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Sanger sequencing

Enhancing the delivery of therapeutics through effective DNA sequencing

What is 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.

Reading the sequence

The labelled DNA fragments are separated by capillary electrophoresis, through a gel-like matrix. Smaller fragments appear first, followed by larger ones, with sufficient resolution to differentiate between fragments with a single base difference in length. The signal decreases as the fragments get larger, due to the number of terminations being favored in smaller fragments as well as the diffusion of larger fragments during the run. This decline is sometimes referred to as the “ski slope,” and for long sequencing runs it must be shallow in nature.

The data quality is better in the early stages due to the sharp peaks and high signal, but gradually drops off to a point below a predefined quality after which collection is stopped. Typically, four different fluorescent dyes are used to label each of the four bases. The emission of the specific ddNTP is then measured and assigned to the fragment length. In this way, the sequence of the original template is read from the fragment pool as each fragment increases by a single base.

Sanger sequencing in practice

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.

Read more here

Sanger sequencing: past successes and current applications

For the last 40 years, Sanger sequencing has been an enduring presence in DNA sequencing. Read our blog to find out how it has remained relevant and continued to help scientists in a world dominated by next-generation sequencing.

Read more...

  • illustra GenomiPhi HY DNA Amplification Kits

    Illustra GenomiPhi HY DNA Amplification Kit offers highly efficient and representative whole-genome amplification with 40 to 50 μg yield from nanogram amounts of DNA sample.

  • illustra ExoProStar

    Enzymatic PCR clean up technology with illustra Exonuclease I and Alkaline Phosphatase to remove unincorporated primers and dNTPs. Comes in two separate tubes for maximum flexibility.

  • illustra TempliPhi Sequence Resolver Kits

    illustra TempliPhi Sequence Resolver Kit uses the highly processive, strand-displacing Phi29 DNA polymerase and modified nucleotides to amplify circular templates for subsequent sequencing.

  • PCR Nucleotide Mix dNTP Set

    Free from DNase, RNase, and nicking enzyme activity, with greater than 99% triphosphate purity (by HPLC), for optimum performance and consistency.

Contact us

Please get in touch if you would like more information on how Cytiva can support your sequencing workflow.

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Thank you for getting in touch to learn more about how GE Healthcare can support your sequencing workflow. A member of our team will reach out to you shortly. In the meantime, please feel free to <a href="/news-center?appa=b6964ebc-f4b8-4dec-9cd0-30c7e238f707">check out our latest sequencing blogs</a>. Sincerely, GE Healthcare Life Sciences
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