Limits...
LOMA: a fast method to generate efficient tagged-random primers despite amplification bias of random PCR on pathogens.

Lee WH, Wong CW, Leong WY, Miller LD, Sung WK - BMC Bioinformatics (2008)

Bottom Line: Wet-lab validation showed that the generated random primers improve the amplification efficiency significantly.The blind use of a random primer with attached universal tag (random-tagged primer) in a PCR reaction on a pathogen sample may not lead to a successful amplification.Thus, the design of random-tagged primers is an important consideration when performing PCR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genome Institute of Singapore, Genome, Singapore. leewhc@gis.a-star.edu.sg

ABSTRACT

Background: Pathogen detection using DNA microarrays has the potential to become a fast and comprehensive diagnostics tool. However, since pathogen detection chips currently utilize random primers rather than specific primers for the RT-PCR step, bias inherent in random PCR amplification becomes a serious problem that causes large inaccuracies in hybridization signals.

Results: In this paper, we study how the efficiency of random PCR amplification affects hybridization signals. We describe a model that predicts the amplification efficiency of a given random primer on a target viral genome. The prediction allows us to filter false-negative probes of the genome that lie in regions of poor random PCR amplification and improves the accuracy of pathogen detection. Subsequently, we propose LOMA, an algorithm to generate random primers that have good amplification efficiency. Wet-lab validation showed that the generated random primers improve the amplification efficiency significantly.

Conclusion: The blind use of a random primer with attached universal tag (random-tagged primer) in a PCR reaction on a pathogen sample may not lead to a successful amplification. Thus, the design of random-tagged primers is an important consideration when performing PCR.

Show MeSH
Application of AES on a RSV sample. An RSV patient sample was amplified separately using primer A1, primer A2 and primer A3. Hybridization signals of probes after amplification by each primer are shown as a heatmap. The probes that have detectable signals above threshold are shown in orange/yellow in the corresponding heatmaps. The graph below the heatmaps shows our AES prediction for the three primers: A1 (orange line), primer A2 (pink line) and primer A3 (dark blue line). Our AES predictions closely matches the actual hybridization results, ie primer A3 performs slightly better than primer A2 but both A3 and A2 performs significantly better than A1 on RSV.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2553803&req=5

Figure 3: Application of AES on a RSV sample. An RSV patient sample was amplified separately using primer A1, primer A2 and primer A3. Hybridization signals of probes after amplification by each primer are shown as a heatmap. The probes that have detectable signals above threshold are shown in orange/yellow in the corresponding heatmaps. The graph below the heatmaps shows our AES prediction for the three primers: A1 (orange line), primer A2 (pink line) and primer A3 (dark blue line). Our AES predictions closely matches the actual hybridization results, ie primer A3 performs slightly better than primer A2 but both A3 and A2 performs significantly better than A1 on RSV.

Mentions: Recall that probes in regions of high AES are expected to be least affected by a poor amplification and thus have the correct high hybridization signals if the pathogen is present in the sample. For all the experiments, we observed that high AES significantly correlates to probe hybridization signal intensity above the detection threshold with a p-value of 2.2 × 10-16 using the Fisher's exact test. About 80% of the probes with high signal intensities (≥ mean + 3 standarad deviation) have high AES values. We also observed that primers A2 and A3 showed a tremendous improvement in overall PCR efficiency in amplifying RSV over primer A1. This increase in PCR efficiency resulted in increased hybridization of DNA to the probes and is reflected in the uniformly higher signal intensities observed using primer A2 and A3. This is illustrated in Figure 3. Further analysis of the RSV experiments revealed that only 20% to 30% of the 1948 RSV probes had signal intensities above detection threshold when primer A1 was used. By contrast, the use of primer A2 resulted in 60% to 71% of probes with signal intensities above detection threshold. Primer A3 fared slightly better than primer A2, resulting in more than 70% of the probes having signal intensities above detection threshold.


