攻无不“克”的无缝克隆为何备受喜爱？

版块：机械制造类型：普通作者：化工产业技术分享查看：78 回复：0 获赞：0 时间：2022-05-18 17:32:13

攻无不“克”的无缝克隆为何备受喜爱？

导读

在阅历了屡次PCR、酶切、衔接、转化后，有些小伙伴仍会面对克隆阳性率低的状况，每一步都认真地完结，但仍然存在不少的问题。今日小翌给我们介绍一下近年来备受科学家们喜爱的“无缝克隆”。比较于传统的酶切克隆，无缝克隆更快更灵敏，不受酶切位点约束，可以在质粒的任何位点进行一个或多个DNA段的刺进。

传统酶切克隆

1.受约束性内切酶的约束

2. 衔接功率低：T4 DNA衔接酶衔接功率具有序列的偏好性

3. 耗时长：酶连过夜

4.多片段克隆困难

图1.传统酶切克隆

无缝克隆

1.无需考虑酶切位点，适用恣意载体、恣意片段

2.阳性率高，可用于50 bp-10 kb的片段

3.衔接快速，20-30 min即可完结

4.可用于多片段克隆、基因突变、多顺反子表达载体构建

图2.无缝克隆

无缝克隆的原理

与传统的双酶切克隆一般，无缝克隆依赖于dsDNA的黏性结尾进行互补配对，但无缝克隆并不运用内切酶来制作黏性结尾，而是经过T5核酸外切酶来发生的。T5核酸外切酶可以沿着5’→3’方向，降解dsDNA，然后发生黏性结尾，可是，T5外切酶并不能确保每一个片段5’端都会酶切成相同长的粘性结尾。因而退火之后，无缝克隆中的DNA聚合酶会针对缺失的碱基进行增加，Taq衔接酶催化碱基之间构成磷酸二酯键，将一切缺口补齐。

