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Publikationstyp: Zeitschriftenaufsatz
Dokumenttyp: Originalarbeit

Jahr: 2002

AutorInnen: Smith, S; Vigilant, L; Morin, PA

Titel: The effects of sequence length and oligonucleotide mismatches on 5" exonuclease assay efficiency.

Quelle: Nucleic Acids Res. 2002; 30(20):e111

Autor/innen der Vetmeduni Vienna:

Smith Steven

Diese Publikation wurde nicht im Namen der Vetmeduni Vienna erstellt und ist deshalb ausschließlich der persönlichen Publikationsliste des/der Autors/Autorin zugeordnet!

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.

Keywords Pubmed: Animals
Base Pair Mismatch
Base Sequence
Binding Sites
Gene Frequency
Genes, myc
Microsatellite Repeats
Oligonucleotide Probes/chemistry*
Oligonucleotide Probes/metabolism
Pan troglodytes/genetics
Phosphodiesterase I
Phosphoric Diester Hydrolases/metabolism*
Polymerase Chain Reaction/methods*
Reproducibility of Results
Templates, Genetic

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