Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA
Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA
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DOI：
10.1093/nar/gnh015
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Borer, PN
中科院分区：
文献类型：
--
作者：
Cavaluzzi, MJ;Borer, PN
紫外光吸收为测定核酸浓度提供了几乎通用的基础。DNA和RNA的紫外线消光系数的数值取决于30-50年前确定的单核苷酸数值。我们发现几乎所有以前发表的核苷-5‘-单磷酸的消光系数都太大，误差高达7%。基于完全水解的浓度和较旧的一组值太低了，对于典型的RNA样品来说，大约是4%，对于典型的DNA样品来说，大约是2-3%。我们还分析了文献中未配对的DNA寡聚体的消光系数。对于具有非重复序列的单链DNA(SsDNA)和40-80%的GC，可以使用38µg/A(260)单位进行稳健的浓度预测。这比目前使用的考虑最近邻频率或基本组成的预测要好。后者导致的浓度对于用作聚合酶链式反应和其他类似技术的引物的典型单链DNA来说太低了10-30%。本文描述了用核磁共振精确测量核苷酸浓度的方法。核磁共振可以准确地测定1%以内的生物分子的浓度(和消光系数)。
Ultraviolet absorption provides the nearly universal basis for determining concentrations of nucleic acids. Values for the UV extinction coefficients of DNA and RNA rely on the mononucleotide values determined 30-50 years ago. We show that nearly all of the previously published extinction coefficients for the nucleoside-5'-monophosphates are too large, and in error by as much as 7%. Concentrations based on complete hydrolysis and the older set of values are too low by similar to4% for typical RNA and 2-3% for typical DNA samples. We also analyzed data in the literature for the extinction coefficients of unpaired DNA oligomers. Robust prediction of concentrations can be made using 38 mug/A(260) unit for single-stranded DNA (ssDNA) having non-repetitive sequences and 40-80% GC. This is superior to currently used predictions that account for nearest-neighbor frequency or base composition. The latter result in concentrations that are 10-30% too low for typical ssDNA used as primers for PCR and other similar techniques. Methods are described here to accurately measure concentrations of nucleotides by nuclear magnetic resonance. NMR can be used to accurately determine concentrations (and extinction coefficients) of biomolecules within 1%.