Low concentration DNA extraction and recovery using a silica solid phase
Low concentration DNA extraction and recovery using a silica solid phase
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DOI：
10.1371/journal.pone.0176848
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发表时间：
2017-05-05
期刊：
影响因子：
3.7
通讯作者：
Klapperich, Catherine M.
中科院分区：
文献类型：
--
作者：
Katevatis, Constantinos;Fan, Andy;Klapperich, Catherine M.
从临床样品中提取DNA通常在离液剂存在下用二氧化硅固相提取柱实现。这些方案的版本已经适用于小型化平台中的床旁（POC）诊断设备，但是商业试剂盒需要大量的输入DNA。因此，当输入的临床样品含有少于1 μ g的总DNA时，在没有用外源载体DNA补充样品的情况下，来自大多数这些方案的靶特异性DNA回收率低。事实上，用于POC诊断开发的许多临床样品通常表现出低至3 ng/mL的靶DNA浓度。随着更广泛的目标，提高产量和效率的核酸为基础的POC装置稀释样品，我们研究了DNA吸附和回收二氧化硅颗粒通过使用1 pg- 1 μ g的DNA与一组吸附和洗脱缓冲液的pH值和离液存在的范围。在吸附方面，我们发现，低pH值和离液序列高的硫氰酸胍（GuSCN）的存在下增强DNA-二氧化硅吸附。当用标准低盐、高pH缓冲液洗脱时，> 70%的DNA是不可回收的，除了当DNA最初用pH 5.2的5 M GuSCN吸附时。未回收的DNA要么最初没有吸附或不可逆地结合在二氧化硅表面上。当用95 ° C甲酰胺和IM NaOH洗脱时，回收率提高，这表明DNA-二氧化硅-离液剂相互作用由疏水相互作用和氢键主导。虽然加热的甲酰胺和NaOH是用于实际POC装置的非理想洗脱缓冲液，但是显著的结果对于工程化一组优化的试剂是重要的，所述试剂可以最大化从微流体DNA-二氧化硅-离液剂系统的核酸回收。
DNA extraction from clinical samples is commonly achieved with a silica solid phase extraction column in the presence of a chaotrope. Versions of these protocols have been adapted for point of care (POC) diagnostic devices in miniaturized platforms, but commercial kits require a high amount of input DNA. Thus, when the input clinical sample contains less than 1 mu g of total DNA, the target-specific DNA recovery from most of these protocols is low without supplementing the sample with exogenous carrier DNA. In fact, many clinical samples used in the development of POC diagnostics often exhibit target DNA concentrations as low as 3 ng/mL. With the broader goal of improving the yield and efficiency of nucleic acid-based POC devices for dilute samples, we investigated both DNA adsorption and recovery from silica particles by using 1 pg- 1 mu g of DNA with a set of adsorption and elution buffers ranging in pH and chaotropic presence. In terms of adsorption, we found that low pH and the presence of chaotropic guanidinium thiocyanate (GuSCN) enhanced DNA-silica adsorption. When eluting with a standard low-salt, high-pH buffer, > 70% of DNA was unrecoverable, except when DNA was initially adsorbed with 5 M GuSCN at pH 5.2. Unrecovered DNA was either not initially adsorbed or irreversibly bound on the silica surface. Recovery was improved when eluting with 95 degrees C formamide and 1 M NaOH, which suggested that DNA-silica- chaotrope interactions are dominated by hydrophobic interactions and hydrogen bonding. While heated formamide and NaOH are non-ideal elution buffers for practical POC devices, the salient results are important for engineering a set of optimized reagents that could maximize nucleic acid recovery from a microfluidic DNA-silica-chaotrope system.