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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 ]
The last 10-12 bases at the 3' end of a primer are sensitive to initiation of polymerase extension and general primer stability on the template binding site. The effect of a single mismatch at these last 10 bases at the 3' end of the primer depends on its position and local structure, reducing the primer binding, selectivity, and PCR efficiency.
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BLAST is one of the most widely used bioinformatics programs for sequence searching. [4] It addresses a fundamental problem in bioinformatics research. The heuristic algorithm it uses is much faster than other approaches, such as calculating an optimal alignment. This emphasis on speed is vital to making the algorithm practical on the huge ...
The necessary primer optimisation should be performed using specialized oligonucleotide design programs. Several solutions for automated primer design for gene synthesis have been presented so far. [12] [13] [14]
A primer dimer (PD) is a potential by-product in the polymerase chain reaction (PCR), a common biotechnological method. As its name implies, a PD consists of two primer molecules that have attached to each other because of strings of complementary bases in the primers.
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For example, oligonucleotides with a hairpin structure cannot act efficiently as a primer. However, after heating the reaction mix to the annealing temperature the primer will undergo a conformation change allowing the primer to form a linear structure instead, which enables the primer to attach to the target segment and begin PCR.
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