图3.无缝克隆原理图

无缝克隆优势详解

TEC传感器 | 猫掌外链

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攻无不“克”的无缝克隆为何备受喜爱？<p><br/></p><section data-mpa-powered-by="yiban.io" style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; visibility: visible; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 10px 8px 0px; padding: 0px; outline: 0px; max-width: 100%; justify-content: flex-start; visibility: visible; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px; padding: 15px; outline: 0px; max-width: 100%; display: inline-block; width: auto; vertical-align: top; background-color: rgb(255, 150, 0); min-width: 10%; height: auto; visibility: visible; box-sizing: border-box !important;"><section powered-by="xiumi.us" style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; color: rgb(255, 255, 255); visibility: visible; box-sizing: border-box !important;"><p style="margin-top: 0px; margin-bottom: 0px; padding: 0px; outline: 0px; max-width: 100%; clear: both; min-height: 1em; visibility: visible; overflow-wrap: break-word !important; font-family: "times new roman", SimHei !important; box-sizing: border-box !important;"><strong style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; visibility: visible; box-sizing: border-box !important;">导读</strong></p></section></section></section><section powered-by="xiumi.us" style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; visibility: visible; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; visibility: visible; box-sizing: border-box !important;"><br style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; visibility: visible; box-sizing: border-box !important;"/></section></section><section powered-by="xiumi.us" style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; visibility: visible; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; visibility: visible; box-sizing: border-box !important;"><span style="margin: 0px; padding: 0px; overflow-wrap: normal !important; font-family: "times new roman", SimHei !important;">在阅历了屡次PCR、酶切、衔接、转化后，有些小伙伴仍会面对克隆阳性率低的状况，每一步都认真地完结，但仍然存在不少的问题。今日小翌给我们介绍一下近年来备受科学家们喜爱的“无缝克隆”。比较于传统的酶切克隆，无缝克隆更快更灵敏，不受酶切位点约束，可以在质粒的任何位点进行一个或多个DNA</span>段的刺进。<br style="overflow-wrap: normal !important; margin: 0px; padding: 0px;"/><br style="overflow-wrap: normal !important; margin: 0px; padding: 0px;"/></section></section><section style="overflow-wrap: break-word !important; margin: 10px 8px; padding: 0px; outline: 0px; max-width: 100%; justify-content: flex-start; visibility: visible; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; display: inline-block; width: auto; vertical-align: top; min-width: 10%; height: auto; border-bottom: 17px solid rgb(255, 230, 199); border-bottom-right-radius: 0px; visibility: visible; box-sizing: border-box !important;"><section powered-by="xiumi.us" style="overflow-wrap: break-word !important; margin: 0px 0px -8px; padding: 0px; outline: 0px; max-width: 100%; visibility: visible; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px; padding: 0px 5px; outline: 0px; max-width: 100%; letter-spacing: 0px; line-height: 1; color: rgb(255, 129, 36); visibility: visible; box-sizing: border-box !important;"><p style="margin-top: 0px; margin-bottom: 0px; padding: 0px; outline: 0px; max-width: 100%; clear: both; min-height: 1em; visibility: visible; overflow-wrap: break-word !important; font-family: "times new roman", SimHei !important; box-sizing: border-box !important;"><span style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; font-size: 15px; visibility: visible; overflow-wrap: break-word !important; box-sizing: border-box !important;"><strong style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; visibility: visible; box-sizing: border-box !important;">传统酶切克隆</strong></span></p></section></section></section></section><section powered-by="xiumi.us" style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; font-size: 15px; line-height: 1.75; letter-spacing: 1px; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;">1.受约束性内切酶的约束</section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;">2. 衔接功率低：T4 DNA衔接酶衔接功率具有序列的偏好性</section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;">3. 耗时长：酶连过夜</section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;">4.多片段克隆困难</section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;"><br style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"/></section></section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; text-align: center; box-sizing: border-box !important;"><img src="uploadfiles/images/1429/20220518/173212_0Y2l79.png" title="1647499200890576.png" alt="1647499200890576.png" width="400" height="409" border="0" vspace="0" style="overflow-wrap: normal !important; margin: 0px; padding: 0px; max-width: 100%; border: 0px; outline-style: none; width: 400px; height: 409px;"/></section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; text-align: center; white-space: normal; line-height: 1.75; letter-spacing: 1px; font-size: 12px; box-sizing: border-box !important;"><strong style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"><strong style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;">图1.</strong>传统酶切克隆</strong><br style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"/></section><section powered-by="xiumi.us" style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;"><br style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"/></section></section><section style="overflow-wrap: break-word !important; margin: 10px 8px; padding: 0px; outline: 0px; max-width: 100%; justify-content: flex-start; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; display: inline-block; width: auto; vertical-align: top; min-width: 10%; height: auto; border-bottom: 17px solid rgb(255, 230, 199); border-bottom-right-radius: 0px; box-sizing: border-box !important;"><section powered-by="xiumi.us" style="overflow-wrap: break-word !important; margin: 0px 0px -8px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px; padding: 0px 5px; outline: 0px; max-width: 100%; letter-spacing: 0px; line-height: 1; color: rgb(255, 129, 36); box-sizing: border-box !important;"><p style="margin-top: 0px; margin-bottom: 0px; padding: 0px; outline: 0px; max-width: 100%; clear: both; min-height: 1em; overflow-wrap: break-word !important; font-family: "times new roman", SimHei !important; box-sizing: border-box !important;"><span style="margin: 0px; padding: 0px; outline: 0px; max-width: 100%; font-size: 15px; overflow-wrap: break-word !important; box-sizing: border-box !important;"><strong style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;">无缝克隆</strong></span></p></section></section></section></section><section powered-by="xiumi.us" style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; line-height: 1.75; letter-spacing: 1px; font-size: 15px; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;">1.