Q：定点突变克隆原理是什么？

A：基于同源重组技术的定点突变系统。使用本试剂盒，目标质粒扩增产物经 DpnI 消化 同源重组环化后直接进行转化即可完成定点突变。

Q：多点突变试剂盒与定点突变试剂盒有什么区别？

多点突变试剂盒可一步实现目标质粒上 3-5 个不连续位点的定点突变，而定点突变试剂盒是适合 1 或2 个不连续位点的定点突变。如：3 个连续的碱基突变算 1 个位点的突变，故定点突变试剂盒即可（相距超过 50 bp 为 1 个不连续位点）。

Q：点突变为什么要选择甲基化的载体？

A：点突变过程需要去除模板质粒，防止假阳性。利用的原理就是限制性内切酶 DpnI 能消化甲基化的模板，若是非甲基化，则无法去除模板。

Q：质粒反向扩增时引物如何设计？

A:正反向扩增引物 5’端包含至少 20 bp 反向互补区域，各引物非互补区域长度至少为 20 bp。互补区尽量选择无重复序列，GC 含量尽量在 40%-60%范围内。

Q：质粒模板的使用量？

A:在不影响扩增产物的情况下，尽可能少的量（建议＜1ng），避免DpnI 消化不完全。

Q：DpnI 处理后要进行回收吗？

A:最好进行胶回收。未消化完全的质粒或其他非特异性条带会影响后续的重组反应。

Q：对于PCR 模板质粒什么要求？

A:PCR 模板应为甲基化质粒，应使用甲基化酶无缺陷型的宿主菌扩增原始质粒（如DH5α，TOP10， JM109 等）。应尽量使用新鲜制备的质粒作为模板，因长期放置，反复冻融可能会导致模板质粒断裂，开环或降解。

Q：适合多大的质粒模板进行突变？

A:小于 20 kb。质粒越大，其他非目的碱基的突变率越高，PCR 更难，重组效率更低。

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