引物分析：Tm、GC 含量、二聚体与发夹
粘贴引物，一次得到 Tm、GC%、热力学参数，以及二聚体和发夹筛查。
做 PCR 之前，把引物粘贴进来：可以是一条寡核苷酸、一对正反向引物，或者整个引物列表。工具按 SantaLucia 1998 最近邻参数计算 Tm、GC 含量和 ΔH / ΔS / ΔG37，并筛查自身二聚体、引物之间的二聚体和发夹结构，每个结构都画成碱基配对图。寡核苷酸、Na⁺、Mg²⁺ 和 dNTP 浓度可以按你的反应体系设置；批量结果可以导出 CSV。
工具界面目前为英文：拖入文件或粘贴序列即可，结果中的数字与图表和英文版完全相同。
One oligo, a primer pair, or a whole list — it reads whichever you paste.
Paste an oligo above for its Tm, GC, hairpin and self-dimer. Two get checked against each other as well; three or more come back as a table.
Tm uses SantaLucia (1998) nearest-neighbor thermodynamics with a salt correction for Na⁺ and a monovalent-equivalent for Mg²⁺/dNTP. Hairpin and dimer ΔG are screening estimates — confirm critical designs with a dedicated secondary-structure tool. A 3′-end dimer/hairpin is the most likely to disrupt PCR.
Check the order against the map before ordering
The commonest wrong primer is a copying error between the design and the order form: a reverse primer reversed but not complemented, the top strand pasted where the reverse primer belongs, a base dropped or doubled. Give the map these were designed from and each oligo is located on it — strand, position, tail, the bases that disagree — and when one binds nowhere, the specific mistake that explains it and the sequence that was meant. It also simulates every product the order can make, and says why a named F/R pair makes none.
A whole set of oligos, every pair
One oligo against itself is the first question; a multiplex panel or an inherited primer list needs every PAIR checked too. This screens up to 384 oligos for cross-dimers, flags duplicate sequences by name, reports the Tm spread across the set, and holds a 3′-end pairing to a stricter threshold than one that only sequesters a primer internally.
▸Conflict thresholds: -6 kcal/mol at a 3′ end · -9 internal
A pairing that occupies an oligo's 3′ end counts as a conflict at a weaker ΔG than one that only sequesters primer internally, because that end is where extension starts. Defaults are −6 and −9.
FASTA headers become the names; one sequence per line gets named oligo_1, oligo_2, … and a “name,sequence” row keeps the name you gave it.
Paste two or more oligos in the box above to screen every pair in the set.
The full folding model, for one oligo or two
The nearest-neighbor screen above finds the most stable pairing. When you want the actual minimum-free-energy structure — the loops, the bulges, the dot-bracket — this folds one oligo or two together with ViennaRNA’s published loop model at your temperature and salt.
Paste an oligo in the box at the top of this page and it is folded here.
One oligo or two, at most 200 nt each — the loop model is for oligos, not templates, and a longer strand is refused.
A whole multiplex panel, not just its oligos
The screens above judge oligos against each other. A multiplex panel also has to agree on ONE annealing temperature and produce amplicons a gel can tell apart, and a pair that dimerises is only one of the ways it fails. This checks the panel as a panel: Tm spread across the targets, product sizes that resolve, and the pairs that cannot go in the same tube at all.
Design, edit, and verify complete constructsSeqStudio combines sequence editing and annotation, plasmid maps, primer/cloning/CRISPR design, Sanger verification, and GenBank/SnapGene files in one workspace.
使用步骤
- 1粘贴一条、一对或多条引物（每行一条）。
- 2按反应体系设置寡核苷酸、Na⁺、Mg²⁺、dNTP 浓度。
- 3查看 Tm、GC%、二聚体和发夹结果；批量时导出 CSV。
常见问题
这里的 Tm 和厂商计算器的结果为什么不一样？
Tm 取决于所用的热力学参数和盐、Mg²⁺、引物浓度。这里使用 SantaLucia 1998 最近邻参数和你设置的浓度；厂商计算器往往针对自家聚合酶和缓冲液，使用不同的规则。
一对引物的 Tm 应该相差多少以内？
通常希望一对引物的 Tm 相差在 2–5 °C 以内，同时避免 3′ 端互补形成引物二聚体。本页会同时显示两条引物的 Tm 和它们之间的二聚体。
更多中文入口见 SeqBench 中文。