无需考虑酶切位点，适用恣意载体、恣意片段</section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;">2.阳性率高，可用于50 bp-10 kb的片段</section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;">3.衔接快速，20-30 min即可完结</section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;">4.可用于多片段克隆、基因突变、多顺反子表达载体构建</section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;"><br style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"/></section></section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; text-align: center; box-sizing: border-box !important;"><img src="uploadfiles/images/1429/20220518/173212_PfrZKl.png" title="1647499219650721.png" alt="1647499219650721.png" width="400" height="366" border="0" vspace="0" style="overflow-wrap: normal !important; margin: 0px; padding: 0px; max-width: 100%; border: 0px; outline-style: none; width: 400px; height: 366px;"/></section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; text-align: center; box-sizing: border-box !important;"><strong style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; font-size: 12px; letter-spacing: 1px; white-space: normal; box-sizing: border-box !important;">图2.无缝克隆</strong></section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; text-align: center; box-sizing: border-box !important;"><br style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"/></section><section powered-by="xiumi.us" style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; line-height: 1.75; letter-spacing: 1px; font-size: 12px; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;"><br style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"/></section></section><section style="overflow-wrap: break-word !important; margin: 10px 8px -30px; padding: 0px; outline: 0px; max-width: 100%; text-align: center; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; display: inline-block; vertical-align: top; overflow: hidden; height: 55px; width: 55px; background-color: rgb(255, 150, 0); box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"><svg viewbox="0 0 1 1" style="float:left;line-height:0;width:0;vertical-align:top;"></svg></section></section></section><section powered-by="xiumi.us" style="overflow-wrap: break-word !important; margin: 0px 0px 20px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; font-size: 18px; line-height: 1; letter-spacing: 0px; text-align: center; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"><strong style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;">无缝克隆的原理</strong></section></section></section><section powered-by="xiumi.us" style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; font-size: 15px; line-height: 1.75; letter-spacing: 1px; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;"><br style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"/></section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;">与传统的双酶切克隆一般，无缝克隆依赖于dsDNA的黏性结尾进行互补配对，但无缝克隆并不运用内切酶来制作黏性结尾，而是经过T5核酸外切酶来发生的。T5核酸外切酶可以沿着5’→3’方向，降解dsDNA，然后发生黏性结尾，可是，T5外切酶并不能确保每一个片段5’端都会酶切成相同长的粘性结尾。因而退火之后，无缝克隆中的DNA聚合酶会针对缺失的碱基进行增加，Taq衔接酶催化碱基之间构成磷酸二酯键，将一切缺口补齐。</section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;"><br style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"/></section></section><section style="overflow-wrap: break-word !important; margin: 10px 8px; padding: 0px; outline: 0px; max-width: 100%; text-align: center; box-sizing: border-box !important;"><img src="uploadfiles/images/1429/20220518/173213_7d90CY.png" title="1647499246530241.png" alt="1647499246530241.png" width="400" height="279" border="0" vspace="0" style="overflow-wrap: normal !important; margin: 0px; padding: 0px; max-width: 100%; border: 0px; outline-style: none; width: 400px; height: 279px;"/></section><section powered-by="xiumi.us" style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; text-align: center; white-space: normal; box-sizing: border-box !important;"><strong style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"><span style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; font-family: "times new roman", SimHei !important; outline: 0px; max-width: 100%; font-size: 12px; box-sizing: border-box !important;">图3.无缝克隆原理图</span></strong></section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;"><br style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"/></section><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;"><br style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"/></section></section><section style="overflow-wrap: break-word !important; margin: 10px 8px -30px; padding: 0px; outline: 0px; max-width: 100%; text-align: center; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; display: inline-block; vertical-align: top; overflow: hidden; height: 55px; width: 55px; background-color: rgb(255, 150, 0); box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"><svg viewbox="0 0 1 1" style="float:left;line-height:0;width:0;vertical-align:top;"></svg></section></section></section><section powered-by="xiumi.us" style="overflow-wrap: break-word !important; margin: 0px 0px 20px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; font-size: 18px; line-height: 1; letter-spacing: 0px; text-align: center; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"><strong style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;">无缝克隆优势详解</strong></section></section></section><section powered-by="xiumi.us" style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"><section style="overflow-wrap: break-word !important; margin: 0px 8px; padding: 0px; outline: 0px; max-width: 100%; white-space: normal; box-sizing: border-box !important;"><br style="overflow-wrap: break-word !important; margin: 0px; padding: 0px; outline: 0px; max-width: 100%; box-sizing: border-box !important;"/></section></section><section style="overflow-wrap: break-word !important; 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