iOligo EazyPrimer calculates basic PCR primer parameters and identifies secondary structures as you type your oligo’s sequence. The sequences length cannot exceed 90 mers.
The following parameters are calculated:
Sequence length, CG content, molecular weight, and Tm with and without salt adjustment. Default salt concentration is set to 50 mM Na+.
The following formulas are used for calculating Tm. If sequences less than 14 nucleotides then the same formula is used for standard and salt adjusted Tm:
Tm = 2*(nA+mT)+4*(pG+qC)
For longer sequences the formulas for standard and salt adjusted Tm are:
Tm=64.9+41*(pG+qC-16.4)/(nA+mT+pG+qC)
Tm=100.5+(41*(pG+qC)/(nA+mT+pG+qC))-(820/(nA+mT+pG+qC))+16.6*log([Na+])
Where n,m,p and q represent the number of respective base.
Secondary structures with stem length > 5 will be identified. A structure is considered a palindrome if loop length < 5 otherwise a hairpin structure is indicated.
Standard nomenclature with square brackets, indicating self-annealing areas, is used. For example, if the sequence AAACCCCCAAAAAGGGGG is entered then its secondry structure will be displayed as AAA[CCCCC]AAAAA[GGGGG]. Similarly, the palindrome sequence AAACCCCCGGGGG will be displayed as AAA[CCCCCGGGGG].
Note that EazyPrimer will find any secondary structure, if there is one. However, for sequences containing multiple structures only the first found will be displayed.