Genotyping Method for Point Mutation Detection in the Intestinal Fatty Acid Binding Protein, Using Fluorescent Probes

The intestinal fatty acid-binding protein (IFABP) is located in the intestine and is involved with long-chain fatty acid transport and metabolism (1). The FABP2 gene at chromosome 4q28-31 encodes IFABP. Genetic variation at this locus could lead to altered fatty acid absorption and energy metabolism. A common point mutation (G→A) in the gene for IFABP, with an allele frequency of the A allele of ∼0.29 (2), generates an amino acid substitution at codon 54 (alanine→threonine), which was found to be associated with insulin resistance and increased fat oxidation in Pima Indians (2). Recently, genetic variation at this locus has been reviewed in terms of plasma lipid response to diet (3). The current evidence indicates that the IFABP threonine variant is often associated with a more deleterious phenotypic expression, i.e., impaired glucose tolerance, obesity, and altered lipid and lipoprotein profiles (3). In vivo studies also support a functional difference between the alanine- and threonine-containing proteins: Caco-2 cells that express the threonine-containing protein transport long-chain fatty acids and secrete triglycerides to a greater extent than cells that express the alanine-containing protein (4). Therefore this variant is an obvious candidate for examining this gene by the use of diet interactions.The established procedure for genotyping the G→A mutation includes restriction digestion with Hha I, which cleaves the natural restriction site in the wild-type (G54) PCR product (2). The restriction enzyme digestion is then followed …