迅速･簡便に網羅的コピー数解析を可能とする全ゲノム増幅を用いた着床前診断法

抄録

Preimplantation genetic diagnosis (PGD) is an established procedure of embryo genetic analysis, allows couples carrying monogenetic diseases to have an unaffected child, without invasive prenatal diagnosis and termination of pregnancy. PGD is performed with nested PCR, involves two sets of primers, used in two successive runs of polymerase chain reaction, the second set intended to amplify a secondary target within the first run product. The scarcity of DNA is a limiting factor in many reseach fields. Multilocus simultaneous analyses from single cell is difficult. DNA microarrays manufactured to date are not able to analyze limited amount of DNA in a single cell. Development of a new gene amplification method which replaces PCR is desirable. Multiple displacement amplification method (MDA) is a non-PCR based whole genome amplification (WGA). We aimed to construct the PGD system using MDA especially for Duchenne muscular dystrophy (DMD) and to examine the presence of bias of amplification and the efficiency rate of amplification. The dilution samples were made with the extracted DNA from the lymphocytes of normal male, DMD patient and carrier, and used for the template of MDA. The amplification by MDA in all nine exons were confirmed from the result of following singleplex PCR and multiplex PCR using multiple primer of Chamberlain. As a result of the gene analyses by using realtime PCR with fluorescence image of TaqMan Probe, the agreement rate of the diagnosis using with 1ng and 100pg of template was 100%(14/14) and 86%(12/14), respectively. The agreement rate of the diagnosis with 10pg of template DNA was 71%(10/14). The MLPA techique was performed for the MDA products using exon-specific probes for the dystrophin gene and amplification of all 79 exons were examined. The amplification were confirmed in almost all exons when 100 pg of DNA was used as the template. Thirty out of 79 exons were not amplified when 10 pg of DNA was used. The bias of relative copy numbers calculated was examined. From the results, the combination of MDA and MLPA methods has not not been effective in analysis of quantification for the duplicated mutation, but effective for simultaneous qualitative analysis of the dystrophin gene. The strong potential of Genome-wide amplification by MDA has been demonstrated for PGD in case of more than 100 pg of template DNA.