Hughesman Curtis, Fakhfakh Kareem, Bidshahri Roza, Lund H Louise, Haynes Charles
Michael Smith Laboratories, University of British Columbia , Vancouver, BC, Canada V6T 1Z4.
Biochemistry. 2015 Feb 17;54(6):1338-52. doi: 10.1021/bi500905b. Epub 2015 Feb 5.
Advances in real-time polymerase chain reaction (PCR), as well as the emergence of digital PCR (dPCR) and useful modified nucleotide chemistries, including locked nucleic acids (LNAs), have created the potential to improve and expand clinical applications of PCR through their ability to better quantify and differentiate amplification products, but fully realizing this potential will require robust methods for designing dual-labeled hydrolysis probes and predicting their hybridization thermodynamics as a function of their sequence, chemistry, and template complementarity. We present here a nearest-neighbor thermodynamic model that accurately predicts the melting thermodynamics of a short oligonucleotide duplexed either to its perfect complement or to a template containing mismatched base pairs. The model may be applied to pure-DNA duplexes or to duplexes for which one strand contains any number and pattern of LNA substitutions. Perturbations to duplex stability arising from mismatched DNA:DNA or LNA:DNA base pairs are treated at the Gibbs energy level to maintain statistical significance in the regressed model parameters. This approach, when combined with the model's accounting of the temperature dependencies of the melting enthalpy and entropy, permits accurate prediction of T(m) values for pure-DNA homoduplexes or LNA-substituted heteroduplexes containing one or two independent mismatched base pairs. Terms accounting for changes in solution conditions and terminal addition of fluorescent dyes and quenchers are then introduced so that the model may be used to accurately predict and thereby tailor the T(m) of a pure-DNA or LNA-substituted hydrolysis probe when duplexed either to its perfect-match template or to a template harboring a noncomplementary base. The model, which builds on classic nearest-neighbor thermodynamics, should therefore be of use to clinicians and biologists who require probes that distinguish and quantify two closely related alleles in either a quantitative PCR or dPCR assay. This potential is demonstrated by using the model to design allele-specific probes that completely discriminate and quantify clinically relevant mutant alleles (BRAF V600E and KIT D816V) in a dPCR assay.
实时聚合酶链反应（PCR）技术的进步，以及数字PCR（dPCR）的出现和有用的修饰核苷酸化学方法，包括锁核酸（LNA），通过其更好地定量和区分扩增产物的能力，为改善和扩展PCR的临床应用创造了潜力，但要充分实现这一潜力，将需要强大的方法来设计双标记水解探针，并根据其序列、化学性质和模板互补性预测其杂交热力学。我们在此提出一种最近邻热力学模型，该模型能够准确预测与完美互补序列或包含错配碱基对的模板双链结合的短寡核苷酸的解链热力学。该模型可应用于纯DNA双链体或其中一条链包含任意数量和模式的LNA取代的双链体。由错配的DNA:DNA或LNA:DNA碱基对引起的双链稳定性扰动在吉布斯能量水平上进行处理，以保持回归模型参数的统计显著性。这种方法，当与模型对解链焓和熵的温度依赖性的考虑相结合时，允许准确预测纯DNA同型双链体或包含一个或两个独立错配碱基对的LNA取代的异型双链体的T(m)值。然后引入考虑溶液条件变化以及荧光染料和淬灭剂末端添加的项，以便该模型可用于准确预测并因此调整与完美匹配模板或包含非互补碱基的模板双链结合时纯DNA或LNA取代的水解探针的T(m)。该模型基于经典的最近邻热力学，因此对于在定量PCR或dPCR分析中需要区分和定量两个密切相关等位基因的探针的临床医生和生物学家应该是有用的。通过使用该模型设计等位基因特异性探针，在dPCR分析中完全区分和定量临床相关的突变等位基因（BRAF V600E和KIT D816V），证明了这种潜力。
