Nuclease P1 digestion/high-performance liquid chromatography, a practical method for DNA quantitation
Nuclease P1 digestion/high-performance liquid chromatography, a practical method for DNA quantitation
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DOI：
10.1016/j.chroma.2006.03.071
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发表时间：
2006-06-09
影响因子：
4.1
通讯作者：
Giese, Roger W.
中科院分区：
文献类型：
--
作者：
Shimelis, Olga;Giese, Roger W.
我们已经开发出一种实用的DNA定量方法。该方法在两个方面是实用的。首先，一个单一的酶被用来将DNA消化成核苷酸，然后在正常条件下用高效液相色谱法进行定量。其次，该方法即使在DNA不纯的情况下也能对其进行量化。在我们的方法‘’核酸酶P1/高效液相色谱‘’中，DNA被核酸酶PI水解，生成的2‘-脱氧核苷5’-单磷酸通过高效液相色谱-紫外检测进行定量。该方法被应用于几种基因组DNA的来源和纯化方法。检测了小牛胸腺DNA(供应商用盐沉淀法提纯)、猪肝DNA(用酚抽提法或使用Qiagen的基因组末端阴离子交换层析提纯)和小鼠皮肤DNA(类似提纯的小鼠皮肤DNA)。在某些情况下，给定的样品通过其中两种方法进行提纯。将我们的方法与其他三种方法的DNA含量进行了比较：酸解/高效液相色谱法(选择作为参考程序)、紫外线吸光度和染料结合。对于19-54微克范围内的DNA量，我们的方法所提供的值与酸水解/高效液相色谱提供的值之间的一致性在10%以内。相比之下，紫外线吸光度和染料结合试验与酸解和我们的方法提供的一致值相比，差异高达30%-40%。总体而言，将10个样品的DNA(胸腺、肝脏、皮肤)浓度通过酸解/高效液相色谱归一化为1.0，得到的相对值和标准偏差如下：1.01+/-0.07(核酸酶P1/高效液相色谱法)，0.8+/-0.17(染料结合)，1.1+/-0.1(UV)。由于人们不能假设任何DNA样本都是纯净的，而且很难确定DNA的纯度，因此建议在需要准确的值时，使用核酸酶P1/高效液相色谱法或酸解/高效液相色谱法来定量DNA，而不是紫外线吸光度或染料结合。(C)2006爱思唯尔B.V.保留所有权利。
We have developed a practical method for quantifying DNA. The method is practical in two ways. First, a single enzyme is used to digest the DNA to nucleotides that are then quantified by HPLC under ordinary conditions. Second, the method quantifies DNA even when it is impure. In our method, '' nuclease P1/HPLC,'' the DNA is hydrolyzed by nuclease PI and the resulting 2'-deoxynucleoside 5'-monophosphates are quantified by HPLC with UV detection. This method was applied to several kinds of genomic DNA in terms of origin and method by which it had been purified. Calf thymus DNA (purified by salt precipitation by the supplier), pig liver DNA (purified by phenolic extraction or by anion-exchange chromatography using a Genomic Tip from Qiagen) and mouse skin DNA (similarly purified) were tested. In some cases a given sample was purified by two of these methods. The values for the amount of DNA by our method were compared with those by three other methods: acid hydrolysis/HPLC (selected as a reference procedure), UV absorbance, and dye binding. Agreement for all DNA samples between the values by our method versus those provided by acid hydrolysis/HPLC was within 10% for amounts of DNA in the 19-54 mu g range. In contrast, UV absorbance and the dye-binding assay gave differences up to 30-40% relative to the consistent values furnished by acid hydrolysis and our method. Overall, normalizing the concentrations of the DNA (thymus, liver, skin) by acid hydrolysis/HPLC in 10 samples to values of 1.0 gave the following, relative values and standard deviations: 1.01 +/- .07 (nuclease P1/HPLC), 0.8 +/- 0.17 (dye binding), and 1.1 +/- 0.1 (UV). Since one cannot assume that any sample of DNA is pure, and determining purity of DNA is difficult, then nuclease P1/HPLC or acid hydrolysis/HPLC is recommended rather than the UV absorbance or dye binding for quantifying DNA whenever an accurate value is important. (c) 2006 Elsevier B.V. All rights reserved.