Mahjabeen Islam

Mahjabeen IslamM.Phil geneticsNucleic Acid Based Diagnostic MethodsIntroductionAmplificationPCR/RT-PCRSteps of PCRApplications of PCRHybridizationSteps of HybridizationDNA SequencingUses of Sequencing

Contents: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.

Nucleic Acid-based DiagnosticsTechniques 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.

Amplification: 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.

PCR/RT-PCRDenaturation (94-98C for 20-30sec)Annealing (50-65C for 20-40sec)Elongation (75-80C)Final elongation (70-75C)

Steps of PCR:Selective DNA isolationDNA sequencinggenetic fingerprintingAmplification and quantification of DNAPCR in diagnosis of diseasesMalignant diseases (leukemia, HIV, tuberculosis)Identification of non-cultivatable or slow-growing microorganisms such as mycobacteria.

Applications of PCR: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.

HybridizationPrepare 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.Steps in Hybridization: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

DNA Sequencing:Molecular biology Evolutionary biology MetagenomicsForensic identificationParental testingExisting ProductsNucleic acid-based diagnostics are widely used in centralized laboratory settings to diagnose both infectious and non-infectious conditions.Use of SequencingThank You