Sequence Analysis
Primer Dimer Check
Says whether a primer set forms self or cross dimers, where they pair, and whether a polymerase can extend from there.
An example primer pair. Start typing and it is replaced.
Examples:
2 primers, 3 combinations
| Combination | Type | ΔG (kcal/mol) | 3′ ΔG | 3′ bases |
|---|---|---|---|---|
| FWD_GAPDH | Self | -1.86 | 0.79 | 2 / 2 |
| REV_GAPDH | Self | -0.30 | 1.46 | 2 / 2 |
| FWD_GAPDH × REV_GAPDH | Cross | -2.20 | 0.54 | 2 / 1 |
ΔG is at 37 °C and 1 M Na⁺, SantaLucia (1998). A negative value means the duplex can form. ⚠️ Mismatches and loops are not part of the calculation, so these values come out more negative than the real ones.
FWD_GAPDH
SelfThe 3′ end is in this one — a polymerase can extend it
5'-ACCACAGTCCATGCCATCAC -3'
|| || ||
3'- CACTACCGTACCTGACACCA-5'ΔG 0.79 kcal/mol · 6 base pairs · longest run 2 · 3′ ends covered 2 and 2
The most stable register
5'-ACCACAGTCCATGCCATCAC -3'
| |||| |
3'- CACTACCGTACCTGACACCA-5'ΔG -1.86 kcal/mol · 6 base pairs · longest run 4 · 3′ ends covered 0 and 0
REV_GAPDH
SelfThe 3′ end is in this one — a polymerase can extend it
5'-TCCACCACCCTGTTGCTGTA -3'
||
3'- ATGTCGTTGTCCCACCACCT-5'ΔG 1.46 kcal/mol · 2 base pairs · longest run 2 · 3′ ends covered 2 and 2
The most stable register
5'-TCCACCACCCTGTTGCTGTA -3'
| || |
3'- ATGTCGTTGTCCCACCACCT-5'ΔG -0.30 kcal/mol · 4 base pairs · longest run 2 · 3′ ends covered 1 and 1
FWD_GAPDH × REV_GAPDH
CrossThe 3′ end is in this one — a polymerase can extend it
5'-ACCACAGTCCATGCCATCAC -3'
| | ||
3'- ATGTCGTTGTCCCACCACCT-5'ΔG 0.54 kcal/mol · 4 base pairs · longest run 2 · 3′ ends covered 2 and 1
The most stable register
5'- ACCACAGTCCATGCCATCAC-3'
| |||| |
3'-ATGTCGTTGTCCCACCACCT -5'ΔG -2.20 kcal/mol · 6 base pairs · longest run 4 · 3′ ends covered 0 and 0
How to use it
- Paste the primer set — one record per primer in FASTA, or a single sequence on its own. Up to twelve.
- Every primer is checked against itself and against every other one, so a set of four gives four self dimers and six pairs.
- Read the 3′ column of the table first. That is the register a polymerase can extend, and it is the one that turns a slow reaction into a competing product.
- The pictures underneath show which bases are stuck to which, which is what a redesign needs — moving one base off the 3′ end is usually enough.
What to know
- ΔG is at 37 °C and 1 M Na⁺, the reference state the nearest-neighbour parameters are quoted in. It is not corrected for your buffer, and correcting it would make a number from a rough model look exact.
- Negative ΔG means the duplex can form, and the more negative it is the more of it there is at equilibrium. ⛔ This tool will not give you a cut-off, because the one that matters depends on primer concentration, annealing temperature and what else is in the tube.
- ⚠️ The model has no mismatch, bulge or loop parameters. A gap between two paired stretches costs nothing here, so values come out more negative than the real ones. If it has to be wrong, that is the better direction — this is a list to check, not a verdict.
- A duplex anywhere along two primers slows a reaction down. A duplex covering a 3′ end is a different event, and it splits in two. If the partner strand carries bases past that 3′ end, the polymerase copies them, and the product competes for primer, dNTP and polymerase for the rest of the run. If the two ends finish flush there is nothing to copy and nothing is extended — but the primer is not going to a template either. ⛔ The first usually clears by moving a base or two at the 3′ end; the second needs a redesign.
- Self dimers are worth as much attention as cross dimers. A primer sits at 200–500 nM and meets another copy of itself far more often than it meets the template.
- The melting temperature of these primers is a separate question, and the primer Tm tool answers it with your actual buffer.
FAQs
There is no number this tool will hand you, and one that claimed to would be making it up. What counts as too much depends on how much primer is in the tube, what the annealing temperature is and how many cycles it runs for. What the table is for is comparison: between the combinations in your own set, and against the duplex the primer is supposed to make with the template, which is tens of kcal/mol.
Because it is the one that turns into a product. Bases stuck together in the middle of two primers hold them up; bases stuck together at a 3′ end give the polymerase somewhere to start, and what it makes then competes for every reagent for the rest of the run. The most stable register is frequently not that one — in the third example on this page it is a twelve-base duplex with neither end in it, while the register that matters is four bases long.
That the only thing that pairs there is too small to hold. Starting a helix costs free energy, and two base pairs of A and T return less than starting them cost. A positive number is the honest answer and it is left on screen rather than hidden, because “the worst these two can do is nothing” is worth reading.
Different tools use different parameter sets and different assumptions. This one is SantaLucia (1998) at 1 M Na⁺ with no mismatch terms, and it says so; another may use an older table, a salt correction, or a search that allows bulges. Numbers from two tools are comparable within each tool and not between them — which is also why this page shows the duplex rather than only the number.
Up to a hundred bases, but the model was built for the short duplexes primers make. A long oligo folds, and folding is a different calculation from sliding two strands past each other.
The other sequence tools
- Primer Tm Calculator — Melting temperature by the nearest-neighbour model
- DNA Reverse Complement — Reverse, complement, or reverse-complement a nucleotide sequence.
- GC Content Calculator — GC and AT content, skew, and GC along the sequence.