Sanger sequencing

Sanger sequencing, also known as dideoxy sequencing, is a chain termination method for determining the nucleotide sequence of DNA. The method was developed by two-time Nobel Laureate Frederick Sanger and his colleagues in 1977, and was the method used exclusively to deliver the first human genome sequence. Sanger sequencing synthesizes DNA fragments from a starting template, typically based on the use of fluorescently labelled dideoxynucleotides (ddNTPs) in addition to the normal nucleotides (NTPs) found in DNA. Dideoxynucleotides lack the 3’ hydroxyl group (OH) required to form the phosphodiester bonds that join the DNA together, so their incorporation results in chain termination. In this way, DNA fragments of increasing length are generated, with each fragment terminated by a specific ddNTP. The fluorescence is then used to identify the terminated base for each fragment, and the sequence is read based on fragment length.

In a recent study an in vivo phage display was used to search for molecular markers of the neurovascular unit, including endothelial cells and astrocytes, in mouse models of Alzheimer’s disease (AD). Using Sanger sequencing, a cyclic peptide, CDAGRKQKC (DAG), that accumulates in the hippocampus of hAPP-J20 mice at different ages was found. Connective tissue growth factor (CTGF), a matricellular protein that is highly expressed in the brain of individuals with AD and in mouse models, was identified as the target of the DAG peptide. DAG could potentially be used as a tool to enhance delivery of therapeutics and imaging agents to sites of vascular changes and astrogliosis in diseases associated with neuroinflammation.