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    Sanger Sequencing

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    manojkhadka.py

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    Sanger Sequencing

    Uploaded by

    manojkhadka.py
    5 views15 pages

    Original Title

    Sanger sequencing

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    5 views15 pages

    Sanger Sequencing

    Uploaded by

    manojkhadka.py

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
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    Sanger sequencing and its application

    Presented by

    Manoj Kumar
    Department of Molecular Biology
    Amrita Hospital, Faridabad
    About ABI ProFlex 8 genetic
    analyzer
    Uses capillary electrophoresis to perform
    Sanger sequencing and fragment length
    analysis (FLA)

    8 capillaries with a 4-plate deck system,


    allows continuous loading and reprioritization
    of urgent samples.
    Principle of Sanger sequencing
    Capillary vs Agarose Gel
    Electrophoresis
    Capillary Gel Agarose Gel

    Resolution single-nucleotide resolution 5 bp in 3% gel w/ <200 bp


    fragment

    Throughput High and automated, 96x4 Low and manual

    Multiplexing Yes, 6 dyes in single tube No

    Cost High Low

    Applications Targeted DNA sequencing, Fragment Fusion, deletion and snv


    length analysis, confirmation of NGS detection of
    variants.
    Tests that can be done
    NPM1, FLT3 and CEBPA mutations in Acute myeloid leukaemia (AML)

    Site-specific germline testing for inherited cancers

    Chimerism monitoring after Bone marrow transplant by FLA

    Ataxia CAG repeats analysis by FLA

    Microsatellite instability by FLA

    MLPA (Multiplex Ligation-dependent Probe Amplification) for BRCA1/2 and DMD

    Orthogonal verification for Next Generation Sequencing


    MSI
    orthogonal verification
    CCAAT/enhancer-binding protein alpha
    (CEBPA) Mutation by Sanger Sequencing

    ~10% incidence in AML

    Mutations across whole gene, two major hotspots i.e. N- terminus


    and C-terminus region

    Favourable prognosis in patients with double mutation


    Frameshift

    P1 Normal P1 Mutant
    Frameshift

    P2 Normal P2 Mutant
    A case of Denovo Li-Fraumeni Syndrome
    Rare genetic disorder, increases risk of developing different cancers
    especially at young age

    Caused by mutation in TP53:chr17:7577121,c.817C>T (p.Arg273Cys)

    Either inherited or can be de-novo (~5%)

    No cure, but regular screening and surveillance can help detect and treat
    cancers early
    Source: clinvar
    Proband, 9 years old, diagnosed with B cell Acute lymphoblastic
    leukemia

    Tested on a NGS Panel


    transactivation motif DNA-binding domain tetramerisation motif

    Parent 1 : Normal Parent 2 : Normal


    transactivation motif DNA-binding domain tetramerisation motif

    G>A

    Proband/Offspring : Mutant, De-novo

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