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    MTPC 140: Molecular Biology and Diagnostics

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    Valdez Francis Zaccheau
    Hybridization assays can be used to detect specific nucleic acid sequences. There are four main types: solid-phase hybridization detects targets bound to a membrane or surface. Liquid-phase hybridization detects targets in solution. In situ hybridization detects targets in intact cells. DNA chip technology detects targets by competitive hybridization to arrays on a chip and can be used for gene profiling and disease classification.

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    MTPC 140: Molecular Biology and Diagnostics

    Uploaded by

    Valdez Francis Zaccheau
    72 views17 pages
    Hybridization assays can be used to detect specific nucleic acid sequences. There are four main types: solid-phase hybridization detects targets bound to a membrane or surface. Liquid-phase hybridization detects targets in solution. In situ hybridization detects targets in intact cells. DNA chip technology detects targets by competitive hybridization to arrays on a chip and can be used for gene profiling and disease classification.

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    Hybridization+Assay

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    Hybridization assays can be used to detect specific nucleic acid sequences. There are four main types: solid-phase hybridization detects targets bound to a membrane or surface. Liquid-phase hybridization detects targets in solution. In situ hybridization detects targets in intact cells. DNA chip technology detects targets by competitive hybridization to arrays on a chip and can be used for gene profiling and disease classification.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    72 views17 pages

    MTPC 140: Molecular Biology and Diagnostics

    Uploaded by

    Valdez Francis Zaccheau
    Hybridization assays can be used to detect specific nucleic acid sequences. There are four main types: solid-phase hybridization detects targets bound to a membrane or surface. Liquid-phase hybridization detects targets in solution. In situ hybridization detects targets in intact cells. DNA chip technology detects targets by competitive hybridization to arrays on a chip and can be used for gene profiling and disease classification.

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    © All Rights Reserved
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    Download as pdf or txt
    of 17

    MTPC 140: Molecular Biology

    and Diagnostics
    MAVERICK V. SUSTIGUER, RMT
    Senior Instructor
    Medical Technology Department
    Notre Dame of Marbel University
    HYBRIDIZATION ASSAYS
    LEARNING OUTCOMES:
    At the end of the class discussion, the students should be
    able to:
    a. Define the basic terms associated to hybridization assays;
    b. List the four types of hybridization assays;
    c. Elucidate the working principle of each type of hybridization
    assays, and
    d. Enumerate the basic applications of these hybridization assays
    in medicine.
    TERM DEFINITION
    TARGET Specific section of DNA/RNA under investigation
    Pairing of complementary strands of nucleic acid T C G T T G T G C C
    HYBRIDIZATION
    DNA:DNA, RNA:RNA, DNA:RNA, RNA:DNA Annealing* A G C A A C A C G G
    HYBRID/DUPLEX Product of hybridization
    Stability of bonding during hybridization (temperature, pH,
    STRINGENCY
    salt concentration and hydrogen disrupters)
    Short strand of DNA/RNA with known base sequence
    From organisms, cloned from bacteria, synthesized
    NUCLEIC ACID Labeled with dyes, enzymes and radioisotopes
    PROBE Complementary base sequence + labeled probe
    Hybridization, RFLP, Conventional and RT-PCR
    A A C A C
    Matching/complementarity
    HOMOLOGOUS Nucleotides are in correct order to match with
    probe/primer
    Determined by the:
    • GC content
    • AT content
    ↓Temperature
    𝑵𝑵𝑵𝑵 Formamide
    ↑ Salt concentration

    A T C G C G A C G
    A G C G C T G

    A T C C C G C C G

    A G C G C T G
    Discourage nonspecific binding to nontarget DNA.

    ↑ Temperature
    ↑ Formamide
    ↓ Salt concentration
    Nucleic acid
    Specimen Specimen Nucleic acid Nucleic acid
    detection or
    collection processing extraction amplification
    analysis

    Gel electrophoresis
    Hybridization with labeled probes*
    Southern or Northern blot*
    Restriction enzyme mapping
    DNA sequencing
    Real-time PCR
    Two Important Features of Hybridization
    1. Hybridization reactions are specific
    2. Hybridization reactions will occur in the presence of large
    quantities of molecules similar but not identical to the target.

    These properties allow you to use hybridization to perform a molecular search for one
    DNA molecule, or one RNA molecule, or one protein molecule in a complex mixture
    containing many similar molecules.

    Basic components: Sample, Probe, Controlled condition permissive to base pairing,


    detection system
    Hybridization Assays

    1 Solid-phase hybridization

    2 Liquid-phase hybridization

    3 In situ hybridization

    4 DNA chip technology


    Sample T C G T T G T G C C

    T C G T T G T G C C
    A G C A A C A C G G A G C A A C A C G G

    A A C A C

    A A C A C
    T C G T T G T G C C A G C A A C A C G G

    A A C A C
    A G C A A C A C G G
    T C G T T G T G C C
    A G C A A C A C G G
    A A C A C
    T C G T T G T G C C
    A A C A C A A C A C
    T C G T T G T G C C T C G T T G T G C C

    A A C A C
    T C G T T G T G C C
    Assay Principle Application
    Dot/slot blot: sample added to the membrane +
    labeled probe → washing → detection Detection of mutations
    Solid-phase Sandwich hybridization: unlabeled probe + associated with genetic
    hybridization membrane + sample → annealing diseases and infectious
    Line probe assay: probe + strips + amplified target diseases
    → washing → detection
    Detection of bacterial RNA in
    Liquid-phase Target nucleic acid and probe interacts in aqueous
    clinical specimens or
    hybridization solution.
    cultures
    In situ Intact cells (target nucleic acid) + probe →
    Cytogenetic studies
    hybridization FLUORESCENCE
    Gene profiling of cancer
    Sample + control nucleic acid label with fluorescent
    DNA chip Classification of leukemia
    tags and loaded to the chip → Competition to
    technology Tumor staging
    hybridize the chip
    Drug resistance in HIV

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