The melting temperature (Tm) is the temperature at which half of a DNA duplex is separated into single strands. Calculating Tm depends on the length and sequence of the DNA molecule. Here are the methods for calculating Tm:
Tm Calculation Methods
There are primarily two methods to calculate the melting temperature (Tm) of DNA, depending on the length of the sequence:
1. For Short Sequences (Less than 14 nucleotides)
This method is a simple approximation:
Formula: Tm = (wA + xT) 2 + (yG + zC) 4
Where:
- w = number of Adenine (A) bases
- x = number of Thymine (T) bases
- y = number of Guanine (G) bases
- z = number of Cytosine (C) bases
Example:
Consider a sequence: ATGC
- A = 1, T = 1, G = 1, C = 1
- Tm = (1 2) + (1 2) + (1 4) + (1 4) = 2 + 2 + 4 + 4 = 12 °C
2. For Longer Sequences (More than 13 nucleotides)
For longer sequences, a more accurate equation is typically used:
Formula: Tm = 64.9 + 41 * (yG + zC - 16.4) / (wA + xT + yG + zC)
Where:
- w = number of Adenine (A) bases
- x = number of Thymine (T) bases
- y = number of Guanine (G) bases
- z = number of Cytosine (C) bases
Example:
Consider a sequence: ATGCGCATGCGCAT
- A = 4, T = 4, G = 6, C = 4
- Tm = 64.9 + 41 * (6 + 4 - 16.4) / (4 + 4 + 6 + 4)
- Tm = 64.9 + 41 * (-6.4) / 18
- Tm = 64.9 + (-14.6)
- Tm = 50.3 °C (approximately)
Factors Affecting Tm
Besides sequence length and base composition, other factors influence Tm:
- Salt Concentration: Higher salt concentrations generally increase Tm because they stabilize the DNA duplex.
- DNA Concentration: Higher DNA concentrations can slightly increase Tm.
- Presence of Mismatches: Mismatches (incorrect base pairings) decrease Tm.
- Formamide Concentration: Formamide destabilizes DNA, decreasing Tm.
Software and Online Tools
Many online tools and software programs can calculate Tm, taking into account these various factors. These tools often provide more accurate Tm predictions than the simple formulas above. A commonly used tool is the OligoAnalyzer tool from IDT (Integrated DNA Technologies).
In summary, calculating Tm involves using formulas specific to sequence length and considering other factors like salt concentration for better accuracy. Online tools are also available for more precise calculations.
