Clinical Chemistry
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Clinical Chemistry 55: 1361-1371, 2009. First published April 30, 2009; 10.1373/clinchem.2008.118661
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(Clinical Chemistry. 2009;55:1361-1371.)
© 2009 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Identification and Prevention of Genotyping Errors Caused by G-Quadruplex– and i-Motif–Like Sequences

Jürgen J. Wenzel1,1, Heidi Rossmann1,a,1, Christian Fottner2, Stefan Neuwirth3, Carolin Neukirch1, Peter Lohse4, Julia K. Bickmann1, Timo Minnemann2, Thomas J. Musholt5, Brigitte Schneider-Rätzke6, Matthias M. Weber2 and Karl J. Lackner1

1 Departments of Clinical Chemistry and Laboratory Medicine; and2 Medicine I, Johannes Gutenberg University Mainz, Mainz, Germany;3 Bundeskriminalamt, Wiesbaden, Germany;4 Institute of Clinical Chemistry – Großhadern, Ludwig-Maximilians University Munich, Munich, Germany;5 Endocrine Surgery; and6 Institute for Human Genetics, Johannes Gutenberg University Mainz, Mainz, Germany.

aAddress correspondence to this author at: Johannes Gutenberg University Mainz, Department of Clinical Chemistry and Laboratory Medicine, Langenbeckstr. 1, 55131 Mainz, Germany. Fax +49-6131-17-6627; e-mail rossmann{at}zentrallabor.klinik.uni-mainz.de.

Background: Reliable PCR amplification of DNA fragments is the prerequisite for most genetic assays. We investigated the impact of G-quadruplex– or i-motif–like sequences on the reliability of PCR-based genetic analyses.

Methods: We found the sequence context of a common intronic polymorphism in the MEN1 gene (multiple endocrine neoplasia I) to be the cause of systematic genotyping errors by inducing preferential amplification of one allelic variant [allele dropout (ADO)]. Bioinformatic analyses and pyrosequencing-based allele quantification enabled the identification of the underlying DNA structures.

Results: We showed that G-quadruplex– or i-motif–like sequences can reproducibly cause ADO. In these cases, amplification efficiency strongly depends on the PCR enzyme and buffer conditions, the magnesium concentration in particular. In a randomly chosen subset of candidate single-nucleotide polymorphisms (SNPs) defined by properties deduced from 2 originally identified ADO cases, we confirmed preferential PCR amplification in up to 50% of the SNPs. We subsequently identified G-quadruplex and i-motifs harboring a SNP that alters the typical motif as the cause of this phenomenon, and a genomewide search based on the respective motifs predicted 0.5% of all SNPs listed by dbSNP and Online Mendelian Inheritance in Man to be potentially affected.

Conclusions: Undetected, the described phenomenon produces systematic errors in genetic analyses that may lead to misdiagnoses in clinical settings. PCR products should be checked for G-quadruplex and i-motifs to avoid the formation of ADO-causing secondary structures. Truly affected assays can then be identified by a simple experimental procedure, which simultaneously provides the solution to the problem. .






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Copyright © 2009 by the American Association for Clinical Chemistry.