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primer3-org / primer3

Primer3 is a command line tool to select primers for polymerase chain reaction (PCR).
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ntthal negative Tm calculations #77

Closed cesimsek closed 2 months ago

cesimsek commented 7 months ago

Hi,

I am using primer3-py, which is an python-abstracted API of primer3.

I had the following issue with the homodimer calculations: https://github.com/libnano/primer3-py/issues/141

Briefly, the primer3 based (ntthal) command is as follows:

$ ./ntthal -mv 50 -dv 1.5 -n 0.6 -d 50 -s1 AGCTGCGTAATGACTAGGTA -s2 AGCTGCGTAATGACTAGGTA -path ./primer3_config/ 
Calculated thermodynamical parameters for dimer:    dS = -127.597   dH = -43000 dG = -3425.8    t = -10.5692
SEQ AGCTGCGTAATGA    GTA----------
SEQ              CTAG             
STR              GATC             
STR           ATG    AGTAATGCGTCGA

Which yielded a negative Tm for the predicted primer dimer. Cannot provide system info as this was based on the primer3-py tool, but in general what could be the reason for obtaining negative Tm?

Thanks!

DougTownsend commented 7 months ago

Melting temperature is calculated with the formula shown in the "Thermodynamics of the two-state model" section of this article https://en.wikipedia.org/wiki/Nucleic_acid_thermodynamics

Melting temperature is just the theoretical temperature at which, for a certain concentration of DNA, half of the DNA will be bound. A negative melting temperature just means that the secondary structure will only exist in very small concentrations at normal temperatures.

untergasser commented 2 months ago

Could not have explained it better. Just one add - the Tm is the temperature where half of the primer is free and the other half is bound in the structure. Higher temperatures shift it to the free form.

Best,

Andreas