Establish A Method Of MDA-PCR-SBT For PGD/HLA-genotyping

Objectives:The first part is to establish the whole genome amplification technology for single blastomere with multiple displacement amplification(MDA). To yield large amounts of DNA of high quality for subsequent preimplantation HLA genotyping. In the second part we developed a new PGD protocol for HLA genotyping in preimplantation embryos by multiple displacement amplification - polymerase chain reaction- sequence based typing (MDA-PCR-SBT).Methods:1～2 blastomeres were removed from the cleavage stage embryos by micromanipulation technique on D3. We use the technology of MDA.The whole blastomere genomic amplification by MDA was achieved using bacteriophage 29 DNA polymerase and random hexamer oligonucleotide primers in a 30℃reaction. To yield large amounts of DNA of high quality for subsequent preimplantation HLA genotyping. The second part of this research was the preimplantation HLA genotyping. The alleles of HLA-A,B,DR loci were detected from the MDA product by the PCR-SSP method. Then the parents'peripheral blood samples were withdrawn and the HLA genotypes were analyzed by the same PCR-SSP protocol. Each genotype of the specific HLA region was evaluated to distinguish the segregation of each haplotype of the family members and the primary HLA matching was done between the preimplantation embryos. Data were analysed with the Statistical Program for Social Sciences (SPSS 16.0).Result:In the first part,we collected 126 cleavage stage embryos which were discarded on D3.1 or 2 blastomeres were selected in the same PCR tube. Be divided into two groups randomly:Single blastomere group, two blastomeres group.50 in Single blastomere group,46 MDA amplified successfuly,Amplification success rate is 92%.76 in two blastomeres group,73 MDA amplified Successfuly,Amplification success rate is 96.1%.The statistical test (χ2=0.944, P>0.05),there was no significant difference of the success rate of MDA amplification in the two groups. It showed that MDA can be used in the whole blastomere genomic amplification.In the second part, nine couples who donated their peripheral blood and discarded embryos voluntarily were collected in this study. There are 76 1PN,2PN,3PN discarded embryos.76 embryos were subjected to MDA and 74 were amplified successfully with the amplification rate was 97.4%.34 in single blastomere group, amplification success rate is 94.1%.40 in two blastomeres group, Amplification success rate is 100%. DQ allele dropout rate1.3%,DR allele dropout rate 0. The positive rate of MDA in group of single blastomere is 100%,DQ allele dropout rate 1.5%,DR allele dropout rate 0. The positive rate of MDA in group of two blastomere is 100%, DQ allele dropout rate 0,DR allele dropout rate 0. Recombination rate of fetal HLA is 20.2%(30/148)。The proportion of embryos HLA matching is 20.3% (15/74).25% lower than the theoretical value, the embryos used in this study may be worse for the classification and abnormal fertilized.Conclusions:1. 1～2 blastomeres were removed from the cleavage stage embryos by micromanipulation techniquesan on D3. We use the technology of MDA. Whole blastomere genomic amplification by MDA was achieved using bacteriophage 29 DNA polymerase and random hexamer oligonucleotide primers in a 30℃reaction. Establishment of MDA was the basis for our follow-up diagnosis of HLA genotypes.2. Preimplantation genetic diagnosis of HLA matching combined with or without a single gene defects in families with previous siblings requiring HLA-identical BMT with the selection and transfer of HLA-identical embryos so that the HLA-compatible baby to be born can be a donor of umbilical cord blood or the bone marrow stem cells to transplant and eventually cure the affected sibling. So the preimplantation HLA matching could be a new tool for couples desiring to conceive a potential donor progeny for transplantation in a sibling with a life-threatening disorder.