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    Sanger Sequencing: A Brief Introduction & Explanation

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    Behram Khan
    Sanger sequencing is a method for DNA sequencing developed by Frederick Sanger. It uses dideoxynucleotides that are labeled with fluorescent dyes and terminate DNA strand elongation to generate fragments that can be read through electrophoresis. While Sanger sequencing was groundbreaking, it has limitations as it is time-consuming and works with radioactive materials. It is being replaced by next-generation sequencing technologies, though Sanger remains the gold standard for validating short sequences.

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    Sanger Sequencing: A Brief Introduction & Explanation

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    Behram Khan
    63 views12 pages
    Sanger sequencing is a method for DNA sequencing developed by Frederick Sanger. It uses dideoxynucleotides that are labeled with fluorescent dyes and terminate DNA strand elongation to generate fragments that can be read through electrophoresis. While Sanger sequencing was groundbreaking, it has limitations as it is time-consuming and works with radioactive materials. It is being replaced by next-generation sequencing technologies, though Sanger remains the gold standard for validating short sequences.

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    Sanger sequencing is a method for DNA sequencing developed by Frederick Sanger. It uses dideoxynucleotides that are labeled with fluorescent dyes and terminate DNA strand elongation to generate fragments that can be read through electrophoresis. While Sanger sequencing was groundbreaking, it has limitations as it is time-consuming and works with radioactive materials. It is being replaced by next-generation sequencing technologies, though Sanger remains the gold standard for validating short sequences.

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    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    63 views12 pages

    Sanger Sequencing: A Brief Introduction & Explanation

    Uploaded by

    Behram Khan
    Sanger sequencing is a method for DNA sequencing developed by Frederick Sanger. It uses dideoxynucleotides that are labeled with fluorescent dyes and terminate DNA strand elongation to generate fragments that can be read through electrophoresis. While Sanger sequencing was groundbreaking, it has limitations as it is time-consuming and works with radioactive materials. It is being replaced by next-generation sequencing technologies, though Sanger remains the gold standard for validating short sequences.

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    Sanger

    Sequencing
    A Brief Introduction &
    Explanation
    DNA
     Blueprint of life
     Chemical building blocks= Nucleotides
     Nucleotides

    i. Phosphate
    ii. Sugar
    iii. Nitrogen Base (A, T, G, C)
     Sequence of base=biological
    instruction.
    Structure
    History of Sequencing
     Watson & Crick identified DNA double-helix
    structure.
     Walter Fieserr= RNA sequencing of
    Bacteriophage MS2
     Maxam & Gilbert developed chemical
    degradation method
     Sanger developed di-deoxy chain termination
    method
     Automation in DNA sequencing by Applied
    Biosystems
    Maxam-Gilbert Method

     Denature DNA to
    get ssDNA

     32
    P as 5’ phosphate

     Cleavage using
    chemicals

     Electrophoresis

     Radiography
    Why Obsolete?
     Time Consuming

     Quite Problematic (Too many steps)

     200-300 bases of DNA every few days

     Working with radioactive material

     More complicated than Sanger


    Sanger Sequencing
     Usingthird form of a Ribose Sugar
    (Dideoxyribose)

     Ribose has OH group on 2’ and 3’ carbon

     Deoxyribose has O removed from 2’


    carbon.

     Dideoxyribose has O removed from 2’ and


    3’carbons.
     ddNTPs are labeled with fluorescent dyes
     Fragments readable through laser light in capillary
    gels
    Conclusion
     15-20% of DNA molecules need to contain the mutation to
    be reliably detected by Sanger. (Rohlin et al., 2009)

     Sanger is being replaced by NGS (Mu et al., 2016)

     NGS technologies have lowered the cost and effort of DNA


    sequencing

     NGS employs massively parallel sequencing of millions of


    DNA fragment simultaneously

     However, Sanger is the gold standard for short fragments


    (Totomoch et al., 2017) & NGS is the future
    References
    Rohlin, A., Wernersson, J., Engwall, Y., Wiklund, L., Björk, J., & Nordling,
    M. (2009). Parallel sequencing used in detection of mosaic mutations:
    Comparison with four diagnostic DNA screening techniques. Human
    Mutation, 30(6), 1012-1020. doi:10.1002/humu.20980

    Mu, W., Lu, H., Chen, J., Li, S., & Elliott, A. M. (2016). Sanger
    Confirmation Is Required to Achieve Optimal Sensitivity and Specificity
    in Next-Generation Sequencing Panel Testing. The Journal of Molecular
    Diagnostics, 18(6), 923-932. doi:10.1016/j.jmoldx.2016.07.006

    Totomoch-Serra, A., Marquez, M. F., & Cervantes-Barragán, D. E.


    (2017). Sanger sequencing as a first-line approach for molecular
    diagnosis of Andersen-Tawil syndrome. F1000Research, 6, 1016.
    doi:10.12688/f1000research.11610.1

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