6. Probe Based Nucleic Acid

Probe Based Nucleic Acid Techniques

Overview

  • The field of techniques involving probes to analyze nucleic acids.

    • Key Techniques:

    • Southern blot

    • Northern blot

    • Western blot

    • Eastern blot

  • Inventor: Sir Edwin Southern

Probes

  • Definition: A probe is a single-stranded sequence of DNA or RNA.

    • Function: Used to search for its complementary sequence in a sample genome.

    • Characteristics:

    • Contain a label for detection (Fluorophore, P32).

    • Example sequence: A G C T C C A T T G C G A A T C G G G T A A C G.

Radioactive Probes (32P)

  • Hazard: 32P is a radioactive isotope; poses radiation hazards.

    • Types of Exposures:

    • External Exposures:

      • Hazards from radiation penetrating deeply into the body, producing X-rays via Bremsstrahlung radiation.

      • Risks to eyes and skin.

    • Internal Exposures:

      • Occur when radiative substances are inhaled, ingested, or absorbed through skin/eyes.

Techniques Using Probes

  • Common techniques:

    • Southern Blot

    • Fluorescence in situ hybridization (FISH)

    • Microarray

  • Emerging techniques (e.g., Bionano).

Southern Blotting

  • Definition: A method for detecting specific sequences within a sample of nucleic acid.

    • Named after Edward Southern.

    • Application: Examine the entire genome for specific sequences using probes.

Steps of Southern Blotting

  1. Preparation of DNA:

    • Break DNA into small fragments using restriction enzymes that cut at specific sequences.

    • Result: Separation of DNA into single strands in gel electrophoresis.

  2. Transfer DNA to Membrane:

    • DNA from gel is transferred to a membrane using capillary action.

  3. Probe Incubation:

    • Membrane treated with a probe designed to bind to specific DNA sequences; detection through fluorescent/radioactive label.

Detailed Steps

1. Quantitation and Normalization of DNA

  • Function: Ensures equal intensity bands on the gel.

2. Digestion with Restriction Enzymes

  • Requirement: Must use a frequent cutter for Southern blotting. This will be detailed in the recombinant DNA section of the course.

3. Gel Loading and Electrophoresis

  • Load quantitated, digested DNA samples into a gel.

  • A large number of fragments (>105) result from whole genomic DNA digestion.

4. Denaturation and Transfer to Membrane

  • Denature dsDNA in the gel using a basic solution (e.g., NaOH) and transfer to a positively charged nylon membrane.

  • Incubate gel to ensure proper transfer and then neutralize.

5. Rinsing and Fixation of DNA on Membrane

  • Rinse the membrane with buffer after transfer; use UV light for cross-linking if necessary.

  • Check efficiency of transfer by staining the gel; should have no color remaining.

6. Blocking Non-special Probe Binding

  • Treat membrane with blocking agents to reduce specific binding artifacts.

  • Components include denatured salmon sperm DNA, SDS, proteins, Denhardt’s reagent, and formamide for lowered Tm.

7. Preparation of Probe

  • Radioactive Probes: 32P dNTP labelled probes require preparation with quick random hexamer elongation on-site.

  • Fluorescent Probes: Usually pre-labelled.

  • Hybridization Solution: Includes blocking agents, buffer, and formamide at lower Tm (65-42 °C).

8. Stringency Washes

  • Wash the membrane to remove unbound probes.

    • Stringency Levels:

    • High Temp/Low Salt = Higher Stringency (more unbound probes washed away).

    • Low Temp/High Salt = Lower Stringency.

9. Visualization of Results

  • Use autoradiography for 32P probes and fluorescent light for fluorescent probes.

  • In autoradiography, X-ray exposure to 32P activates silver halide crystals.

Practical Application and Analysis

Quantification Data

  • DNA fragment size: Important for identifying repeats that cause disorders (e.g., CGG expansions leading to fragile X syndrome).

  • Techniques Summary (for Southern Blot):

    • Sample quantitation: 2-10μg.

    • Restriction Enzyme (RE) digest: Frequent cutter needed.

    • Electrophoresis: 0.6-0.8% gel, constant voltage overnight.

    • Transfer and pre-hybridization outlined with specific reagents and conditions.

  • FISH and Northern Blot Comparisons:

    • FISH is used to stain DNA directly within cells for chromosomal analysis.

    • Northern Blotting focuses on RNA without digestion because RNA is already short.

Fluorescence In Situ Hybridization (FISH)

  • Definition: Diagnostic technique for chromosomal abnormalities with a resolution of 1-5 kb.

  • Capable of detecting deletions, amplifications, translocations, and more; typically used in cytogenetics labs.

  • Applications include BCR-ABL fusion detection in Chronic myeloid leukemia and rapid aneuploidy detection.

FISH Procedure

  1. Cell Preparation:

    • Cells grown in culture and prepared for probing.

  2. Slide Preparation:

    • Cells fixed to slides and pre-treated for probe application.

  3. Probe Application and Denaturation:

    • Apply probes and denature, hybridizing overnight.

  4. Washing and Counterstaining:

    • Wash slides to remove excess probes, followed by counterstaining (e.g., DAPI).

FISH Controls

  • Importance of using controls:

    • Internal Control: Additional probes that are not the region of interest (e.g., centromere probes).

    • Negative Control: Shows background levels.

    • Positive Control: Verifies probe functioning.

Advantages and Disadvantages of FISH

  • Advantages:

    • Quick, high resolution, can be used on uncultured cells.

    • Can confirm results from G-banding analysis.

  • Disadvantages:

    • Higher costs, limited information, technical challenges with fluorescence.

DNA Microarrays

  • Definition: Devices consisting of ordered sequences of genes immobilized on a solid support for genomic analysis.

  • Applications include gene expression analysis, comparative genomic hybridization (CGH), and SNP detection.

Microarray Analysis Procedure

  1. Sample Labeling:

    • Patient DNA is labeled with one fluorophore (red) and control DNA with another (green).

  2. Hybridization:

    • Mixture is added to microarray slide, allowing binding.

  3. Result Analysis:

    • Interpret binding patterns based on fluorescence intensity (e.g., yellow indicates equal expression).

Applications of Microarray Technology

  • Identifying genetic mutations in cancer tissues, predicting therapies, and detecting abnormalities in genetic disorders.

Clinical Example: Williams Syndrome

Characteristics and Diagnosis

  • Definition: Genetic condition with cardiovascular issues, developmental delays, and unique social personality traits.

  • Genetic Analysis: Microdeletion on chromosome 7 related to elastin (ELN) gene.

  • Diagnostic Techniques: FISH probes targeting deletion regions for specific genotype identification.

Conclusion

  • This document encapsulates comprehensive information on probe-based techniques to analyze nucleic acids. Techniques include Southern blotting, FISH, microarrays, and their applications in genetic diagnostics. Their comparative advantages and challenges enhance our understanding of molecular genetics and diagnostics.