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A strip of eight PCR tubes, each containing a 100 μL reaction mixture Placing a strip of eight PCR tubes into a thermal cycler. The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed study.
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 ]
Annealing of the 3' end of one primer to itself or the second primer may cause primer extension, resulting in the formation of so-called primer dimers, visible as low-molecular-weight bands on PCR gels. [15] Primer dimer formation often competes with formation of the DNA fragment of interest, and may be avoided using primers that are designed ...
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.
The specificity of fluorescent reporter probes also prevents interference of measurements caused by primer dimers, which are undesirable potential by-products in PCR. However, fluorescent reporter probes do not prevent the inhibitory effect of the primer dimers, which may depress accumulation of the desired products in the reaction.
The primer design for all primers pairs has to be optimized so that all primer pairs can work at the same annealing temperature during PCR. Multiplex-PCR was first described in 1988 as a method to detect deletions in the dystrophin gene. [1] It has also been used with the steroid sulfatase gene. [2]
The polymerase chain reaction is the most widely used method for in vitro DNA amplification for purposes of molecular biology and biomedical research. [1] This process involves the separation of the double-stranded DNA in high heat into single strands (the denaturation step, typically achieved at 95–97 °C), annealing of the primers to the single stranded DNA (the annealing step) and copying ...
A primer binding site is a region of a nucleotide sequence where an RNA or DNA single-stranded primer binds to start replication. The primer binding site is on one of the two complementary strands of a double-stranded nucleotide polymer , in the strand which is to be copied, or is within a single-stranded nucleotide polymer sequence.