McGraw R A, Steffe E K, Baxter S M
Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens 30602.
Biotechniques. 1990 Jun;8(6):674-8.
We were interested in developing a better method to predict the thermal stability of specific oligonucleotide-target duplexes. Recognizing that the base sequence can have important effects, we investigated the use of a simple parameter based on nearest-neighbor stacking interactions, the mean stacking temperature. We took values for doublet stabilities from the literature and used a computer program to calculate mean stacking temperatures for all oligonucleotides of specified length and G + C content in the M13 phage genome. As expected, the program predicted a fairly broad range of stabilities for different sequences of equal G + C content. We selected 20-mer sequences representing the highest and lowest mean stacking temperatures at 25%, 50% and 75% G + C and synthesized them for use as probes against M13 DNA immobilized on filters. By hybridizing and washing at different temperatures, we demonstrated that mean stacking temperatures correlate well with observed stabilities. Relative stabilities of the six oligos were predicted correctly in every case. We used conditions appropriate to oligonucleotide probing and polymerase chain reaction and we were able to derive simple linear equations relating the empirical data and mean stacking temperature for both. These observations should be useful in planning experiments with oligonucleotides.
我们希望开发一种更好的方法来预测特定寡核苷酸-靶标双链体的热稳定性。认识到碱基序列可能具有重要影响，我们研究了使用基于最近邻堆积相互作用的简单参数——平均堆积温度。我们从文献中获取了双链体稳定性的值，并使用计算机程序计算了M13噬菌体基因组中指定长度和G + C含量的所有寡核苷酸的平均堆积温度。正如预期的那样，该程序预测了具有相同G + C含量的不同序列的相当广泛的稳定性范围。我们选择了在25%、50%和75% G + C时代表最高和最低平均堆积温度的20聚体序列，并合成它们用作针对固定在滤膜上的M13 DNA的探针。通过在不同温度下杂交和洗涤，我们证明平均堆积温度与观察到的稳定性密切相关。在每种情况下，六种寡核苷酸的相对稳定性都被正确预测。我们使用了适合寡核苷酸探测和聚合酶链反应的条件，并且能够推导出将经验数据与两者的平均堆积温度相关联的简单线性方程。这些观察结果对于规划寡核苷酸实验应该是有用的。
