Panjkovich Alejandro, Melo Francisco
Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile.
Bioinformatics. 2005 Mar;21(6):711-22. doi: 10.1093/bioinformatics/bti066. Epub 2004 Oct 22.
The overall performance of several molecular biology techniques involving DNA/DNA hybridization depends on the accurate prediction of the experimental value of a critical parameter: the melting temperature Tm. Till date, many computer software programs based on different methods and/or parameterizations are available for the theoretical estimation of the experimental Tm value of any given short oligonucleotide sequence. However, in most cases, large and significant differences in the estimations of Tm were obtained while using different methods. Thus, it is difficult to decide which Tm value is the accurate one. In addition, it seems that most people who use these methods are unaware about the limitations, which are well described in the literature but not stated properly or restricted the inputs of most of the web servers and standalone software programs that implement them.
A quantitative comparison on the similarities and differences among some of the published DNA/DNA Tm calculation methods is reported. The comparison was carried out for a large set of short oligonucleotide sequences ranging from 16 to 30 nt long, which span the whole range of CG-content. The results showed that significant differences were observed in all the methods, which in some cases depend on the oligonucleotide length and CG-content in a non-trivial manner. Based on these results, the regions of consensus and disagreement for the methods in the oligonucleotide feature space were reported. Owing to the lack of sufficient experimental data, a fair and complete assessment of accuracy for the different methods is not yet possible. Inspite of this limitation, a consensus Tm with minimal error probability was calculated by averaging the values obtained from two or more methods that exhibit similar behavior to each particular combination of oligonucleotide length and CG-content class. Using a total of 348 DNA sequences in the size range between 16mer and 30mer, for which the experimental Tm data are available, we demonstrated that the consensus Tm is a robust and accurate measure. It is expected that the results of this work would be constituted as a useful set of guidelines to be followed for the successful experimental implementation of various molecular biology techniques, such as quantitative PCR, multiplex PCR and the design of optimal DNA microarrays.
涉及DNA/DNA杂交的几种分子生物学技术的整体性能取决于一个关键参数实验值的准确预测：解链温度Tm。到目前为止，有许多基于不同方法和/或参数化的计算机软件程序可用于理论估计任何给定短寡核苷酸序列的实验Tm值。然而，在大多数情况下，使用不同方法时获得的Tm估计值存在很大且显著的差异。因此，很难确定哪个Tm值是准确的。此外，似乎大多数使用这些方法的人并未意识到其局限性，这些局限性在文献中有详细描述，但在大多数实现它们的网络服务器和独立软件程序中未得到恰当说明或对输入进行了限制。
报告了对一些已发表的DNA/DNA Tm计算方法之间异同的定量比较。对一大组长度在16至30个核苷酸之间的短寡核苷酸序列进行了比较，这些序列涵盖了整个CG含量范围。结果表明，所有方法都存在显著差异，在某些情况下，这些差异以复杂的方式取决于寡核苷酸长度和CG含量。基于这些结果，报告了寡核苷酸特征空间中各方法的共识区域和分歧区域。由于缺乏足够的实验数据，目前还无法对不同方法的准确性进行公平和全面的评估。尽管存在这一局限性，但通过对来自两种或更多种对寡核苷酸长度和CG含量类别特定组合表现出相似行为的方法所获得的值进行平均，计算出了具有最小误差概率的共识Tm。使用总共348个长度在16聚体至30聚体之间且有实验Tm数据的DNA序列，我们证明了共识Tm是一种可靠且准确的度量。预计这项工作的结果将构成一套有用的指导方针，用于成功实验实施各种分子生物学技术，如定量PCR、多重PCR和优化DNA微阵列的设计。
