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Non-specific binding of degenerate primers is also common. Manipulation of annealing temperature and magnesium ion concentration may be used to increase specificity. For example, lower concentrations of magnesium or other cations may prevent non-specific primer interactions, thus enabling successful PCR. A "hot-start" polymerase enzyme whose ...
Hot start PCR reduces the amount of non-specific binding through limiting reagents until the heating steps of PCR – limit the reaction early by limiting Taq DNA polymerase in a reaction. Non-specific binding often leads to primer dimers and mis-primed/false primed targets. [11] These can be rectified through modified methods such as:
This allows amplification for a low number of runs in the first round, limiting non-specific products. The second nested primer set should only amplify the intended product from the first round of amplification and not non-specific product. This allows running more total cycles while minimizing non-specific products.
In allele-specific PCR the opposite is done. At least one of the primers is chosen from a polymorphic area, with the mutations located at (or near) its 3'-end. Under stringent conditions, a mismatched primer will not initiate replication, whereas a matched primer will. The appearance of an amplification product therefore indicates the genotype.
For example, chain-termination-based kits are commercially available that contain the reagents needed for sequencing, pre-aliquoted and ready to use. Limitations include non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA sequence, and DNA secondary structures affecting the fidelity of the sequence.
DNA is bisulfite-converted, and bisulfite-specific primers are annealed to the sequence up to the base pair immediately before the CpG of interest. The primer is allowed to extend one base pair into the C (or T) using DNA polymerase terminating dideoxynucleotides, and the ratio of C to T is determined quantitatively.
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. [2]
If there is a high likelihood of non-specific oligonucleotide retention in the reaction conditions, non specific competitors, which are small molecules or polymers other than the SELEX library that have similar physical properties to the library oligonucleotides, can be used to occupy these non-specific binding sites. [13]