Download Molecular Testing and Clinical Diagnosis

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
Gen-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
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
HPA Sample Preparation
RNA
RNA RNA RNA
RNA
RNA
RNA
Sample
containing
cells
RNA
RNA
Cel
lCell
Lysis
RNA RNA
RNA
RNA
Target
Ribosomal
RNA
Advantages of Ribosomal RNA Targets
 Absolute specificity for target organism is
achieved through targeting of unique
sequences
 Sensitivity increased through “biological
amplification”
rRNA “Biological Amplification”
One bacterial cell contains:
RNA
RNA RNA
DNA
RNA
RNA
RNA
One or several
copies of DNA
AN
D
RNA RNA
RNA
RNA
Up to 10,000
Copies
Ribosomal RNA
HPA
Hybridization Protection Assay
Hybridizatio
RNA RNA RNA
n
RNA RNA RNA
RNA
o
60 C
15 minutes
Hybridize
RNA
RNA
Labeled DNA
Probe
Chemiluminescence from an Acridinium
Ester
RNA
RNA
RNA
RNA
RNA
Detected Light
HPA Hybridization Protection Assay
Selection/Detection
RNA
RNA
Light
Hybridized Probe
No Light
Unhybridized Probe
Light is generated if hybridization has occurred.
Amount of light is proportional to the amount of
original target sequence.
Hybridization Protection Assay (HPA)
Summary
• Ribosomal RNA Targets
– High sensitivity and specificity
• Chemiluminescence detection
• Solution phase separation/detection
• No wash steps
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 )
In situ Hybridization:
Clinical Applications
• Gene mapping
• Chromosomal abnormalities
• Detection of microorganisms in
tissue/cells
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 sheet
National Institutes of Health –
Information Sheet
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.
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
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