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          Institute: MPI für evolutionäre Anthropologie     Collection: Publications MPI for Evolutionary Anthropology     Display Documents

ID: 21659.0, MPI für evolutionäre Anthropologie / Publications MPI for Evolutionary Anthropology
The effects of sequence length and oligonucleotide mismatches on 5 ' exonuclease assay efficiency
Authors:Smith, S.; Vigilant, Linda; Morin, Phillip A.
Date of Publication (YYYY-MM-DD):2002-10-15
Title of Journal:Nucleic Acids Research
Journal Abbrev.:Nucleic Acids Res.
Issue / Number:20
Start Page:e111
End Page:e111
Sequence Number of Article:e111
Review Status:Peer-review
Audience:Not Specified
Abstract / Description: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.
Comment of the Author/Creator:Date: 2002, OCT 15
External Publication Status:published
Document Type:Article
Communicated by:Gisela Lausberg
Affiliations:MPI für evolutionäre Anthropologie/Department of Primatology
MPI für evolutionäre Anthropologie/Department of Evolutionary Genetics
Identifiers:ISI:000178826700008 [ID No:1]
ISSN:0305-1048 [ID No:2]
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