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    Animal Biotech Assignmnt

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    Noorshan
    Nucleic acid-based diagnostic methods can detect pathogens by amplifying their DNA or RNA using techniques like PCR or hybridization to probe for specific genes. PCR works by replicating DNA many times, allowing minute pathogen traces to be found. It involves denaturing DNA, annealing primers, and elongating new strands in cycles. Hybridization uses probes that bind directly to pathogen DNA or RNA present on filters. These techniques are highly sensitive and used in central labs to diagnose diseases from amplified nucleic acids.

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    Animal Biotech Assignmnt

    Uploaded by

    Noorshan
    28 views13 pages
    Nucleic acid-based diagnostic methods can detect pathogens by amplifying their DNA or RNA using techniques like PCR or hybridization to probe for specific genes. PCR works by replicating DNA many times, allowing minute pathogen traces to be found. It involves denaturing DNA, annealing primers, and elongating new strands in cycles. Hybridization uses probes that bind directly to pathogen DNA or RNA present on filters. These techniques are highly sensitive and used in central labs to diagnose diseases from amplified nucleic acids.

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    Nucleic acid-based diagnostic methods can detect pathogens by amplifying their DNA or RNA using techniques like PCR or hybridization to probe for specific genes. PCR works by replicating DNA many times, allowing minute pathogen traces to be found. It involves denaturing DNA, annealing primers, and elongating new strands in cycles. Hybridization uses probes that bind directly to pathogen DNA or RNA present on filters. These techniques are highly sensitive and used in central labs to diagnose diseases from amplified nucleic acids.

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    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    28 views13 pages

    Animal Biotech Assignmnt

    Uploaded by

    Noorshan
    Nucleic acid-based diagnostic methods can detect pathogens by amplifying their DNA or RNA using techniques like PCR or hybridization to probe for specific genes. PCR works by replicating DNA many times, allowing minute pathogen traces to be found. It involves denaturing DNA, annealing primers, and elongating new strands in cycles. Hybridization uses probes that bind directly to pathogen DNA or RNA present on filters. These techniques are highly sensitive and used in central labs to diagnose diseases from amplified nucleic acids.

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    MAHJABEEN ISLAM

    M.PHIL GENETICS

    Nucleic Acid Based


    Diagnostic Methods

    Contents:
    Introduction
    Amplification
    PCR/RT-PCR
    Steps of PCR
    Applications of PCR
    Hybridization
    Steps of Hybridization
    DNA Sequencing
    Uses of Sequencing

    Nucleic Acid-based Diagnostics

    It detects the presence of a pathogen either by


    directly detecting the presence of DNA or RNA
    nucleic acids in the host or by first amplifying the
    pathogen DNA or RNA.
    It is a standard central laboratory technique, used to
    detect a specific gene or the expression of a gene
    associated with disease.
    Detect DNA or RNA from the infecting organism.

    Amplification:

    Techniques take tiny amounts of DNA or RNA,


    replicate them many times.
    Detect minute traces of an organism in a specimen,
    avoiding the need for culture.
    Useful for organisms that are difficult to culture or
    identify using other methods.
    Highly sensitive, false-negative results sometimes
    occurs.

    PCR/RT-PCR

    Polymerase chain reaction (PCR)


    Used to generate thousands to millions of copies of a
    particular DNA sequence.
    DNA (or RNA reverse transcribed to cDNA) is
    amplified to detect the presence of a pathogen or host
    gene of interest.
    Components: DNA template, two primers, Taq
    polymerase, dNTPs, buffer solution, bivalent cations,
    magnesium or manganese ions, monovalent cation.

    Steps of PCR:

    Denaturation (94-98C for 20-30sec)


    Annealing (50-65C for 20-40sec)
    Elongation (75-80C)
    Final elongation (70-75C)

    Applications of PCR:
    Selective

    DNA isolation
    o DNA sequencing
    o genetic fingerprinting
    Amplification and quantification of DNA
    PCR in diagnosis of diseases
    o Malignant diseases (leukemia, HIV, tuberculosis)
    Identification of non-cultivatable or slow-growing
    microorganisms such as mycobacteria.

    Hybridization
    DNA probes bind directly to DNA of pathogen or host
    gene of interest.
    Types:
    Southern hybridization (filter bound DNA)
    Northern hybridization (filter bound RNA)
    Probes (Plasmid clones): is a clone developed by
    inserting DNA into a vector.

    Steps in Hybridization:

    Prepare a probe by nick translation or rando.


    Add the probe to a filter (nylon or nitrocellulose) to
    which single-stranded nucleic acids are bound.
    Hybridize the single-stranded probe to the filter-bound
    nucleic acid for 24 hr.
    Wash the filter to remove non-specifically bound probe.
    Expose the filter.

    DNA Sequencing:
    Determine the precise order of nucleotides within a
    DNA molecule.
    Determine the order of the four basesadenine,
    guanine, cytosine, and thymine.
    Provide detailed information about the original and
    unique characteristics of the pathogen in a specific
    patient.
    Not practical for low-resource settings

    Use of Sequencing

    Molecular biology
    Evolutionary biology
    Metagenomics
    Forensic identification
    Parental testing
    Existing Products
    Nucleic acid-based diagnostics are widely used in
    centralized laboratory settings to diagnose both
    infectious and non-infectious conditions.

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