Casas Veronica, Rohwer Forest
Center for Microbial Sciences, San Diego State University, San Diego, CA, USA.
Methods Enzymol. 2007;421:259-68. doi: 10.1016/S0076-6879(06)21020-6.
The vast majority of novel DNA sequences deposited in the databases now comes from environmental phage DNA sequences. Methods are presented for the cloning and sequencing of phage DNA that might otherwise be lethal to bacterial host vectors or contain modified DNA bases that prevent standard cloning of such sequences. In addition, methods are presented for the isolation of viral particles directly from soil and sediment environmental samples or from large volumes of environmental water samples. The viral particles are then purified by cesium-chloride density centrifugation followed by DNA extraction. This purified viral metagenomic DNA is then used for cloning and sequencing.
目前数据库中绝大多数新的DNA序列都来自环境噬菌体DNA序列。本文介绍了噬菌体DNA的克隆和测序方法，这些噬菌体DNA序列可能对细菌宿主载体具有致死性，或者含有修饰的DNA碱基，从而阻止此类序列的标准克隆。此外，本文还介绍了直接从土壤和沉积物环境样本或大量环境水样中分离病毒颗粒的方法。然后通过氯化铯密度离心法纯化病毒颗粒，随后进行DNA提取。接着，将这种纯化的病毒宏基因组DNA用于克隆和测序。
