利用Linker adaptor 與MDA 兩種整體染色體放大的技術比較單一細胞基因組倍增的效率

Abstract

Preimplantation genetic diagnosis (PGD) of single gene disorders relies on PCR-based tests performed on single cells (polar bodies or blastomeres). Single cell PCR protocols are subject to serious difficulties, including contamination, amplification failure, and preferential amplification or the complete absence of one allele (allele dropout, ADO) in heterozygous loci, but it remains the only tool for detecting specific mutation in PGD. The DNA content of single cell was limited. Different whole genome amplification (WGA) techniques have been developed to increase the DNA quantities from clinical samples with limited DNA contents. Therefore, the utilization of genomic amplification methods will be a useful tool in single-cell genetic diagnosis. In this study, the complete genome DNA of all chromosome could be non-specific amplify by two WGA methods, Linker adaptor and the multiple displacement amplification (MDA) method, without the loss of genomic regions or preferential amplification of genomic loci or alleles. The WGA technique was not only increase the original DNA content, but also retained the integrity of whole genome DNA. The two common SNP of β-thalassemia was amplified from the longer or shorter primer set from these amplified genome DNA. DNA sequencing of the PCR product was performed to evaluate the ADO effect during two WGA methods. Total of 222 single cells were picked up and performed the Linker-adaptor and MDA method to amplify the whole genome DNA, respectively. DNA sequencing results of the PCR product from 174 samples found that 91 samples had the complete allele, and 83 samples had the allele drop-out (ADO) effect. The amplification efficiency of linker adaptor and MDA two methods are 90 ％. The ADO rate was 26.67 ％ and 28.89 ％, respectively. These two methods still had higher than 25 ％ of ADO rate. Therefore, the ADO problem was the biggest obstacle for single cell PCR and diagnosis of single-gene disorders.