关于chromosome walking染色体行走的问题Q:Chromosome walking is a technique used to clone a gene from its known closest markers.How would you know whether the mutation is upstream or downstream of the known marker?And whether you have found t
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关于chromosome walking染色体行走的问题Q:Chromosome walking is a technique used to clone a gene from its known closest markers.How would you know whether the mutation is upstream or downstream of the known marker?And whether you have found t
关于chromosome walking染色体行走的问题
Q:Chromosome walking is a technique used to clone a gene from its known closest markers.How would you know whether the mutation is upstream or downstream of the known marker?And whether you have found the mutation that confers the mutant phenotype or not?
关于chromosome walking染色体行走的问题Q:Chromosome walking is a technique used to clone a gene from its known closest markers.How would you know whether the mutation is upstream or downstream of the known marker?And whether you have found t
1、为判断在上游还是在下游,应该在上游与下游同时分别寻找次近的其他已知marker（或按自己设计的新marker）,分别设计pcr引物,然后分别与那条已知的最近的marker设计的引物进行pcr扩增,最后在较浓的胶中跑电泳观察.含有突变基因的扩增产物通常在大小上会与正常基因相应的扩增产物大小不同,电泳条带也就会有不同大小.或是含有突变基因的基因组扩增产物除了会得到与正常基因大小相同的条带外,还有另一条大小有差异的带.看看有差异的带是用哪对引物扩增出的就确定了；
但是如果附近没有其他marker,且该物种基因组序列未知时,只能双向walking；
如果是序列已知的插入片段（比如病毒）导致突变,则通常插入位置就是目标基因的内部,直接双向walking分别测序即可
2、得到基因后,如果该物种基因组序列已知,测序扩增的片段并与已知序列比较即可初步确认；
最确凿的证据是互补实验得到的.如果突变是显性的,用怀疑是突变基因的基因片段转基因互补正常生物体（或细胞）,看是否出现突变表型；
若突变是隐性的,则用正常片段转基因互补隐性纯合突变体,看突变表型是否被回复