BCH5413 – Lecture 3 Notes

Lecture Overview

  • Instructor: Dr. Michelle Gumz

  • Departments: Physiology, Biochemistry, Molecular Biology

  • Lecture Topic: DNA, RNA, and Protein Blotting

    • Introduction of the lecture with the painting "Girl with a Pearl Earring."

    • Outline of today's lecture: Types of blots (Southern, Northern, Western), experimental controls, labeling, and detection methods.

    • Lecture style emphasizes key terms and relevant textbook readings.

Gel Electrophoresis

  • Definition: Electrophoresis is a process used to separate charged molecules in an electrical field.

  • Importance: Discusses separation techniques for DNA, RNA, and proteins.

  • Types of Gels:

    • Agarose: Typically used for DNA and RNA samples.

    • Polyacrylamide: Often used for protein gels; may be toxic and requires caution in preparation.

  • Equipment:

    • Uses power supply to induce an electrical field.

    • Gel apparatus can be horizontal or vertical.

  • Buffers: Variations in salt content, neutrality, and whether they are denaturing based on application.

  • Movement of samples:

    • Negatively charged DNA/RNA moves towards the positive electrode.

    • Separation by size occurs because smaller fragments move faster through the gel.

Examples of Gel Electrophoresis

  • Cartoon of Gel Electrophoresis:

    • Depicts double-stranded DNA restriction fragments from genomic DNA preparation subjected to restriction enzymes.

    • Visualization of size separation in agarose gels.

  • Specific Example:

    • BAC Cloning:

    • Bacterial Artificial Chromosome (150,000 base pairs from mouse chromosome 11).

    • Digestion with EcoR1 produces varying fragment sizes.

  • Preparation for Gel:

    • Example: Run 10 µL of 1 µg/µL of BAC DNA in 50 µL total volume with EcoR1 buffer and water.

    • Use of carcinogenic ethidium bromide for DNA visualization under UV light.

Southern Blot Analysis

  • Overview:

    • Method to determine specific DNA presence in a gel.

    • Transfer DNA fragments from agarose gel to a nylon membrane using capillary action.

  • Equipment & Process:

    • Use wicking paper along with absorbent paper to draw buffer and DNA upwards into the membrane.

    • DNA must be single-stranded for probe hybridization.

  • Steps:

    • Denature DNA in the gel:

      • Soak in dilute acid (depurination) followed by sodium hydroxide to convert double-stranded DNA to single-stranded DNA.

    • Transfer apparatus setup consists of multiple layers: gel, membrane, blotting paper, and weight.

  • Detection:

    • Use labeled probes to identify DNA of interest.

    • Signal detection on film to visualize bands of interest.

  • Controls:

    • Positive controls ensure that the DNA of interest is present and will bind to the probe.

Probe Preparation for Southern Blot

  • Methods to Generate Probes:

    • Random Priming:

    • Start with template DNA. Denature and incubate with Random Primers (Hexamers or Decamers).

    • Use Klenow fragment of DNA polymerase to synthesize labeled DNA probes using radioactively labeled nucleotides (e.g., $ ext{α-}P^{32}dCTP$).

    • End Labeling:

    • Label short syntheses commercially by cleaving five-prime phosphates with Calf Intestinal Alkaline Phosphatase and then adding radioactive labels.

    • Non-radioactive methods (e.g., DIG):

    • Use DIG labeled nucleotides for probe preparation and detect with specific antibodies.

Hybridization Process

  • Hybridization Conditions:

    • Probes incubated with nylon membrane in controlled conditions.

    • Optimize washing conditions for maximum specific signal over background noise.

  • Stringency:

    • Refers to conditions determining mismatches between probe and target DNA.

    • Low stringency: higher background noise.

    • High stringency: precise detection with fewer mismatches.

Transition to Northern Blots

  • Definition: Applied to RNA, hence "Northern" blots.

  • RNA characteristics: Complex structures necessitate denaturation via formaldehyde.

    • Key difference from Southern: Northern blots assess RNA expression levels, not just presence.

  • Example: Using GAPDH as a control to evaluate mRNA levels across different tissues in a Northern blot.

Experimental Controls for Northern Blot

  • Importance: Ensures accuracy in mRNA expression comparisons.

  • Loading Controls: Compare intensity of ribosomal RNA bands on gel.

  • Positive Controls: Validate that expected responses occur in treated cells compared to controls.

In Situ Hybridization

  • Method Overview: Apply hybridization directly in tissue samples.

  • Example: Use RNA scope on mouse kidney samples to visualize mRNA quantity between wild type and knockout.

Introduction to Western Blotting

  • Definition: Focuses on protein detection.

  • Denaturation Steps:

    • Use sodium dodecyl sulfate (SDS) and beta-mercaptoethanol to coat proteins and disrupt disulfide bonds before electrophoresis.

  • Transfer Methods: Protein transfer to membrane requires electrical field rather than capillary action.

Detection in Western Blots

  • Antibody Usage: Requires primary and secondary antibodies to bind to specific proteins and visualize using enzyme-linked detection.

  • Signal Verification: Look for chemiluminescence as a result of enzyme-substrate reaction post-blotting.

Example of Western Blotting Analysis

  • Case Study: Investigate the effect of hormone treatment on Per1 protein levels in kidney cells, using both control and treated samples.

  • Loading Controls: Use housekeeping proteins for normalization of protein levels.

Summary and Conclusion

  • Final Remarks: Emphasis on the necessity of controls in every type of blotting. Confirmation bias comparison using the story of the forged Vermeer paintings, stressing the importance of maintaining scientific integrity in hypothesis testing.

  • Farewell: End of lecture and introduction of the next topic on PCR monitoring.