https://scholars.lib.ntu.edu.tw/handle/123456789/159867
標題: | 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 Comparison with Linker adaptor and MDA Two Methods of Whole Genome Amplification for Single Cell Genome Amplification Efficiency |
作者: | 李黛君 Lee, Tai-Chun |
關鍵字: | 整體染色體放大技術;單一細胞聚合酶;連;鎖反應;放大失敗;胚胎著床前診斷;Whole Genome Amplification;Linker-adaptor;MDA;ADO;Amplification efficiency failure | 公開日期: | 2007 | 摘要: | 針對單一基因疾病的胚胎著床前診斷,主要的方法以聚合酶連鎖反應為主,將特定基因片段放大,進而偵測單一基因突變點。而單一細胞聚合酶連鎖反應步驟中會出現許多問題,像是污染、放大失敗、或是只優先放大其中一股,而另一股完全沒有被放大,造成ADO 的情形,但是單一細胞聚合酶連鎖反應,仍是作為胚胎著床前偵測單一基因突變的唯一方法。單一細胞的DNA 含量是很有限的,使用整體染色體放大技術,可以增加DNA 的含量,並且能將整段染色體完整的放大出來。因此,整體染色體放大技術對於單一細胞基因診斷,是一種非常好用的方法。本論文主要利用兩種整體染色體放大的方法: Linker-adaptor 方法與MDA 方法,利用整體染色體放大技術,將染色體中的基因體DNA,進行非專一性的放大,再使用有乙型海洋性貧血基因常見的兩個SNP 個體中淋巴球的整體DNA,針對這兩個SNP 設計專一性引子,進行聚合酶連鎖反應和DNA 定序,評估整體染色體放大技術的過程中造成ADO 的現象。本實驗共做了222 個檢體,分別使用Linker-adaptor 和MDA 方法放大整體染色體後,利用聚合酶連鎖反應放大乙型海洋性貧血常見的兩個SNP 基因,共有174 個檢體被放大出來,DNA 序列分析結果顯示有91 個檢體兩股allele 皆有放大出來,83 個檢體有ADO的情形。Linker-adaptor和MDA 方法倍增速率皆為90 %,ADO 比率則分別是26.67 %和28.89 %。這兩種整體染色體放大方法仍然有超過25 % ADO 情形。因此,ADO 對於單一基因疾病診斷所使用的單一細胞聚合酶連鎖反應,仍然是一個主要的問題,需要更進一步去克服它。 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. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/51338 | 其他識別: | zh-TW |
顯示於: | 分子醫學研究所 |
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