引物設計軟件都可以給出Tm,與引物長度、堿基組成、引物使用緩沖的離子強度有關。
長度為25mer以下的引物,Tm計算公式為:Tm = 4℃(G + C)+ 2℃(A + T)
對于更長的寡聚核苷酸,Tm計算公式為:
Tm = 81.5 + 16.6 x Log10[Na+] + 0.41 (%GC) - 600/size
公式中,Size = 引物長度。
Tm的定義:Tm = Temperature at which 50% of a given oligonucleotide is hybridized to its complementary strand. In the absence of destabilizing agents, like formamide or urea, Tm will depend on 3 major parameters: The sequence: a GC-rich sequence has a higher melting temperature. The strand concentration: high oligonucleotide concentrations favor hybrid formation, which results in a higher melting temperature. The salt concentration: high ionic strength results in a higher Tm as cations stabilize the DNA duplexes.
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原創作者:上海恒遠生物技術發展有限公司