Laura Poliseno (ed.), Pseudogenes: Functions and Protocols, Methods in Molecular Biology, vol. 1167, DOI 10.1007/978-1-4939-0835-6_18, © Springer Science+Business Media New York 2014 Chapter 18 Discrimination of Pseudogene and Parental Gene DNA Methylation Using Allelic Bisulfite Sequencing Luke B. Hesson and Robyn L. Ward Abstract Determining the methylation status of genes with pseudogenes can be technically challenging due to sequence homology. High sequence homology can result in the amplification of both pseudogene and parental gene alleles, potentially leading to data misinterpretation. Allelic bisulfite sequencing allows for detection of the methylation status of individual alleles at nucleotide resolution and represents the most reliable method for discriminating pseudogene and parental gene sequences. Here, we discuss important points that should be considered when investigating pseudogene and parental gene methylation status and we describe the method of allelic bisulfite sequencing, including assay design. Key words Pseudogene, Epigenetics, Methylation, Bisulfite, Sequencing 1 Introduction Pseudogenes are ancestral nonfunctional copies of protein coding genes that have lost the potential to give rise to a protein product [1]. Pseudogenes that arise by genomic duplication are called unprocessed pseudogenes, whereas those formed by retrotranspo- sition through reverse transcription of an mRNA intermediate and reintegration into the genome are known as processed. Pseudogenes are not restricted by the same selective pressures as functional parental genes and accumulate deleterious sequence changes over time, usually resulting in stop codons that render the “open read- ing frame” nonfunctional. Pseudogenes are ubiquitous in the human genome with current estimates indicating that there are over 17,000 pseudogenes [2]. Given the close similarity between pseudogenes and almost all coding genes, it is challenging to develop molecular analyses that are specific for the gene of interest rather than pseudogenes [3]. Most notably, amplification of DNA sequences using PCR can be problematic if the target region is not unique in the genome. Pseudogenes with high sequence homology can therefore be a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32