Molecular Testing and Clinical Diagnosis

Direct Nucleic Acid Testing

Non-amplified Probe Assays • Three main steps for direct nucleic acid

testing1. Sample preparation2. Probe hybridization

• In solution• On a solid surface

3. Detection

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

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

Sample containing cells

Target Ribosomal

RNA

CellCellLysis

RNA

RNARNA

RNARNA

RNARNARNA

RNARNARNA

RNA

RNA

Advantages of Ribosomal RNA Targets

Absolute specificity for target organism is achieved through targeting of unique sequences

Sensitivity increased through “biological amplification”

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

RNA

HPAHybridization Protection Assay

Hybridization

60 Co

15 minutesHybridize

Labeled DNA Probe

RNA

RNA

RNA

RNA

RNA

RNARNA

RNA

Chemiluminescence from an Acridinium Ester

RNA

RNARNA

RNARNA

Detected Light

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

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 sheetNational 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