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How do DNA methylation and non B-DNA interact to cause genotyping failure during PCR?

A 2018/2019 Summer Studentship research project

Our data suggests that allelic drop-out occurs across a variety of technical platforms that are used to analyze DNA. Such DNA amplification and sequencing techniques are widely used in medical diagnostic laboratories, but this type of failure is difficult to detect and could produce misleading results. This research will substantially increase our understanding of the mechanisms underlying this failure of PCR, and provide better tools for predicting when it might occur.

Student: Eva Mulder
Supervisors: Dr Aaron Stevens, Professor Martin Kennedy
Sponsor: TBC

Project brief


This studentship builds on an interesting observation that a widely used genetic technique for amplifying and visualizing DNA fails to provide reliable results at certain regions of the human genome [1, 2]. Although similar observations of “allelic drop-out” have been documented to occur during polymerase chain reaction (PCR) [3, 4], our observation is unique in that it occurs in a parent-of-origin manner, whereby the maternally inherited DNA copy fails to amplify.

Our previous research has characterized several conditions that contribute to this failure of PCR, but the main cause appears to be enhanced formation of secondary structures in the DNA referred to as “G-quadruplexes”, due to the presence of methylated cytosine bases. In situations where the pattern of cytosine methylation is exclusively inherited from one parent, this promotes preferential G-quadruplex formation on one copy of the DNA. G-quadruplex formation then inhibits Taq polymerase from progressing along the DNA molecule, which prevents exponential DNA amplification of the methylated DNA during PCR. This methylated copy then fails to be detected during visualization of PCR products. In order to further our understanding of how G-quadruplex structures and DNA methylation interact, there are several factors in this process which still require investigation. With overseas collaborators we have identified several regions of the human genome that behave in this manner, and this project will study some of these newly identified regions to help build a better model for predicting the likelihood that such events can occur in a given DNA sequence.


This studentship will primarily examine the role that the location of methylated cytosine within the G-quadruplex forming DNA sequence has on causing allelic drop-out during PCR.


Several methods can be applied to explore structural features of specific DNA sequences [5, 6], and to evaluate the role of methylation in formation of such structures [7, 8].  In order to fit within a 10 week period, we have selected some relatively simple approaches that should yield valuable data. Experiments will be carried out using artificial DNA constructs that can contain methylated cytosine at different positions of interest. These constructs will be treated with M.SssI methyltransferase to methylate all CpG dinucleotides, and then mixed with unmethylated constructs of the same sequence. The methylated and non-methylated DNA will be differentiated by a single nucleotide change, which will enable the quantification of methylated vs non-methylated DNA after amplification. By changing the location and abundance of methylated cytosine in these sequences we will perform comparisons of the structural behaviour of the G-quadruplex and also the propensity of the methylated DNA to “drop-out” during visualization.

Student researcher’s component of the study

  • Aid in design of artificial DNA constructs
  • Carry out DNA modification techniques (DNA ligation, and treatment with M.SssI methyltransferase)
  • Carry out laboratory assays (PCR and Sanger Sequencing)
  • Document the results


  1. Stevens AJ, Taylor MG, Pearce FG, Kennedy MA. Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci. G3: Genes, Genomes, Genetics 7(3), 1019-1025 (2017).
  2. Stevens AJ, Stuffrein-Roberts S, Cree SL et al. G-Quadruplex Structures and CpG Methylation Cause Drop-Out of the Maternal Allele in Polymerase Chain Reaction Amplification of the Imprinted MEST Gene Promoter. Plos One 9(12), 1-24 (2014).
  3. Boan F, Blanco MG, Barros P, Gonzalez AI, Gomez-Marquez J. Inhibition of DNA synthesis by K+-stabilised G-quadruplex promotes allelic preferential amplification. FEBS Lett 571(1-3), 112-118 (2004).
  4. Woodford KJ, Howell RM, Usdin K. A novel K(+)-dependent DNA synthesis arrest site in a commonly occurring sequence motif in eukaryotes. J Biol Chem 269(43), 27029-27035 (1994).
  5. Sun D, Hurley LH. Biochemical techniques for the characterization of G-quadruplex structures: EMSA, DMS footprinting, and DNA polymerase stop assay. Methods Mol Biol 608 65-79 (2010).
  6. Wenzel JJ, Rossmann H, Fottner C et al. Identification and prevention of genotyping errors caused by G-quadruplex- and i-motif-like sequences. Clin Chem 55(7), 1361-1371 (2009).
  7. Diede SJ, Tanaka H, Bergstrom DA, Yao MC, Tapscott SJ. Genome-wide analysis of palindrome formation. Nat Genet 42(4), 279 (2010).
  8. Hardin CC, Corregan M, Brown BA, 2nd, Frederick LN. Cytosine-cytosine+ base pairing stabilizes DNA quadruplexes and cytosine methylation greatly enhances the effect. Biochemistry 32(22), 5870-5880 (1993).