What are PCR artifacts?
What are PCR artifacts?
The following are common PCR artifacts:
-Primer dimers
Primer dimers are formed through self-complementarity at the 3' end of the amplification primers. Primer dimers are suspected if product is produced in a template-free reaction (negative control). To avoid primer dimers, primers shouldn't have complementarity at their 3' ends.
-Chimeric PCR products
Chimeric PCR products can be caused by incompletely extended template. In other words, single-stranded template that was not completely replicated due to premature polymerase termination can anneal to partially homologous template. This creates chimeric PCR products. To minimize chimeras, use the fewest possible PCR amplification cycles.
-PCR bias
PCR bias occurs when some sequences are amplified more efficiently than others due to preferential binding by PCR primers. If one sequence is amplified 10% more than another in one cycle, it will be 17.4-times more abundant after 30 cycles. To reduce PCR bias, use a high ramp rate between the denaturation and annealing steps and use low annealing temperatures. Long extension times (>180 sec) should be avoided.
-PCR drift
PCR drift is due to stochastic fluctuation in the interactions of PCR reagents, particularly in the early cycles when a very low template concentration exists. This artifact is observed in multiplex assays, where a loss of sensitivity is caused by the interactions between different sets of primers. It is important to carefully design primers for these types of assays.
-PCR generates high-molecular-weight products that barely migrate through the agarose gel
There is no good explanation for this artifact. Most researchers assume that this is caused by overcycling, since in the later stages of PCR, both single- and double-stranded molecules accumulate. Accumulation of such single-stranded molecules can create heteroduplexes by competing with the primers. Incomplete denaturation in later stages, when there is a high concentration of PCR products, prevents DNA strand separation, and thus a newly formed amplicon may remain bound to the previously made template. This process could repeat, trapping PCR products in a network of molecules.Another explanation for the origin of high-molecular-weight smears is the partial extension of templates during initial PCR cycles. Partial extensions could be generated by jumping artifacts—when a primer or single-stranded DNA anneals and extends from one priming site, then anneals partially to a homologous segment elsewhere (see Chimeric PCR products, above). Partially extended molecules can act as new primers, since they contain a free 3'-OH, and could generate chimeric molecules that combine the initial priming site and the ""jump"" site. Finally, this type of artifact can also be generated when a crude template is used for PCR. Products amplified directly from animal or plant tissues can become trapped in cell debris, which prevents them from migrating in the gel.
This problem can be solved by Proteinase K digestion of the amplified PCR product:
-Add 15 µl of loading buffer containing Proteinase K to the entire 50-µl PCR reaction.
OR
-Before loading your samples onto a gel, add 1 µl of the loading buffer containing Proteinase K to 4 µl of the PCR reaction.