Question

引物的纯化方式及选择

Answer 1

  寡核苷酸是当今许多生物学研究、药物发现和诊断应用的起点。这些高端技术应用需要高品质寡核苷酸才能获得成功。我们的定制 DNA 寡核苷酸采用标准氰基 - 乙基亚磷酰胺在高度自动化的计算机控制系统上合成。通过三苯甲基分析，在每个寡核苷酸的合成过程中监控偶联效率，不仅仅监控最终产物，更确保整个工艺流程的质量。对于合成后质量控制，对短寡核苷酸进行质谱分析，对长寡核苷酸进行毛细管电泳（CE）分析，确保产品质量。

  根据下游分析应用的性质，选择最适合您分析应用的合成规模和纯化选项（表 2）。该表旨在帮助您为您的分析应用选择适合的寡核苷酸和纯化方法。

  通常，小柱纯化快速、经济，但依照寡核苷酸序列可能影响纯化或产量（具有 5’G 的序列可能出现问题）。HPLC 更耗时，但可提供高达 55 个碱基的卓越纯度。对于 55 个以上的碱基，将全长产物与 n-1 个失败序列区分开来变得越来越困难。PAGE 纯化即使对于非常长的寡核苷酸也能提供出色的分辨率，但通常会因产品品质牺牲产量。

Your choice of oligonucleotide purification should be based on several factors, including:

PCR or sequencing : Standard desalting provided with every oligo order is sufficient for most of these applications, as truncations and deletions will not affect your results appreciably. Deletion products are very rare compared to full length oligo, with truncations occurring on the 5’ end of the growing strand. The 3’ end will always remain intact, which is the most important consideration for PCR-based applications.

Cloning, mutagenesis, and gel shift : For these applications, full length product is of utmost importance, and PAGE purification should be strongly considered. PAGE purification will result in the highest purity level of full length product—routinely achieving greater than 85% full length product.

Note that PAGE purification can sometimes result in lower yields than HPLC purification. If you need a relatively pure product, but also need a higher yield, you should consider HPLC.

Modified oligos : The urea used in PAGE pu­rification can damage certain modifications including many fluorophores and some modifications used for attachment. PAGE purification should be avoided for modifi­cations including: any fluorophore, amino modifiers, digoxigenin, I-linker, or thiol modifiers. HPLC is the purification method of choice for these modifications.

寡核苷酸纯化 ^[4]