search

primer3-org / primer3

Primer3 is a command line tool to select primers for polymerase chain reaction (PCR).
GNU General Public License v2.0
218 stars 62 forks source link

ntthal negative Tm calculations #77

Open cesimsek opened 3 months ago

cesimsek commented 3 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 3 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.