Development of RFLP and qPCR Assays to Detect the Human C/T‐13910 SNP, which Is Associated with Lactase Persistence

Abstract

Human infants, like those of other mammals, express the lactase enzyme to digest the lactose sugar in mother’s milk. After weaning, the lactase‐phlorizin hydrolase gene (LCT) ceases to produce lactase, resulting in the lactase non‐persistence (LNP) phenotype. However, in 35% of the world’s population of humans, expression of the lactase enzyme continues into adulthood, known as lactase persistence (LP). The LP phenotype occurs when an individual has one of more than 20 single nucleotide polymorphisms (SNPs) located near the lactase gene. The most commonly studied SNP is the “European” SNP, C/T‐13910, which occurs in an enhancer region upstream of the LCT gene in intron 13 of the neighboring MCM6 gene. The T allele correlates with LP, as it results in a higher expression of lactase due to increased binding of the Oct‐1 regulatory transcription factor and/or prevention of gene silencing. Several studies have been conducted in non‐European, isolated populations to determine the frequency of LP within the ethnic group. However, fewer studies have been conducted in ethnically diverse locations such as the US. To lay the groundwork for such a study, we have successfully implemented a restriction fragment length polymorphism (RFLP) assay reported in Morales et al. (2011, doi:10.1136/bmjopen‐2011‐000125) to detect the C/T‐13910 SNP in human cheek cell lysate, confirming the results by direct sequencing of the DNA. We used the TOPO cloning technique to generate control plasmids containing inserts with either the “C” or “T” genotype to use as positive controls in future assays. We confirmed that the sequence of the control plasmid inserts match the original target sequence. We are using the control plasmids to optimize a SYBR green quantitative polymerase chain reaction (qPCR) genotyping assay based on Weinlander et al. (2010, doi:10.1002/bmb.20357) as an alternate method of genotyping. Finally, we have curated a list of SNPs from the literature and public databases that are known to affect the LP phenotype in different world populations.Support or Funding InformationLoyola Marymount University Summer Undergraduate Research Program (S.M.C., A.N.W.), Loyola Marymount University Honors Summer Research Fellowship (S.M.C.)