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Nucleic Acids Research, 2002, Vol. 30, No. 20 e111
© 2002 Oxford University Press

The effects of sequence length and oligonucleotide mismatches on 5' exonuclease assay efficiency

Steve Smith, Linda Vigilant1 and Phillip A. Morin*

Laboratory for Conservation Genetics and 1 Department of Primatology, Max Planck Institute for Evolutionary Anthropology, Inselstrasse 22, 04103 Leipzig, Germany

*To whom correspondence should be addressed. Tel: +49 341 995 2538; Fax: +49 341 995 2555; Email: morin{at}raredna.com
+AF519445–AF519458

Although increasingly used for DNA quantification, little is known of the dynamics of the 5' exonuclease assay, particularly in relation to amplicon length and mismatches at oligonucleotide binding sites. In this study we used seven assays targeting the c-myc proto-oncogene to examine the effects of sequence length, and report a marked reduction in efficiency with increasing fragment length. Three of the assays were further tested on 15 mammalian species to gauge the effect of sequence differences on performance. We show that the effects of probe and primer binding site mismatches are complex, with single point mutations often having little effect on assay performance, while multiple mismatches to the probe caused the greatest reduction in efficiency. The usefulness of the assays in predicting rates of ‘allelic dropout’ and successful polymerase chain reactions (PCRs) in microsatellite genotyping studies is supported, and we demonstrate that the use of a fragment more similar in size to typical microsatellites (190 bp) is no more informative than a shorter (81 bp) fragment. The assays designed for this study can be used directly for quantification of DNA from many mammalian species or, alternatively, information provided here can be used to design unique sequence-specific assays to maximise assay efficiency.


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