Quick and clean cloning: a ligation-independent cloning strategy for selective cloning of specific PCR products from non-specific mixes.
Quick and clean cloning: a ligation-independent cloning strategy for selective cloning of specific PCR products from non-specific mixes.
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DOI：
10.1371/journal.pone.0020556
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Marillonnet S
中科院分区：
文献类型：
--
作者：
Thieme F;Engler C;Kandzia R;Marillonnet S
我们已经开发了一种有效的策略克隆PCR产品，其中包含一个未知区域的两侧是一个已知的序列。与非连接克隆一样，该策略基于存在于载体和插入物中的序列之间的同源性。然而，与非连接克隆相比，克隆载体只与用于插入物扩增的两个引物中的一个同源。线性化克隆载体的另一端与插入物中存在的序列具有同源性，但与用于扩增的基因特异性引物嵌套且不重叠。由于只有特异性产物含有该序列，而非特异性产物均不含有，因此只能克隆特异性产物。克隆使用一步反应进行，只需要在T4 DNA聚合酶存在的室温下孵育10分钟，就可以在载体和插入物的末端产生单链延伸。然后将反应混合物直接转化为大肠杆菌，在大肠杆菌中修复和连接退火的载体插入复合物。我们已经测试了这种我们称之为快速清洁克隆（QC克隆）的方法，用于克隆非霍奇金淋巴瘤肿瘤样本中表达的免疫球蛋白的可变区。该方法也可用于鉴定DNA元件的侧翼序列，如T-DNA或转座子插入，或用于克隆任何具有高特异性的PCR产物。
We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. The other side of the linearized cloning vector has homology with a sequence present in the insert, but nested and non-overlapping with the gene-specific primer used for amplification. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. Cloning is performed using a one-step reaction that only requires incubation for 10 minutes at room temperature in the presence of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. The reaction mix is then directly transformed into E. coli where the annealed vector-insert complex is repaired and ligated. We have tested this method, which we call quick and clean cloning (QC cloning), for cloning of the variable regions of immunoglobulins expressed in non-Hodgkin lymphoma tumor samples. This method can also be applied to identify the flanking sequence of DNA elements such as T-DNA or transposon insertions, or be used for cloning of any PCR product with high specificity.
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影响因子：
3.7
作者：
Engler, Carola;Kandzia, Romy;Marillonnet, Sylvestre
通讯作者：
Marillonnet, Sylvestre
影响因子：
50.5
作者：
Bendandi, M.;Marillonnet, S.;Gleba, Y.
通讯作者：
Gleba, Y.
影响因子：
14.9
作者：
ASLANIDIS, C;DEJONG, PJ
通讯作者：
DEJONG, PJ
DOI：
10.1073/pnas.85.23.8998
发表时间：
1988-12-01
影响因子：
11.1
作者：
FROHMAN, MA;DUSH, MK;MARTIN, GR
通讯作者：
MARTIN, GR
影响因子：
1.6
作者：
Lee, Jonas;Kim, Sung-Hou
通讯作者：
Kim, Sung-Hou