Burtt Noël P
Cold Spring Harb Protoc. 2011 Jan 1;2011(1):pdb.prot5552. doi: 10.1101/pdb.prot5552.
The cornerstones of any genetic analysis study are the quality and quantity of the DNA samples. DNA is a precious limited resource, and in human disease studies the accessibility of sample DNA is often governed by the isolation method and the human source. Additionally, forensic analysis and archaeological research are generally infeasible without intact sample DNA. Therefore, mechanisms to preserve or enhance the quantity of the DNA stock are crucial to the success of these studies. Historically, to preserve and maintain DNA stocks, costly and labor-intensive Epstein-Barr-virus-transformed cell lines were produced. The creation of cell lines can be valuable for a number of reasons in addition to creating a renewable resource of DNA, but the cost and effort to create them, as well as the requirement of intact cells to begin with, limit the utility of this approach. More recently, whole-genome amplification (WGA), utilizing the unique property of the enzyme Φ29 DNA polymerase, has been used to generate robust high-fidelity copies of the genome. As described in this protocol, WGA using Φ29 DNA polymerase allows unbiased representation of the genome via multiple-strand displacement, followed by rolling-circle amplification on random primers.
任何基因分析研究的基石都是DNA样本的质量和数量。DNA是一种珍贵的有限资源,在人类疾病研究中,样本DNA的可获取性通常取决于分离方法和人类来源。此外,没有完整的样本DNA,法医分析和考古研究通常是不可行的。因此,保存或增加DNA储备量的机制对于这些研究的成功至关重要。从历史上看,为了保存和维持DNA储备,人们生产了成本高昂且劳动密集的爱泼斯坦-巴尔病毒转化细胞系。创建细胞系除了能创造可再生的DNA资源外,还有许多其他原因可能很有价值,但创建它们的成本和精力,以及一开始对完整细胞的要求,限制了这种方法的实用性。最近,利用酶Φ29 DNA聚合酶的独特特性进行的全基因组扩增(WGA)已被用于生成基因组的强大高保真拷贝。如本方案所述,使用Φ29 DNA聚合酶的WGA通过多链置换实现基因组的无偏代表性,随后在随机引物上进行滚环扩增。