Nucleic acid contamination of glycogen used in nucleic acid precipitation and assessment of linear polyacrylamide as an alternative co-precipitant
Nucleic acid contamination of glycogen used in nucleic acid precipitation and assessment of linear polyacrylamide as an alternative co-precipitant
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DOI：
10.2144/000113276
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发表时间：
2009-12-01
期刊：
影响因子：
2.7
通讯作者：
Neufeld, Josh D.
中科院分区：
文献类型：
--
作者：
Bartram, Andrea K.;Poon, Calvin;Neufeld, Josh D.
分子级糖原被广泛用于回收纳克或皮克量的DNA和RNA，这些DNA和RNA跨越生命科学中的分子生物学应用。因此，其纯度对于获得可靠的结果至关重要。使用琼脂糖凝胶电泳，我们检测到pg/μ L（DNA）到ng/μ L（RNA）的核酸浓度在两个从商业供应商获得的9个糖原样品。16 S rRNA基因PCR扩增产物的变性梯度凝胶电泳表明，另外两个样品含有可检测的污染。我们还测试了合成聚合物共沉淀剂，线性聚丙烯酰胺（LPA）;用LPA测试的四个样品中没有一个被检测到污染。与贝类来源的糖原的污染样品相关的部分16 S rRNA基因序列与先前从贻贝中分离的卢菲放线菌的序列几乎相同。通过用多种沉淀剂和模拟实验条件测试低纳克量DNA的回收率，我们证明LPA是用于敏感方案的优选共沉淀剂。
Molecular-grade glycogen is widely used to recover nanogram or picogram quantities of DNA and RNA across molecular biology applications in the life sciences. As a result, its purity is critical to obtain reliable results. Using agarose gel electrophoresis, we detected pg/mu L (DNA) to ng/mu L (RNA) concentrations of nucleic acid in two of the nine glycogen samples obtained from commercial suppliers. Denaturing gradient gel electrophoresis of 16S rRNA gene PCR-amplified products indicated that an additional two samples contained detectable contamination. We also tested a synthetic polymer co-precipitant, linear polyacrylamide (LPA); none of the four samples tested with LPA were detectably contaminated. The partial 16S rRNA gene sequence associated with the contaminated samples of the shellfish-derived glycogen was nearly identical to the sequence of Actinobacteria lwoffii, which has been isolated from mussels previously. By testing the recovery of low-nanogram amounts of DNA with multiple precipitants and simulated experimental conditions, we demonstrated that LPA was a preferable co-precipitant for sensitive protocols.