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

Wah Heng Lee12, Christopher W Wong1, Wan Yee Leong1, Lance D Miller1 and Wing Kin Sung12*

Author Affiliations

1 Genome Institute of Singapore, 60 Biopolis Street #02-01, Genome, Singapore

2 Department of Computer Science, National University of Singapore, 10 Kent Ridge Crescent, Singapore

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BMC Bioinformatics 2008, 9:368  doi:10.1186/1471-2105-9-368

Published: 10 September 2008

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.