Single-base extension


Single-base extension is a method for determining the identity of a nucleotide base at a specific position along a nucleic acid. The method is used to identify a single-nucleotide polymorphism.
In the method, an oligonucleotide primer hybridizes to a complementary region along the nucleic acid to form a duplex, with the primer’s terminal 3’-end directly adjacent to the nucleotide base to be identified. Using a DNA polymerase, the oligonucleotide primer is enzymatically extended by a single base in the presence of all four nucleotide terminators; the nucleotide terminator complementary to the base in the template being interrogated is incorporated and identified. The presence of all four terminators suppresses misincorporation of non-complementary nucleotides. Many approaches can be taken for determining the identity of an incorporated terminator, including fluorescence labeling, mass labeling for mass spectrometry, isotope labeling, and tagging the base with a hapten and detecting chromogenically with an anti-hapten antibody-enzyme conjugate.
The method was invented by Philip Goelet, Michael Knapp, Richard Douglas and Stephen Anderson while working at the company Molecular Tool. This approach was designed for high-throughput SNP genotyping and was originally called "Genetic Bit Analysis". Illumina, Inc. utilizes this method in their Infinium technology to measure DNA methylation levels in the human genome.