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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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