Sequence-specific purification of DNA oligomers in hydrophobic interaction chromatography using peptide nucleic acid amphiphiles: extended dynamic range.
Sequence-specific purification of DNA oligomers in hydrophobic interaction chromatography using peptide nucleic acid amphiphiles: extended dynamic range.
复制标题
使用肽核酸两亲物在疏水相互作用层析中对 DNA 寡聚物进行序列特异性纯化：扩展的动态范围。
DOI：
10.1002/bit.21242
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发表时间：
2007
影响因子：
3.8
通讯作者：
Schneider,JamesW
中科院分区：
文献类型：
--
作者：
Savard,JeffreyM;Schneider,JamesW
我们提出了对先前报道的方法（Vernille JP，Schneider JW. 2004. Biotechnol Prog 20（6）：1776-1782）来纯化DNA寡聚体，通过将肽核酸两亲物（PNAA）连接到寡聚体上的特定序列，然后使用疏水相互作用色谱（HIC）将它们与未结合的寡聚体分离。使用烷基改性的HIC介质（丁基和辛基琼脂糖凝胶）优于苯基改性的介质（苯基琼脂糖凝胶）减少了未结合DNA的洗脱时间，同时不影响PNAA/DNA复合物的洗脱时间。将PNAA的烷烃尾长从C12改变为C18，PNAA/DNA双链体的保留略有增加。通过结合这两种改进，我们表明，即使当PNAA烷烃连接到靶链的中心时，也可以以高分辨率实现长度为60个碱基或更多的DNA寡聚体的序列特异性纯化。PNAA/DNA双链体结合对HIC介质选择的不敏感性似乎是由于表面诱导的聚集现象，该现象在未标记的DNA的情况下不会发生。我们还报告了使用批HIC作为线性梯度HIC中洗脱曲线的充分预测因子，以及其通过应用阶梯梯度大大减少纯化时间的潜力。Biotechnol. Bioeng. 2007;97：367-376.© 2006 Wiley Periodicals，Inc.
We present improvements on a previously reported method (Vernille JP, Schneider JW. 2004. Biotechnol Prog 20(6):1776–1782) to purify DNA oligomers by attachment of peptide nucleic acid amphiphiles (PNAA) to particular sequences on the oligomers, followed by their separation from unbound oligomers using hydrophobic interaction chromatography (HIC). Use of alkyl‐modified HIC media (butyl and octyl sepharose) over phenyl‐modified media (phenyl sepharose) reduced the elution time of unbound DNA while not affecting the elution time of the PNAA/DNA complex. Modifying the alkane tail length for PNAA from C12to C18increased slightly the retention of PNAA/DNA duplexes. By combining these two refinements, we show that sequence‐specific purifications of DNA oligomers 60 bases in length or more can be achieved with high resolution, even when the PNAA alkane is attached to the center of the target strand. The insensitivity of the PNAA/DNA duplex binding to choice of HIC media appears to be due to a surface‐induced aggregation phenomenon that does not occur in the case of untagged DNA. We also report on the use of batch HIC as an adequate predictor of elution profiles in linear gradient HIC, and its potential to considerably reduce purification times by applying step gradients. Biotechnol. Bioeng. 2007;97: 367–376. © 2006 Wiley Periodicals, Inc.