如何确认观察到的表型是由于病毒特定的突变引起的？

In genetic analysis of viruses, mutations are made in vitro by a variety of techniques, all of which can introduce unexpected changes. 

Errors can be introduced during cloning, from PCR, during sequencing, and when the viral DNA or plasmid DNA is introduced into the eukaryotic cell. With these potential problems in mind, how can it be concluded that a phenotype arises from the planned mutation? 

1. Repeat the construction. It is unlikely that an unlinked mutation with the same phenotype would occur twice.

2. Look for marker rescue. Replace the mutation and all adjacent DNA with parental DNA. If the mutation indeed causes the phenotype, the wild-type phenotype should be restored in the rescued virus.

3. Allow synthesis of the wild-type protein in the mutant background. If the wild-type phenotype is restored (complemented), then the probability is high that the phenotype arises from the mutation. 

4. Each of these approaches has limitations, and it is therefore prudent to use more than one.

Forward Genetics  vs  Reverse Genetics

病毒研究中的“正向遗传学”与“反向遗传学”

 Methods for coronavirus reverse genetics

冠状病毒的反向遗传学方法/策略