Zhao H, Arnold F H
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA.
Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):7997-8000. doi: 10.1073/pnas.94.15.7997.
We describe a convenient method for distinguishing functional from nonfunctional or deleterious mutations in homologous genes. High fidelity in vitro gene recombination ("DNA shuffling") coupled with sequence analysis of a small sampling of the shuffled library exhibiting the evolved behavior allows identification of those mutations responsible for the behavior in a background of neutral and deleterious mutations. Functional mutations are expected to occur in 100% of the sequenced screened sample; neutral mutations are found in 50% on average, and deleterious mutations do not appear at all. When used to analyze 10 mutations in a laboratory-evolved gene encoding a thermostable subtilisin E, this method rapidly identified the two responsible for the observed protease thermostability; the remaining eight were neutral with respect to thermostability, within the precision of the screening assay. A similar approach, coupled with selection for growth and survival of the host organism, could be used to distinguish adaptive from neutral mutations.
我们描述了一种区分同源基因中功能性突变与非功能性或有害突变的简便方法。高保真体外基因重组（“DNA 改组”）与对表现出进化行为的改组文库的小样本进行序列分析相结合，能够在中性和有害突变的背景下识别出导致该行为的那些突变。预计在 100%的测序筛选样本中会出现功能性突变；平均 50%的样本中会发现中性突变，而有害突变根本不会出现。当用于分析编码热稳定枯草杆菌蛋白酶 E 的实验室进化基因中的 10 个突变时，该方法迅速识别出了两个导致观察到的蛋白酶热稳定性的突变；在筛选测定的精度范围内，其余八个突变对于热稳定性而言是中性的。一种类似的方法，结合对宿主生物体生长和存活的选择，可以用于区分适应性突变和中性突变。
