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Multi-template polymerase chain reaction

View Article: PubMed Central - PubMed

ABSTRACT

PCR is a formidable and potent technology that serves as an indispensable tool in a wide range of biological disciplines. However, due to the ease of use and often lack of rigorous standards many PCR applications can lead to highly variable, inaccurate, and ultimately meaningless results. Thus, rigorous method validation must precede its broad adoption to any new application. Multi-template samples possess particular features, which make their PCR analysis prone to artifacts and biases: multiple homologous templates present in copy numbers that vary within several orders of magnitude. Such conditions are a breeding ground for chimeras and heteroduplexes. Differences in template amplification efficiencies and template competition for reaction compounds undermine correct preservation of the original template ratio. In addition, the presence of inhibitors aggravates all of the above-mentioned problems. Inhibitors might also have ambivalent effects on the different templates within the same sample. Yet, no standard approaches exist for monitoring inhibitory effects in multitemplate PCR, which is crucial for establishing compatibility between samples.

No MeSH data available.


Limit of detection in single and multi-template PCR. In the single-template PCR limit of detection (LOD) varies depending on the type of microorganism, background DNA, level of PCR inhibition and other factors. At present there is a lack of experimental evidences confirming that all types of the homologous targets have the same LOD in the multi-template PCR. On the contrary, there are experimental data supporting the idea that LODs of the different targets within the mixed sample differ (for discussion see the text).
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fig0015: Limit of detection in single and multi-template PCR. In the single-template PCR limit of detection (LOD) varies depending on the type of microorganism, background DNA, level of PCR inhibition and other factors. At present there is a lack of experimental evidences confirming that all types of the homologous targets have the same LOD in the multi-template PCR. On the contrary, there are experimental data supporting the idea that LODs of the different targets within the mixed sample differ (for discussion see the text).

Mentions: In multi-template assays, amplicons are sorted by fractionation and each amplicon type is treated as a direct reference to a particular template in the original sample. In microbial ecology studies, distinct types of amplicons are called operational taxonomic units (OTUs) and are treated as directly corresponding to a particular microorganism in the studied community. Based on this assumption, diversity indices for a particular community are estimated [125]. How justified is this approach? The particular traits and the quality of multi-template PCR should be considered before treating the results of an assay like this as a direct inventory of the original sample. The first intrinsic attribute of the multi-template PCR is the formation of artifactual products. As described above, PCR amplification of such complex samples almost inevitably leads to formation of artifacts and with current protocols, false signals cannot be distinguished from correct ones. The second obstacle is the unknown detection limit of multi-template PCR. This question has not been properly analyzed so far. The limit of detection is the minimum amount of target DNA sequence that can be detected in a sample with a given level of confidence [126]. Imagine a sample where homologous templates A, B, C, D and F are present in 10,000, 1000, 100, 10 and 1 copies respectively. Will templates D and F be amplified in a mix with the more abundant A, B and C homologs? Where does the low detection limit lie? Are the detection limits equal for all targets in the mixture or are they individual (Fig. 3)? How can detection limits be estimated for each particular assay?


Multi-template polymerase chain reaction
Limit of detection in single and multi-template PCR. In the single-template PCR limit of detection (LOD) varies depending on the type of microorganism, background DNA, level of PCR inhibition and other factors. At present there is a lack of experimental evidences confirming that all types of the homologous targets have the same LOD in the multi-template PCR. On the contrary, there are experimental data supporting the idea that LODs of the different targets within the mixed sample differ (for discussion see the text).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121205&req=5

fig0015: Limit of detection in single and multi-template PCR. In the single-template PCR limit of detection (LOD) varies depending on the type of microorganism, background DNA, level of PCR inhibition and other factors. At present there is a lack of experimental evidences confirming that all types of the homologous targets have the same LOD in the multi-template PCR. On the contrary, there are experimental data supporting the idea that LODs of the different targets within the mixed sample differ (for discussion see the text).
Mentions: In multi-template assays, amplicons are sorted by fractionation and each amplicon type is treated as a direct reference to a particular template in the original sample. In microbial ecology studies, distinct types of amplicons are called operational taxonomic units (OTUs) and are treated as directly corresponding to a particular microorganism in the studied community. Based on this assumption, diversity indices for a particular community are estimated [125]. How justified is this approach? The particular traits and the quality of multi-template PCR should be considered before treating the results of an assay like this as a direct inventory of the original sample. The first intrinsic attribute of the multi-template PCR is the formation of artifactual products. As described above, PCR amplification of such complex samples almost inevitably leads to formation of artifacts and with current protocols, false signals cannot be distinguished from correct ones. The second obstacle is the unknown detection limit of multi-template PCR. This question has not been properly analyzed so far. The limit of detection is the minimum amount of target DNA sequence that can be detected in a sample with a given level of confidence [126]. Imagine a sample where homologous templates A, B, C, D and F are present in 10,000, 1000, 100, 10 and 1 copies respectively. Will templates D and F be amplified in a mix with the more abundant A, B and C homologs? Where does the low detection limit lie? Are the detection limits equal for all targets in the mixture or are they individual (Fig. 3)? How can detection limits be estimated for each particular assay?

View Article: PubMed Central - PubMed

ABSTRACT

PCR is a formidable and potent technology that serves as an indispensable tool in a wide range of biological disciplines. However, due to the ease of use and often lack of rigorous standards many PCR applications can lead to highly variable, inaccurate, and ultimately meaningless results. Thus, rigorous method validation must precede its broad adoption to any new application. Multi-template samples possess particular features, which make their PCR analysis prone to artifacts and biases: multiple homologous templates present in copy numbers that vary within several orders of magnitude. Such conditions are a breeding ground for chimeras and heteroduplexes. Differences in template amplification efficiencies and template competition for reaction compounds undermine correct preservation of the original template ratio. In addition, the presence of inhibitors aggravates all of the above-mentioned problems. Inhibitors might also have ambivalent effects on the different templates within the same sample. Yet, no standard approaches exist for monitoring inhibitory effects in multitemplate PCR, which is crucial for establishing compatibility between samples.

No MeSH data available.