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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.

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Molecular Testing and Clinical Diagnosis

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  1. Molecular Testing and Clinical Diagnosis Direct Nucleic Acid Testing

  2. 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

  3. 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

  4. 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

  5. HPA Sample Preparation RNA RNA RNA RNA RNA RNA RNA RNA Cell CellLysis RNA RNA RNA RNA RNA Sample containing cells Target Ribosomal RNA

  6. Advantages of Ribosomal RNA Targets • Absolute specificity for target organism is achieved through targeting of unique sequences • Sensitivity increased through “biological amplification”

  7. rRNA “Biological Amplification” One bacterial cell contains: DNA RNA RNA RNA RNA RNA RNA RNA RNA RNA RNA One or several copies ofDNA Up to 10,000 Copies Ribosomal RNA AND

  8. HPA Hybridization Protection Assay Hybridization RNA RNA RNA RNA RNA RNA RNA RNA RNA o 60 C 15 minutes Hybridize Labeled DNA Probe

  9. Chemiluminescence from an Acridinium Ester RNA RNA RNA RNA RNA Detected Light

  10. HPA Hybridization Protection Assay Selection/Detection Light Hybridized Probe RNA RNA No Light Unhybridized Probe Light is generated if hybridization has occurred. Amount of light is proportional to the amount of original target sequence.

  11. Hybridization Protection Assay (HPA) Summary • Ribosomal RNA Targets • High sensitivity and specificity • Chemiluminescence detection • Solution phase separation/detection • No wash steps

  12. 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 )

  13. In situ Hybridization: Clinical Applications • Gene mapping • Chromosomal abnormalities • Detection of microorganisms in tissue/cells

  14. 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

  15. 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.

  16. 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

  17. 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

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