Thermo Fisher Scientific的引物純化選項(xiàng)
為您的分析應(yīng)用選擇適合的 DNA 寡核苷酸[1]
??寡核苷酸是當(dāng)今許多生物學(xué)研究、藥物發(fā)現(xiàn)和診斷應(yīng)用的起點(diǎn)。這些高端技術(shù)應(yīng)用需要高品質(zhì)寡核苷酸才能獲得成功。我們的定制 DNA 寡核苷酸采用標(biāo)準(zhǔn)氰基 - 乙基亞磷酰胺在高度自動(dòng)化的計(jì)算機(jī)控制系統(tǒng)上合成。通過(guò)三苯甲基分析，在每個(gè)寡核苷酸的合成過(guò)程中監(jiān)控偶聯(lián)效率，不僅僅監(jiān)控最終產(chǎn)物，更確保整個(gè)工藝流程的質(zhì)量。對(duì)于合成后質(zhì)量控制，對(duì)短寡核苷酸進(jìn)行質(zhì)譜分析，對(duì)長(zhǎng)寡核苷酸進(jìn)行毛細(xì)管電泳（CE）分析，確保產(chǎn)品質(zhì)量。
??根據(jù)下游分析應(yīng)用的性質(zhì)，選擇最適合您分析應(yīng)用的合成規(guī)模和純化選項(xiàng)（表 2）。該表旨在幫助您為您的分析應(yīng)用選擇適合的寡核苷酸和純化方法。
??通常，小柱純化快速、經(jīng)濟(jì)，但依照寡核苷酸序列可能影響純化或產(chǎn)量（具有 5’G 的序列可能出現(xiàn)問(wèn)題）。HPLC 更耗時(shí)，但可提供高達(dá) 55 個(gè)堿基的卓越純度。對(duì)于 55 個(gè)以上的堿基，將全長(zhǎng)產(chǎn)物與 n-1 個(gè)失敗序列區(qū)分開(kāi)來(lái)變得越來(lái)越困難。PAGE 純化即使對(duì)于非常長(zhǎng)的寡核苷酸也能提供出色的分辨率，但通常會(huì)因產(chǎn)品品質(zhì)犧牲產(chǎn)量。
Integrated DNA Technologies 的引物純化選項(xiàng)[2]
Your choice of oligonucleotide purification should be based on several factors, including:
- the application in which you will be using the oligo
- the length of the oligo
- whether the oligo contains any modifications
- how much yield is needed
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 pu-rification can damage certain modifications including many fluorophores and some modifications used for attachment. PAGE purification should be avoided for modifi-cations including: any fluorophore, amino modifiers, digoxigenin, I-linker, or thiol modifiers. HPLC is the purification method of choice for these modifications.
生工的引物純化方式選擇[3]
寡核苷酸純化[4]
參考資料：
- https://www.thermofisher.com/cn/zh/home/life-science/oligonucleotides-primers-probes-genes/custom-dna-oligos/standard-oligos.html
- https://sg.idtdna.com/pages/products/optional-services/page-hplc-purification
- https://www.sangon.com/faq_selection_of_primer_purification_methods.html
- https://www.sigmaaldrich.cn/CN/zh/technical-documents/technical-article/genomics/dna-and-rna-purification/best-purification
