol, and deceted its effectiveness through some measurement indeicators.First we collected single human lymphocyte by micromanipulation technique, then divided them in to four group on the basis of the number of cells,each group contains 16 samples.And then, each sample was lysed by alkaline, the whole genomic amplification by MDA was achieved using bacteriophage 29 DNA polymerase and random hexamer oligonucleotide primers in a 30℃reaction.The length of MDA products was greater than 10kb.There were no differences of amplification successful rates were found between groups.Then MDA products were analysed of 16 STR loci genotyping using fluorescent PCR.The loci amplification successful rates were higher in 5 cells group and 10 cells group than 1 cell group and 2 cells group(75%,87.5%vs 100%,100%,P<0.05).The accuracy rate of STR genotyping increased with increased cell number(79.37% vs 93.83% vs 98.91%,99.46%, P<0.05). It is demonstrated that MDA could be successfully used to amplify single lymphocyte,so we can use single cell MDA follow up fluorescent PCR protocol to detect several loci simultaneously.In the second part, we used the protocol developed in the first part to detect gender of preimplanted embryos.We removed 20 single blastomeres from 20 embryos,and proceeded whole genomic amplification using MDA,then detect SRY, AMEL, XHPRT, X22 makers using fluorescent PCR to determine sex. Blastomeres from the same embryo were proceeded fluorescent in situ hybridization(FISH) at the same time as positive control.All the 20 blastomeres were successful amplification and 19 of them achieved gender determination outcome,12 of them were male,and 8 of them were femal. Compared with FISH, the accuracy rate of fluorescent PCR was 95%.Therefore this was an efficient and accurate protocol for preimplantation gender determination using SRY,XHPRT,AMEL and X22 makers by fluorescent PCR afer multiple displacement amplification.