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The design of appropriate short or long primer pairs is only one goal of PCR product prediction. Other information provided by in silico PCR tools may include determining primer location, orientation, length of each amplicon , simulation of electrophoretic mobility, identification of open reading frames , and links to other web resources.
The free NCBI tool Primer-BLAST integrates primer design and BLAST search into one application, [6] as do commercial software products such as ePrime and Beacon Designer. Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design by giving melting and annealing temperatures, etc. [7]
Primer dimer formation often competes with formation of the DNA fragment of interest, and may be avoided using primers that are designed such that they lack complementarity—especially at the 3' ends—to itself or the other primer used in the reaction. If primer design is constrained by other factors and if primer-dimers do occur, methods to ...
A real-time polymerase chain reaction (real-time PCR, or qPCR when used quantitatively) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real time), not at its end, as in conventional PCR.
The kit adapted for carrying out the method includes a pair of primers to amplify the locus and optionally polymerase chain reaction reagents. [17] LCR can be used to diagnose tuberculosis. [18] The sequence containing protein antigen B is targeted by four oligonucleotide primers—two for the sense strand, and two for the antisense strand. The ...
Primer Premier is a bioinformatics software used for various PCR applications. It supports the design of degenerate primers for amplifying a related set of nucleotide sequences for the detection of common traits amongst organisms, as well as to determine heredity. [1] The software also designs tagged and nested primers for multiplex PCR ...
For this reason, it is critical to accurately predict the melting curve of PCR products when designing primers that will distinguish sequence variants. Specialty software, such as uMelt [13] and DesignSignatures, [14] are available to help design primers that will maximize melting curve variability specifically for high-resolution melting assays.
This includes the full set of conditions used for the reaction, the volume of both the reaction and the cDNA, the concentrations for the probes, magnesium ions, and dNTPs, what kind of polymerase was used and its concentration, what kit was used and its manufacturer, what additives to the reaction were used, who manufactured the qPCR machine ...