molecular testing and clinical diagnosis
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Molecular Testing and Clinical Diagnosis. Direct Nucleic Acid Testing. Non-amplified Probe Assays. Three main steps for direct nucleic acid testing 1. Sample preparation 2. Probe hybridization In solution On a solid surface 3. Detection. DNA Probe – An Example. - PowerPoint PPT PresentationTRANSCRIPT
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Molecular Testing and Clinical Diagnosis
Direct Nucleic Acid Testing
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Non-amplified Probe Assays • Three main steps for direct nucleic acid
testing1. Sample preparation2. Probe hybridization
• In solution• On a solid surface
3. Detection
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DNA Probe – An ExampleGen-Probe Assay HPA: Hybridization Protection
Assay Procedure
Three main steps: Sample preparation releases target rRNA Hybridization of DNA probe with rRNA Detection of chemiluminescent label on DNA probe
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Hybridization Protection Assay (HPA) Overview
DNA probe labeled with chemiluminescent molecule (acridinium ester)
Probe hybridizes with rRNA of organism
Separation of hybridized from unhybridized probes occurs in solution phase
No wash steps or solid substrate
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HPA Sample Preparation
Sample containing cells
Target Ribosomal
RNA
CellCellLysis
RNA
RNARNA
RNARNA
RNARNARNA
RNARNARNA
RNA
RNA
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Advantages of Ribosomal RNA Targets
Absolute specificity for target organism is achieved through targeting of unique sequences
Sensitivity increased through “biological amplification”
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One or several copies of DNA
Up to 10,000 Copies
Ribosomal RNA
rRNA “Biological Amplification”One bacterial cell contains:
AND
DNA
RNA RNA
RNA
RNA
RNA RNA
RNA RNA RNA
RNA
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RNA
HPAHybridization Protection Assay
Hybridization
60 Co
15 minutesHybridize
Labeled DNA Probe
RNA
RNA
RNA
RNA
RNA
RNARNA
RNA
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Chemiluminescence from an Acridinium Ester
RNA
RNARNA
RNARNA
Detected Light
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HPA Hybridization Protection AssaySelection/Detection
Light is generated if hybridization has occurred. Amount of light is proportional to the amount of original target sequence.
RNA RNA
Hybridized Probe
Light
Unhybridized Probe
No Light
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Hybridization Protection Assay (HPA) Summary
• Ribosomal RNA Targets
– High sensitivity and specificity
• Chemiluminescence detection
• Solution phase separation/detection
• No wash steps
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In situ Hybridization• DNA or RNA probes can be used• Detects DNA or RNA in fixed tissue• Determines if target is present & its
distribution within cells• Requires tissue sections, probe and
visualization system• If fluorescent tag used = fluorescent in
situ hybridization (FISH )
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In situ Hybridization: Clinical Applications
• Gene mapping• Chromosomal abnormalities• Detection of microorganisms in
tissue/cells
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Biochips - Microarray• DNA fragments (probes) are anchored to
a glass or silicon chip. • DNA probes within the microarray can
measure the gene expression of thousands of genes in a single RNA sample
• Click on link for information sheetNational Institutes of Health – Information Sheet
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Biochips - Microarray• Two common detection systems have
been developed. – On glass slides, hybridization can be
detected by fluorescence and spot color detection by a microarray scanner.
– The silicone chip consists of electrodes, independently addressable via an electronic control system. Hybridization is detected by changes in resistance.
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Biochips-Microarrays• Feasibility
– increasing as more genes are characterized – Human Genome Project and studies have identified
expression patterns characteristic of diseases and disorders• Applications:
– Infectious disease– Gene mutations – Cancer– Screening blood products
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Summary• Direct probes measure the presence of
target sequence (DNA or RNA)• Three easy steps
– sample preparation, hybridization, detection• Detection systems allow the visualization
of hybridization reactions• Easy to automate