An important parameter in the design of PCR primers is the melting temperature (Tm). This is the temperature at which 50% of the primers and complementary sequences behave as double-stranded DNA molecules. Tm is necessary to set the PCR annealing temperature. Ideally, the annealing temperature is low enough to ensure efficient annealing of the primers to the desired sequence while still being high enough to reduce non-specific binding. Reasonable annealing temperatures range from 55 ° C to 70 ° C. The annealing temperature is generally set to be 5 ° C lower than the Tm of the primer. There are several formulas. Table 4 lists the two most common methods for determining the primer Tm. The first formula is derived from hybridization in a high salt solution and is suitable for primers of less than 18 bases. The second formula estimates Tm based on GC content. The most reliable method for determining the primer Tm is the proximity analysis method. This method predicts the hybridization stability of the primers from the sequence primary structure and the properties of adjacent bases. Most computer programs use neighborhood analysis. The Tm will vary greatly depending on the formula used and the sequence of the primers. Since most of the formulas provide an estimated Tm value, all annealing temperatures are only a starting point. The specificity can be improved by analyzing several reactions that gradually increase the annealing temperature. Start below the estimated Tm 5 ° C and gradually increase the annealing temperature in 2 ° C increments. Higher annealing temperatures reduce the formation of primer dimers and non-specific products. For best results, the two primers should have an approximate Tm value. If the Tm difference of the primer pair exceeds 5 ° C, the primer will exhibit a significant erroneous onset using a lower annealing temperature in the cycle. If the two primers Tm are different, the annealing temperature is set to be 5 ° C lower than the lowest Tm. Alternatively, to increase specificity, 5 cycles can be performed first at an annealing temperature designed according to a higher Tm, and then the remaining cycle is performed at an annealing temperature designed according to a lower Tm. This allows partial copies of the target template to be obtained under more stringent conditions. 5-Layer FFP2 Particulate Respirator 5 Layers Filter FFP2 Mask,5 Layers Particulate Respirators,5 Layers Filter Disposable Ffp2 Mask,Nonwoven 5 Layers Particulate Respirators Henan Aklly Filter Engineering Co., Ltd , https://www.akllyfilter.com
An important parameter in the design of PCR primers is the melting temperature (Tm). This is the temperature at which 50% of the primers and complementary sequences behave as double-stranded DNA molecules. Tm is necessary to set the pcr annealing temperature. Ideally, the annealing temperature is low enough to ensure efficient annealing of the primers to the desired sequence while still being high enough to reduce non-specific binding. Reasonable annealing temperatures range from 55 ° C to 70 ° C. The annealing temperature is generally set to be 5 ° C lower than the Tm of the primer.