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Nucleic acid thermodynamics is the study of how temperature affects the nucleic acid structure of double-stranded DNA (dsDNA). The melting temperature (Tm) is defined as the temperature at which half of the DNA strands are in the random coil or single-stranded (ssDNA) state. Tm depends on the length of the DNA molecule and its specific ...
The result is a stem-loop primer that excludes annealing involving shorter overlaps, but permits annealing of the primer to its fully complementary sequence in the target. Chimeric primers: some DNA bases in the primer are replaced with RNA bases, creating a chimeric sequence. The melting temperature of a chimeric sequence with another chimeric ...
The annealing temperature at the initial cycles is usually a few degrees (3–5 °C) above the T m of the primers used, while at the later cycles, it is a few degrees (3–5 °C) below the primer T m. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the ...
GC-content. Nucleotide bonds showing AT and GC pairs. Arrows point to the hydrogen bonds. In molecular biology and genetics, GC-content (or guanine-cytosine content) is the percentage of nitrogenous bases in a DNA or RNA molecule that are either guanine (G) or cytosine (C). [1] This measure indicates the proportion of G and C bases out of an ...
The annealing temperature in the early cycles is usually 3–5 °C above the standard T m of the primers used, while in the later cycles it is a similar amount below the T m. The initial higher annealing temperature leads to greater specificity for primer binding, while the lower temperatures permit more efficient amplification at the end of ...
COLD-PCR (co -amplification at l ower d enaturation temperature PCR) is a modified polymerase chain reaction (PCR) protocol that enriches variant alleles from a mixture of wildtype and mutation -containing DNA. The ability to preferentially amplify and identify minority alleles and low-level somatic DNA mutations in the presence of excess ...
The annealing temperature during a polymerase chain reaction determines the specificity of primer annealing. The melting point of the primer sets the upper limit on annealing temperature. At temperatures just below this point, only very specific base pairing between the primer and the template will occur. At lower temperatures, the primers bind ...
NetPrimer is a gratis web-based tool used for analysing primers used in PCR to amplify a DNA sequence. The software predicts the melting temperature of the primers using the nearest neighbor thermodynamic algorithm. The accurate prediction of the melting temperature (Tm) is one of the most important factors that governs the success of a PCR ...