Size-selective DNA separation: Recovery spectra help determine the sodium chloride (NaCl) and polyethylene glycol (PEG) concentrations required
Size-selective DNA separation: Recovery spectra help determine the sodium chloride (NaCl) and polyethylene glycol (PEG) concentrations required
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DOI：
10.1002/biot.201400234
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发表时间：
2014-10-01
影响因子：
4.7
通讯作者：
Gu, Hongchen
中科院分区：
文献类型：
--
作者：
He, Zhangyong;Xu, Hong;Gu, Hongchen
在氯化钠（NaCl）存在的情况下，可以通过改变聚乙二醇（PEG）的最终浓度来选择性地分离DNA片段。这种分离策略结合使用顺磁性颗粒提供了一个有价值的平台，用于实现所需的DNA大小区间，这在高通量DNA测序的自动化文库制备中是重要的。在这里，我们报告的DNA片段，使确定合适的NaCl和PEG浓度的大小选择性分离的恢复光谱的建立。首先，在给定的NaCl浓度下，通过使用逻辑函数拟合DNA回收率与PEG浓度以确定所需参数来获得回收方程。其次，通过对恢复方程的一阶导数进行推导，得到了恢复方程的斜率函数。因此，可以使用基于这些参数的斜率函数来生成恢复谱。根据不同长度DNA片段的回收谱，通过计算其分辨值和回收率，分别确定合适的NaCl和PEG浓度。该策略有效地应用于大小选择性分离的532-，400-和307-bp的片段在选定的试剂浓度与回收率分别为96.9%，64.7%和85.9%，分别。我们的方法能够很好地预测NaCl和PEG浓度的大小选择性DNA分离。
In the presence of sodium chloride (NaCl), DNA fragments can be size-selectively separated by varying the final concentration of polyethylene glycol (PEG). This separation strategy in combination with the use of paramagnetic particles provides a valuable platform for achieving the desired DNA size interval, which is important in automated library preparation for high-throughput DNA sequencing. Here, we report the establishment of recovery spectra of DNA fragments that enable the determination of suitable NaCl and PEG concentrations for size-selective separation. Firstly, at a given NaCl concentration, the recovery equation was obtained by fitting the DNA recovery ratios versus the PEG concentrations using the logistic function to determine the required parameters. Secondly, the slope function of the recovery equation was achieved by deducing its first derivative. Therefore, the recovery spectrum can be generated using the slope function based on those parameters. According to the recovery spectra of different length DNA fragments, suitable NaCl and PEG concentrations can be determined, respectively, by calculating their resolution values and recovery ratios. The strategy was effectively applied to the size-selective separation of 532-, 400-, and 307-bp fragments at the selected reagent concentrations with recoveries of 96.9, 64.7, and 85.9%, respectively. Our method enables good predictions of NaCl and PEG concentrations for size-selective DNA separation.