Jiang S W, Trujillo M A, Eberhardt N L
Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA.
Nucleic Acids Res. 1996 Aug 15;24(16):3278-9. doi: 10.1093/nar/24.16.3278.
Tandemly repeated DNA sequences generated from single synthetic oligonucleotide monomers are useful for many purposes. With conventional ligation procedures low yields and random orientation of oligomers makes cloning of defined repeated sequences difficult. We solved these problems using 2 bp overhangs to direct orientation and random incorporation of linkers containing restriction sites during ligation. Ligation products are amplified by PCR using the linker oligonucleotides as primers. Restriction digestion of the PCR products generate multimer distributions whose length is controlled by the monomer/linker ratio. The concatenated DNA fragments of defined length, orientation and spacing can be directly used for subcloning or other applications without further treatment.
由单个合成寡核苷酸单体产生的串联重复DNA序列有多种用途。采用传统的连接方法时，寡聚物的低产量和随机取向使得克隆特定的重复序列很困难。我们通过使用2个碱基对的突出端来指导取向，并在连接过程中随机掺入含有限制性酶切位点的接头，解决了这些问题。连接产物使用接头寡核苷酸作为引物通过聚合酶链反应（PCR）进行扩增。PCR产物的限制性酶切产生多聚体分布，其长度由单体/接头比例控制。具有确定长度、取向和间距的串联DNA片段无需进一步处理即可直接用于亚克隆或其他应用。
