INVESTIGATION Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci Aaron J. Stevens,* Millie G. Taylor,* Frederick Grant Pearce,† and Martin A. Kennedy*,1 *Department of Pathology, University of Otago, Christchurch 8140, New Zealand and †Biomolecular Interaction Centre, School of Biological Sciences, University of Canterbury, Christchurch 8140, New Zealand ORCID IDs: 0000-0001-9148-8045 (A.J.S.); 0000-0002-6445-8526 (M.A.K.) ABSTRACT Loss of one allele during polymerase chain reaction (PCR) amplification of DNA, known as KEYWORDS allelic dropout, can be caused by a variety of mechanisms. Allelic dropout during PCR may have profound G-quadruplex implications for molecular diagnostic and research procedures that depend on PCR and assume biallelic PCR amplification has occurred. Complete allelic dropout due to the combined effects of cytosine methylation allelic dropout and G-quadruplex formation was previously described for a differentially methylated region of the human methylation imprinted gene, MEST. We now demonstrate that this parent-of-origin specific allelic dropout can poten- polymerase chain tially occur at several other genomic regions that display genomic imprinting and have propensity for reaction G-quadruplex formation, including AIM1, BLCAP, DNMT1, PLAGL1, KCNQ1, and GRB10. These findings demonstrate that systematic allelic dropout during PCR is a general phenomenon for regions of the ge- nome where differential allelic methylation and G-quadruplex motifs coincide, and suggest that great care must be taken to ensure biallelic amplification is occurring in such situations. DNA amplification by polymerase chain reaction (PCR) is an enzymatic Saunders et al. 2010; Wenzel et al. 2009). ADO is an insidious problem technique for the in vitro synthesis of targeted DNA regions, mediated that is difficult to recognize because the PCR appears successful, but by thermally stable DNA polymerases. PCR is a prerequisite for most half of the genetic information is missing. ADO can have significant experimental procedures that involve DNA detection, sequencing, implications in both research and clinical applications, where there is a cloning, and genotyping (Mullis and Faloona 1987; Saiki et al. 1988; requirement for high sensitivity and accurate PCR genotyping. Incor- Bevan et al. 1992), and is of widespread application in genetic research rect genotyping can have substantial negative consequences and may and molecular diagnostic applications (Desforges and Eisenstein 1990). result in the misdiagnosis of genetic diseases, loss of the ability to Despite extensive optimization and near ubiquitous usage, PCR is still differentiate between individuals, and false assumptions about parent- prone to failure under certain circumstances. For diploid organisms, age or genetic diversity (Boán et al. 2004; Landsverk et al. 2012; the failure of one allele to amplify can result in allelic dropout (ADO), Saunders et al. 2010; Tomaz et al. 2010; Wenzel et al. 2009). causing apparent homozygosity (Askree et al. 2011; Boán et al. 2004; We previously characterized a novel mechanism of ADO that Lam and Mak 2013; Landsverk et al. 2012; Piyamongkol et al. 2003; occurred during genotyping of the imprinted human gene, MEST (Stevens et al. 2014). Extensive PCR analysis of a short region in the Copyright © 2017 Stevens et al. MEST promoter invariably led to non-Mendelian genotype patterns for doi: 10.1534/g3.116.038687 three single nucleotide polymorphisms (SNPs), which could not be Manuscript received December 22, 2016; accepted for publication January 23, resolved by primer redesign or standard PCR optimization strategies. 2017; published Early Online January 30, 2017. We established that both cytosine methylation and DNA structures This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/ known as G-quadruplexes (G4s) in this region contributed to licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction ADO, leading to incorrect genotyping (Stevens et al. 2014). G4 in any medium, provided the original work is properly cited. are secondary DNA structures that can form in G-rich regions Supplemental material is available online at www.g3journal.org/lookup/suppl/ due to the self-association of guanine through Hoogsteen bonds. Four doi:10.1534/g3.116.038687/-/DC1. 