Chimeras

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Chimeras

A chimera is a spurious gene sequence derived from more than one DNA template (Meyerhans et al., 1990; Shuldiner et al., 1989). Chimeras form during PCR amplification, where sequence synthesis starts from one template, is interupted (for example, through damage or degradation of the template; Pääbo et al., 1990), and then continues from another template sharing a degree of localised homology to the original. In this way, a chimera can form from two or more phylogenetically distinct gene fragments, derived from different parental templates. Clearly, chimera formation has the potential to occur when there is more than one DNA template available within the PCR mixture; as is the case when PCR amplification is performed on pure DNA samples contaminated with foreign genetic material, or, as is increasingly common, on DNA extracted from environmental or medical samples (e.g., Kopczynski et al., 1994).

Note...

Chimera formation is the most frequent 16S rDNA artifact currently displayed by new submissions to the public repositories (Ashelford et al., 2005). Chimeras tend also to be the most insidious artifacts, since if undetected, they can be responsible for spurious phylogenetic reconstructions, inaccurate taxonomic identifications and over-estimations of microbial diversity.

Chimera formation becomes more likely, the greater the amount of the homology that exists between the different DNA templates present in the PCR mixture. All 16S rDNA sequences share a degree of homology due to the highly conserved nature of the 16S rRNA gene; so much so, chimeras straddling different phyla can be found within clone libraries with surprising frequency (43.1% of chimeras surveyed by Ashelford et al., 2005). Chimeras are even more likely to form when different templates within the PCR mixture derive from the same phylum, and the likelihood of chimera formation increases as evolutionary distance between templates decreases. Unfortunately, this also means that the most common chimeras will be those formed from the most closely related species and thus the most difficult to detect.

Wang and Wang (1996) were able to show that up to 30% of clones within a clone library of 16S rRNA will be chimeric - depending on methodology used. Factors known to increase the likelihood of chimera formation include the number of PCR cycles used, the stringency of the reaction conditions, and the harshness of the DNA extraction method employed. Chimeras have been recognised as a problem for almost as long as PCR amplification has been used, and in that time a number of methods for detecting chimeras have been developed, including Chimera_Check, Bellerophon and ChimeraBuster. Recently, the programs Pintail and Mallard have been made available.

Finally it should be noted that, whilst chimera formation discussed here is clearly an artifical recombination event, there is some evidence to suggest that natural recombination between 16S rDNA, presumably as a consequence of horizontal gene transfer, does occur, leading to the formation of naturally occurring chimera-like sequences, thought this would appear to be a rare event and it is unclear whether such genes are functional.

Synonyms

Jumping PCR products
Shuffle-genes
In vitro recombination products
Recombination