LOMA: a fast method to generate efficient tagged-random primers despite amplification bias of random PCR on pathogens.

Lee WH, Wong CW, Leong WY, Miller LD, Sung WK - BMC Bioinformatics (2008)

Application of AES on a RSV sample. An RSV patient sample was amplified separately using primer A1, primer A2 and primer A3. Hybridization signals of probes after amplification by each primer are shown as a heatmap. The probes that have detectable signals above threshold are shown in orange/yellow in the corresponding heatmaps. The graph below the heatmaps shows our AES prediction for the three primers: A1 (orange line), primer A2 (pink line) and primer A3 (dark blue line). Our AES predictions closely matches the actual hybridization results, ie primer A3 performs slightly better than primer A2 but both A3 and A2 performs significantly better than A1 on RSV.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553803&req=5

Figure 3: Application of AES on a RSV sample. An RSV patient sample was amplified separately using primer A1, primer A2 and primer A3. Hybridization signals of probes after amplification by each primer are shown as a heatmap. The probes that have detectable signals above threshold are shown in orange/yellow in the corresponding heatmaps. The graph below the heatmaps shows our AES prediction for the three primers: A1 (orange line), primer A2 (pink line) and primer A3 (dark blue line). Our AES predictions closely matches the actual hybridization results, ie primer A3 performs slightly better than primer A2 but both A3 and A2 performs significantly better than A1 on RSV.
Mentions: Recall that probes in regions of high AES are expected to be least affected by a poor amplification and thus have the correct high hybridization signals if the pathogen is present in the sample. For all the experiments, we observed that high AES significantly correlates to probe hybridization signal intensity above the detection threshold with a p-value of 2.2 × 10-16 using the Fisher's exact test. About 80% of the probes with high signal intensities (≥ mean + 3 standarad deviation) have high AES values. We also observed that primers A2 and A3 showed a tremendous improvement in overall PCR efficiency in amplifying RSV over primer A1. This increase in PCR efficiency resulted in increased hybridization of DNA to the probes and is reflected in the uniformly higher signal intensities observed using primer A2 and A3. This is illustrated in Figure 3. Further analysis of the RSV experiments revealed that only 20% to 30% of the 1948 RSV probes had signal intensities above detection threshold when primer A1 was used. By contrast, the use of primer A2 resulted in 60% to 71% of probes with signal intensities above detection threshold. Primer A3 fared slightly better than primer A2, resulting in more than 70% of the probes having signal intensities above detection threshold.

Bottom Line: Wet-lab validation showed that the generated random primers improve the amplification efficiency significantly.The blind use of a random primer with attached universal tag (random-tagged primer) in a PCR reaction on a pathogen sample may not lead to a successful amplification.Thus, the design of random-tagged primers is an important consideration when performing PCR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genome Institute of Singapore, Genome, Singapore. leewhc@gis.a-star.edu.sg

ABSTRACT

Background: Pathogen detection using DNA microarrays has the potential to become a fast and comprehensive diagnostics tool. However, since pathogen detection chips currently utilize random primers rather than specific primers for the RT-PCR step, bias inherent in random PCR amplification becomes a serious problem that causes large inaccuracies in hybridization signals.

Results: In this paper, we study how the efficiency of random PCR amplification affects hybridization signals. We describe a model that predicts the amplification efficiency of a given random primer on a target viral genome. The prediction allows us to filter false-negative probes of the genome that lie in regions of poor random PCR amplification and improves the accuracy of pathogen detection. Subsequently, we propose LOMA, an algorithm to generate random primers that have good amplification efficiency. Wet-lab validation showed that the generated random primers improve the amplification efficiency significantly.

Conclusion: The blind use of a random primer with attached universal tag (random-tagged primer) in a PCR reaction on a pathogen sample may not lead to a successful amplification. Thus, the design of random-tagged primers is an important consideration when performing PCR.

Show MeSH