1Corresponding author: Department of Pathology, University of Otago, guanine nucleotides can adopt a square planar arrangement, referred Christchurch, P.O. Box 4345, Christchurch, 8140 New Zealand. E-mail martin. to as a G-quartet, and multiple G-quartets can then stack upon one [email protected] another to form a G4 (Sen and Gilbert 1988; Sundquist and Klug Volume 7 | March 2017 | 1019 1989). G4 formation is stabilized by the integration of a cation, like potassium (Ambrus et al. 2005; Biffi et al. 2012; Maizels 2015; Rhodes and Lipps 2015; Simonsson 2001), making PCR buffer an optimal environment for G4 formation. G4 structures may then act as a steric block to Taq polymerase (Boán et al. 2004; Chambers et al. 2015; Saunders et al. 2010; Weitzmann et al. 1996), an effect that is exac- erbated when the G4 region is methylated (Stevens et al. 2014). During amplification of the imprinted MEST promoter region, we observed consistent ADO of the maternally inherited, methylated allele. Correct genotypes from this locus were only obtained using extraordi- nary modifications of PCR, including methylation-specific PCR, allele- specific enzymatic digestion of genomic DNA, and PCR buffers lacking potassium. We demonstrated that ADO resulted from the combination of both cytosine methylation and guanine Hoogsteen bonds in the template DNA, which can form G4 structures, and that neither factor in isolation was sufficient to cause complete ADO (Stevens et al. 2014). The novel form of ADO observed at the MEST promoter region was Figure 1 Synthetic template ADO assay (A) Two templates (black bars) intriguing and problematic, but it was unclear if it was a phenomenon were generated by targeted PCR amplification of genomic DNA (gray). restricted solely to this genomic region or a more general occurrence These assay templates differed by a single base pair, which was introduced throughout the genome. In this report, we describe the design of an into one template (right) through primer-directed mutagenesis (small assay and its application to test for the potential occurrence of ADO green arrows and enlarged view). (B) An aliquot of each template was during amplification of differentially methylated DNA. The templates then methylated (blue circles) with M.SssI, and reciprocal mixing exper- used in this assay were generated by PCR from a range of imprinted iments were performed on each template combination as shown. In total, genes, and in vitro methylation with the enzyme M.SssI was used to three template mixing experiments were performed per amplicon, for vs. vs. mimic differentially methylated alleles. After demonstrating potential example: (1) methylated G nonmethylated A; (2) methylated A vs. ADO using the assay on multiple synthetic model templates, we then nonmethylated G; and (3) nonmethylated A nonmethylated G. demonstrated that this type of ADO could be observed in genomic DNA analysis of an imprinted human locus other than MEST. Sequencing reaction products were run on an AB3130xl fragment anal- ysis system equipped with a 50-cm capillary, using POP7 polymer. MATERIALS AND METHODS Synthetic DNA templates Selection of G4s for analysis Selected G4-containing amplicons (Figure S1 in File S1)wereamplified Imprinted genes with a confirmed parent-of-origin methylation status by PCR, and as described below (ADO assay on synthetic templates), a (Jirtle 2012; Morison et al. 2001) were analyzed for G4-forming motifs single nucleotide difference was introduced to allow alleles to be dis- using the bioinformatic software, QGRS Mapper (Kikin 2006). G4 tinguished. In vitro methylation and digestion experiments were per- motifs that contained runs of three or more guanines and a loop length formed on PCR products generated from genomic DNA, using between 0 and 7 nt were considered for analysis, as these were most enzymes purchased from New England Biolabs Inc., (Ipswich, MA). likely to adopt G4 structure. This corresponded with a QGRS mapper In vitro methylation was carried out by incubation with M.SssI for score of at least 37, which was therefore selected as an arbitrary thresh- 120 min at 37°, followed by heat inactivation at 65° for 20 min, as old for G4 cutoff. PCR amplicons of 300 bp were designed from these recommended by the manufacturer’s protocol. To assess the extent of regions, to contain a single G4 motif and a CCGG endonuclease rec- in vitro methylation achieved, each methylated DNA template was ognition sequence for HpaII and MspI endonucleases (Supplemental then incubated with the restriction enzyme MspIanditsmethylation- Material, Figure S1 and Table S1 in File S1). sensitive isoschizomer HpaII. The resulting digestion products were analyzed by gel electrophoresis and compared, to ensure successful PCR methylation (data not shown). PCR was carried out in a Mastercycler pro thermal cycler (Eppendorf, Stevenage, UK) with Fisher Taq-ti polymerase (Fisher Biotec, WA, ADO assay on synthetic templates Australia). The initial denaturation step consisted of 95° for 2 min, The assay for modeling methylation specific ADO