title: Identification and prevention of genotyping errors caused by G-quadruplex- and i-motif-like sequences.
abstract: 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.
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Mainz, Mainz, Germany.
authors.forename: Jürgen J
authors.initials: JJ
authors.lastname: Wenzel
authors.forename: Heidi
authors.initials: H
authors.lastname: Rossmann
authors.forename: Christian
authors.initials: C
authors.lastname: Fottner
authors.forename: Stefan
authors.initials: S
authors.lastname: Neuwirth
authors.forename: Carolin
authors.initials: C
authors.lastname: Neukirch
authors.forename: Peter
authors.initials: P
authors.lastname: Lohse
authors.forename: Julia K
authors.initials: JK
authors.lastname: Bickmann
authors.forename: Timo
authors.initials: T
authors.lastname: Minnemann
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Musholt
authors.forename: Brigitte
authors.initials: B
authors.lastname: Schneider-Rätzke
authors.forename: Matthias M
authors.initials: MM
authors.lastname: Weber
authors.forename: Karl J
authors.initials: KJ
authors.lastname: Lackner
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000235:genetics; D054856:G-Quadruplexes; D005838:Genotype; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D011518:Proto-Oncogene Proteins / Q000235:genetics
chemical_list: C105373:MEN1 protein, human; D011518:Proto-Oncogene Proteins; D004247:DNA