What is melting temperature of a primer? - primer melting temperature calculation
This means that if were the temperature at which 50% of the same type of DNA molecule forms a stable double helix and 50% separated molecules from a single thread.
Sunday, January 24, 2010
Primer Melting Temperature Calculation What Is Melting Temperature Of A Primer?
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A primer is a nucleic acid chain or a related molecule that serves as a starting point for DNA replication. An introduction is necessary because most DNA polymerases, enzymes that catalyze DNA replication can not start a new synthesis of DNA strand from scratch, but you can add an existing string nucleotides.
Melting point: melting temperature (TM) By definition, the temperature at which half of the duplex DNA to be separated single-stranded and duplex stability is suggested. Primers with melting temperatures of about 52-58 ° C, usually achieve better results. Primers with melting temperatures above 65oC have a tendency for secondary annealing. The GC content of the sequence gives a good overview of the MT. All our products are accepted by using the nearest neighbor thermodynamic theory as a superior method of estimation, which is regarded as the youngest and disposal.
Formula for calculating the Tm:
The melting temperature Tm (OK) = (delta H / delta S + R ln (C)), or the melting temperature Tm (° C) = (delta H / delta SR + ln (C))-273.15where
Delta H (kcal / mol): H is the enthalpy. The enthalpy is the amount of heat they possess substances. Delta H is the enthalpy change. In the above formula, the delta H is by getting all the pairs of nucleotide di-enthalpy values of each base pair nearest neighbors.
Delta S (kcal / mol): S is the amount of the exposure system disorder is called entropy. Delta S is the entropy change. Here are by getting all the pairs of di-nucleotide entropy values of each base pair closest neighbors. An update more salt than a near neighbor, the parameters were carried out from studies of DNA melting in 1M Na + buffer and is the standard condition used for all calculations.
Delta S (salt corrected) = delta S (1M NaCl) + 0.368 x N x ln ([Na +])
Where
N is the number of pairs of nucleotides in the first (first length -1).
[Na +] is the salt of the equivalent value in mm.
[Na +] Calculation:
[Na +] = concentration of monovalent ions 4 x Mg 2 + free.
3.Primer temperature annealing: The primer mergeEffort is to estimate the stability of DNA-DNA hybrid and essential in setting the annealing temperature. Too high for her is enough hybrid model, which is initially in a lower yield of PCR products. Ta may be too low, can not cause-specific products by a large number of base pairs caused discrepancies. Tolerance imbalance shows that the greatest influence on the specificity of PCR.
Ta = 0.3 XT (first) + 0.7 tons (total) - 14.9
where,
Tm (first) = melting temperature of the primers
T (product) = melting temperature of the product
shakira
http://www.newton.dep.anl.gov/askasci/bi ...
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