Received on October 1, 2008
Accepted on March 24, 2009

Molecular Diagnostics and Genetics

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

¹ Department of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Mainz, Mainz, Germany
² Department of 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, Johannes Gutenberg University Mainz, Mainz, Germany
⁶ Institute for Human Genetics, Johannes Gutenberg University Mainz, Mainz, Germany

^* To whom correspondence should be addressed. 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)]. Bioinformatics 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 genome-wide 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.