G-Quadruplex Structures and CpG Methylation Cause Drop-Out of the Maternal Allele in Polymerase Chain Reaction Amplification of the Imprinted MEST Gene Promoter

Abstract

We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5′ end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs) in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a T m of greater than 99°C in polymerase chain reaction (PCR) buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings.

Structural Analysis of G-Quadruplex Formation at the Human MEST Promoter

“…All three motifs had high thermal stability and displayed characteristics of G4 formation, however, methylation appeared to decrease the G4 stability as measured by CD, which was contrary to our initial hypothesis [19]. It is unclear how cytosine methylation impacts on G4 formation or stability, but this analysis showed that both factors were required to cause ADO and neither G4 formation or DNA methylation in isolation was sufficient.…”

“…(2014) [19]. Non B-DNA formation was characterised using fluorescence analysis of dimethyl sulfate footprinting assay (FADFA) and fluorescence analysis of nuclease footprinting assay (FANFA), as previously described by Stevens et al .…”

“…Previously, we reported persistent allelic drop-out (ADO) during polymerase chain reaction (PCR) analysis of the human MEST promoter region (Fig 1). The MEST gene is a maternally imprinted locus that displays paternal expression, with heavy methylation on the maternal allele [15–19], and this allele consistently failed to amplify during PCR. This very robust ADO phenomenon was attributed to the combined effect of non B-DNA structure and DNA methylation in the template DNA [19].…”

“…First, potassium, which is required for stable formation of G4 in DNA [20, 21], was found to promote ADO of the MEST amplicon. Second, 7-deaza dGTP, which prevents formation of the Hoogsteen bonds, alleviated ADO when it was incorporated into synthetic templates prior to PCR [19]. …”

In silico analysis of regulatory and structural motifs of the ovine HSP90AA1 gene

“…G4 motifs are enriched at the TSS, the 5′-UTR, and the 5′ end of the first intron and depleted in coding regions (Maizels and Gray 2013). In the promoters of several eukaryotic and prokaryotic genes, G-rich sequences with potential to form G4 have been identified (Rankin et al 2005;Rawal et al 2006;Stevens et al 2014). The potential for G4 structures to inhibit DNA synthesis has long been recognized (Han et al 1999;Boán et al 2004), as has its ability to act as a transcriptional repressor (Siddiqui-Jain et al 2002;Lin et al 2013).…”