Use of a DNA toolbox for the characterization of mutation scanning methods. I: Construction of the toolbox and evaluation of heteroduplex analysis

W. Edward Highsmith, Qian Jin, Arun J. Nataraj, Jacquelyn M. O'Connor, Valerie D. Burland, Wendy R. Baubonis, Foner P. Curtis, Noriko Kusukawa, Mark M. Garner

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

A systematic characterization of the effects of important physical parameters on the sensitivity and specificity of methods in searching for unknown base changes (mutations or single nucleotide polymorphisms) over a relatively long DNA segment has not been previously reported. To this end, we have constructed a set of molecules of varying G+C content (40, 50, and 60% GC) having all possible base changes at a particular location - the 'DNA toolbox'. Exhaustive confirmatory sequencing demonstrated that there were no other base changes in any of the clones. Using this set of clones as polymerase chain reaction (PCR) templates, amplicons of various lengths with the same base mutated to all other bases were generated. The behavior of these constructs in manual and automated heteroduplex analysis was analyzed as a function of the size and overall base content of the fragment, the nature and location of the base change. Our results show that in heteroduplex analysis, the nature of the mismatched base pair is the overriding determinant for the ability to detect the mutation, regardless of fragment length, GC content, or the location of the mutation.

Original languageEnglish (US)
Pages (from-to)1186-1194
Number of pages9
JournalElectrophoresis
Volume20
Issue number6
DOIs
StatePublished - Jun 16 1999

Keywords

  • Heteroduplex analysis
  • Mutation scanning

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

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    Highsmith, W. E., Jin, Q., Nataraj, A. J., O'Connor, J. M., Burland, V. D., Baubonis, W. R., Curtis, F. P., Kusukawa, N., & Garner, M. M. (1999). Use of a DNA toolbox for the characterization of mutation scanning methods. I: Construction of the toolbox and evaluation of heteroduplex analysis. Electrophoresis, 20(6), 1186-1194. https://doi.org/10.1002/(SICI)1522-2683(19990101)20:6<1186::AID-ELPS1186>3.0.CO;2-6