Spits C, Le Caignec C, De Rycke M, Van Haute L, Van Steirteghem A, Liebaers I, Sermon K
Research Centre for Reproduction and Genetics, Academisch Ziekenhuis, Vrije Universiteit Brussel, Brussels, Belgium.
Hum Mutat. 2006 May;27(5):496-503. doi: 10.1002/humu.20324.
The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minute quantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research.
基因组DNA的稀缺可能是某些遗传研究领域的一个限制因素。为克服这一困难而开发的方法之一是全基因组扩增（WGA）。最近，多重置换扩增（MDA）已被证明在小DNA样本和细胞群体的WGA中非常有效，该反应由phi29或Bst DNA聚合酶催化。本研究的目的是开发一种在单细胞水平上可靠、高效且快速的MDA方案。我们首先比较了phi29和Bst聚合酶对DNA样本和单细胞的扩增效率。phi29聚合酶能在短时间内从单个细胞准确地产生足够的DNA用于大量检测，而Bst酶效率低且错误率高。使用phi29聚合酶优化了单细胞方案，并在60个单细胞上进行了评估；通过22个位点特异性PCR对获得的DNA进行了评估。这种新方案可用于许多涉及微量起始材料的应用，如法医DNA分析、产前和植入前基因诊断或癌症研究。
