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Primer3 is a command line tool to select primers for polymerase chain reaction (PCR).
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ntthal alignment not complete. Incomplete thermodynamic calculation? #24

Closed Permafacture closed 5 years ago

Permafacture commented 5 years ago

ntthal alignment does not show all matching bases as paired. I am unsure if the thermodynamic calculation considers the matched bases or not either.

After pulling master, make clean and make, I see this behavior

./ntthal -s1 GCTCGCGTCGCCTGCTGCTTTTGGCAGGGTAAAAGA -s2 GCTCGCGTCGCCTGCTGCTTTTGGCAGGGTAAAAGA
Calculated thermodynamical parameters for dimer:        dS = -297.472   dH = -99200     dG = -6939.1    t = 24.1818
SEQ     GCTCGCGTCG     TGCTTTTG     GTAAAAGA--
SEQ               CCTGC        GCAGG
STR               GGACG        CGTCC
STR       AGAAAATG     GTTTTCGT     GCTGCGCTCG

The last two 3bases (for example) on both strands are complementary but not displayed as matches. There's another T/A match 6 back from the 3 end that is also not shown as a match. I see this behavior more often than not (I.E. most heterodimers I look at have this happening).

Does this visualization reflect the thermodynamic calculations? If so, are those matches intentionally excluded? If so, why?

Thanks for reading

(edited get formatting correct)

Permafacture commented 5 years ago

This sequence makes it clear to me that this is a bug: tgcgagctataaataatgaaagct

./ntthal -s1 tgcgagctataaataatgaaagct -s2 tgcgagctataaataatgaaagct
Calculated thermodynamical parameters for dimer:    dS = -69.5073   dH = -24400 dG = -2842.31   t = -42.2412
SEQ TGCG    ATAAATAATGAAAGCT----
SEQ     AGCT
STR     TCGA
STR         AAGTAATAAATATCGAGCGT
triinuk commented 5 years ago

No, this and previous structures are correct ones. You are not familiar with two-state melting and thermodynamics of DNA duplexes. Besides Primer3 articles, do read article "The thermodynamics of DNA structural motifs" by SantaLucia and Hicks, 2004