Probe based Lecture part 2

Overview of Molecular Techniques

  • Understanding reagents and steps in molecular techniques is crucial.

Southern Blotting

  • Reagents and Purpose: Knowledge of reagents, DNA samples, and their roles in southern blotting is essential for troubleshooting.

  • Common Questions: Troubleshooting questions related to southern blotting may appear on exams.

Troubleshooting Common Errors

  • Adding Too Much Sample DNA:

    • Issue: Can lead to increased noise and reduced resolution in results.

    • Results:

    • Bands appear too thick or smudged.

    • Difficulty in distinguishing between bands.

    • Increased likelihood of observing off-target or random binding events (more bands).

  • Not Digesting DNA with Restriction Enzyme:

    • Issue: Without a restriction enzyme, large genomic DNA segments remain intact.

    • Results:

    • Appearance of a high molecular weight band at the top of the gel.

    • Lack of specific probe binding leads to inaccurate results or no identifiable bands.

  • Not Incubating Gel in NaOH:

    • Purpose: NaOH denatures DNA, allowing probes to bind.

    • Results:

    • Absence of probe binding.

    • No results or specific binding patterns visible.

  • Insufficient Incubation Time in NaOH:

    • Issue: Limited denaturation leads to ineffective binding.

    • Results:

    • Weak and possibly non-specific probe binding.

  • Not Removing Air Bubbles from Filter Paper:

    • Issue: Air bubbles prevent capillary action that is essential for blotting.

    • Results:

    • Possible blank spots on the blot where DNA is not transferred.

  • Inadequate Transfer Time:

    • Issue: Insufficient duration leads to limited transfer of DNA.

    • Results:

    • Weak, but specific bands may be observed.

  • Forgetting Formamide in Prehybridization:

    • Purpose: Formamide helps to denature DNA during hybridization.

    • Results:

    • No probe binding or specific bands detected due to lack of denaturation.

  • Insufficient Blocking with Prehybridization Solution:

    • Results:

    • Non-specific binding of the probe leads to inaccurate results.

  • Insufficient Hybridization Time with Probe:

    • Results:

    • Weak, specific bands may result from insufficient probe interaction.

  • High Stringency Washing Conditions:

    • Issue: Stringency is too high, washing away all bound probes.

    • Results:

    • No bands detected or weak binding evident from overly stringent conditions.

Final Exam Preparation

  • Students should note these troubleshooting elements for southern blotting for exams as comparable to those from PCR.

Northern Blotting

  • Similar to southern blotting, but focuses on RNA analysis. Minimal details required for this method identified.

Fluorescence In Situ Hybridization (FISH)

  • FISH is crucial for detecting chromosomal abnormalities and is applicable in clinical settings.

    • In situ: Allows probing of DNA within cells without extraction.

    • Higher resolution than southern blotting but lower than sequencing.

Types of Mutations Detected

  • Common Mutations:

    • Deletions, amplifications, and translocations are targeted.

    • Different mutations are relevant in inherited diseases and cancers.

Process for FISH Technique

  1. Fixation of Cells: Cells are fixed with formalin to create protein-nucleic acid crosslinks.

  2. Probe Design: Probes must be complementary to the regions of interest.

    • Short double-stranded RNA or DNA probes are synthesized using random DNase cuts.

    • Fluorophores are attached to nucleotides during probe synthesis for later visualization.

  3. Hybridization:

    • Denature target DNA in the cells by heating.

    • Allow the labeled probe to bind to the target DNA under specific conditions.

  4. Washing: Unbound probes are removed through washing under controlled stringency conditions.

  5. Visualization: A fluorescence microscope is used to analyze results. Fluorescent signals indicate successful hybridization, while absent signals denote mutations.

Applications of FISH

  • Commonly used for:

    • Hematologic malignancies (e.g., chronic myeloid leukemia identified via BCR-ABL fusion).

    • Evaluating solid tumors and various cancers (e.g., breast, lung cancers).

    • Detecting chromosomal abnormalities such as aneuploidy and microdeletions (e.g., Williams syndrome).

Controls in FISH

  • Importance of controls in ensuring probe efficacy:

    • Internal Control: Probes bind to non-target regions (e.g., centromeres) to confirm the process is working.

    • Negative Control: Ensures reliability by using a probe that should not bind under normal conditions.

    • Positive Control: Additional probes to confirm expected results.

Advantages of FISH Over Southern Blotting

  • Higher resolution and specificity; applicable on uncultured cells.

  • Less time-consuming compared to southern blot procedures.

Summary of Different Probes

  • Whole Chromosome Paint Probes: Bind to entire chromosomes.

  • Unique Sequence Probes: Target specific genes for mutations.

  • Repetitive Sequence Probes: Useful in assessing diseases with repetitive DNA sequences.

Closing Remarks

  • FISH demonstrates advanced diagnostic capabilities in comparison to traditional methodologies like Southern blotting.