How does NimbleGen calculate Tm of probes?
If an isothermal design is requested, we screen probes with one of two algorithms, depending on probe length, for a target Tm of 76C. For probes greater than or equal to 36 nucleotides we calculate the melting temperature of substr(seq, start, length) using the formula from Bolton and McCarthy, PNAS 84:1390 (1962) as presented in Sambrook, Fritsch and Maniatis, Molecular Cloning, p 11.46 (1989, CSHL Press). Tm = 81.5 + 16.6(log10([Na+])) + .41*(%GC) – 600/length. Where [Na+] is the molar sodium concentration, (%GC) is the percent of Gs and Cs in the sequence, and length is the length of the sequence. For shorter oligos, we use the melting temperature of the given oligo, as calculated using eqn(ii) in Rychlik, Spencer, Roads, Nucleic Acids Research, vol 18, no 21, page 6410, with tables of nearest-neighbor thermodynamics for DNA bases as provided in Breslauer, Frank, Bloecker, and Markey, Proc. Natl. Acad. Sci. USA, vol 83, page 3748.
