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Oligonucleotide Tm Predictor

It is a melting temperature predicter for short DNA sequences based on a simple phenomenological model of DNA denaturation. It takes into account the stacking and hydrogen bonding interaction (as DNA strength parameter). Length of the sequences, the salt concentration (taken as [Na+]) and nucleotide strand concentration (taken in M) to predict the melting temperature.

    




Read the DNA sequence from one end to the other & add up
the DNA strength parameter (Given in table 1).

↓

Calculate the strength parameter per base (E) as:
E = DNA strength parameter ⁄ Length of sequence

↓

Substitute the value of E, the length of sequence , the Na+ ion concentration and the nucleotide strand concentration of the solution in the given equation
Tm (°C) = (7.35 ×E) + [ 17.34 ×ln(Len) ] + [4.96 ×ln(Conc)] + [0.89× ln(DNA)] - 25.42


Figure:Flow chart to calculate the melting temperature


Table 1. DNA strength parameter value for each dinucleotide step

      Stack →        H-bond
↓
5
3
3
2
RY
YY
RR
YR
4+4
GC =13
CC =11
GG =11
CG =10
1+4
AC = 10
TC = 8
AG = 8
TG = 7
4+1
GT = 10
CT = 8
GA = 8
CA = 7
1+1
AT = 7
TT = 5
AA = 5
TA = 4
                                        

Example :-

Consider a 15 bp long sequence GACGAAGACCGCG , taken at 0.22M concentration whose Tm is reported experimentally to be 64.4 °C. The Melting temperature for this ia calculated as follows:

Read the sequence from 5' end to 3'end and add up the DNA strength parameter given in the Table1 for each dinucleotide step, moving one base at a time as :
GA =8, AC =10, CG =10, GA =8, AC = 10, CA=7 & so on.(For the given sequence of 15 base pairs, 14 dinucleotide step are obtained)

The DNA strength parameter for the given sequence is :
8+10+10+8+10+7+5+8+8+10+11+10+11+10+13+10=128

The strength parameter per base (E) is Calculated as: 128/15 = 8.53

Subsititute all the values in the above equation :
   Tm (°C)= (7.35 × 8.53) + [17.34 × ln(15)] + [4.96 × ln(0.22)] + [0.89 × ln(0.00002)] - 25.42

Predicted Tm = 65.04 °C