Blotting & Probing

While newer techniques like qPCR and sequencing have taken center stage for many applications, understanding Blotting and Probing is fundamental. It’s like learning classical mechanics before quantum physics – it lays the groundwork for how we detect specific nucleic acid sequences after they’ve been separated

Think of it this way: You’ve run a gel (agarose or polyacrylamide) to separate a complex mixture of DNA or RNA fragments by size. Now you want to know, “Is my specific gene or sequence of interest present in this mess, and if so, which band is it?” That’s where blotting and probing come in

Blotting and Probing: Finding Your Needle in the Haystack

The overall process involves two main phases:

Blotting Transferring the separated nucleic acid fragments from the fragile gel onto a solid support membrane (making them accessible)
Probing (Hybridization) Using a labeled piece of nucleic acid (the probe), which has a sequence complementary to your target, to find and bind to that specific target sequence on the membrane

The Blotting Process: Moving from Gel to Membrane

Why move it? Gels are difficult to handle, fragile, and probes don’t easily diffuse into them. Membranes (like nitrocellulose or nylon) are durable and bind nucleic acids tightly, making probing much easier

Step 1: Electrophoresis: First, you separate your DNA (for Southern) or RNA (for Northern) fragments by size using gel electrophoresis (agarose is common)
Step 2: Pre-Treatment (Important for Southern Blotting!)
- Depurination (Optional, for large DNA): Treat the gel with dilute acid (HCl) to nick large DNA fragments, making them transfer more efficiently
- Denaturation (Essential for DNA): Treat the gel with an alkaline solution (like NaOH). This denatures the double-stranded DNA into single strands. Why? The probe is single-stranded, and hybridization requires base pairing (A-T, G-C) which can only happen if the target DNA is also single-stranded. Note: RNA is typically already single-stranded, so this harsh denaturation step isn’t needed for Northern blots, although denaturing conditions are used during the RNA gel electrophoresis itself to prevent secondary structures
- Neutralization: Bring the pH back to neutral with a buffer (like Tris-HCl) to prepare for transfer
Step 3: The Transfer (“Blotting”): This is the physical movement of the nucleic acids from the gel to the membrane, preserving the separation pattern achieved in the gel. Common methods:
- Capillary Transfer: The classic method. A stack is created: buffer reservoir -> wick (filter paper) -> gel -> membrane -> dry filter paper -> paper towels -> weight. Buffer is drawn up through the wick, gel, and membrane by capillary action, carrying the nucleic acids from the gel and depositing them onto the membrane where they bind. Simple, but can be slow (overnight)
- Vacuum Blotting: Uses suction to pull the buffer and nucleic acids through the gel and onto the membrane. Faster than capillary transfer
- Electroblotting: Uses an electric current to drive the negatively charged nucleic acids from the gel towards a positive electrode, with the membrane placed in between. Fast and efficient, especially common for proteins (Western blotting) but also used for nucleic acids
Step 4: Fixation: The nucleic acids need to be permanently attached to the membrane so they don’t wash off during probing
- Baking: Heating the membrane (e.g., 80°C in a vacuum oven)
- UV Crosslinking: Exposing the membrane (especially nylon) to UV light causes covalent links to form between the nucleic acids and the membrane. Quick and efficient

The Probing Process: Finding the Target with Hybridization

Now that your target sequences are immobilized on the membrane, you need to find them

Step 1: Prehybridization (Blocking)
- Purpose: Membranes can non-specifically bind single-stranded nucleic acids (the probe!). You need to block these non-specific binding sites to prevent the probe from sticking everywhere, which would cause high background noise
- How: Incubate the membrane in a “prehybridization solution.” This solution typically contains blocking agents like Denhardt’s solution, sheared salmon sperm DNA (to saturate non-specific DNA binding sites), and detergents (like SDS)
Step 2: Hybridization
- Introduce the Probe: The labeled probe (a specific DNA, RNA, or oligonucleotide sequence complementary to your target) is added to a fresh hybridization solution (often similar to the prehyb solution)
- Incubation: The membrane is incubated in the probe solution for several hours (or overnight) at a specific temperature. During this time, the probe diffuses and finds its complementary sequence on the membrane, binding via hydrogen bonds (A=T, G≡C)
- Probe Labeling: The probe must be labeled so you can detect where it has bound. Labels can be:
  - Radioactive: Traditionally ³²P incorporated into the probe. Highly sensitive but requires special handling and disposal. Detected by autoradiography (exposing X-ray film)
  - Non-radioactive: More common now. Uses enzymes (like alkaline phosphatase or horseradish peroxidase) or haptens (like Biotin or Digoxigenin - DIG) attached to the probe. Detected using chemiluminescence or colorimetric substrates. Fluorescent dyes can also be used
Step 3: Washing (The Crucial Step for Specificity!)
- Purpose: To wash away unbound probe and probe that has bound non-specifically or weakly (e.g., to sequences with partial similarity), while leaving the probe that is specifically bound to the target sequence. This step determines the signal-to-noise ratio of your experiment
- The Concept of Stringency: This is the key! Stringency refers to the combination of conditions (temperature, salt concentration) used during the washes that dictate how perfectly matched the probe and target sequences must be to remain hybridized (bound together)
  - High Stringency: Conditions that promote the dissociation of weakly bound probes (e.g., high temperature, low salt concentration). Only perfectly (or very nearly perfectly) matched probe-target hybrids will remain bound. Used to detect highly specific targets
  - Low Stringency: Conditions that allow even partially mismatched probe-target hybrids to remain bound (e.g., lower temperature, higher salt concentration). Used if you are looking for related sequences (e.g., homologous genes in different species) or if your probe isn’t a perfect match
- How Washing is Done: Typically involves a series of washes, starting with lower stringency (higher salt, lower temp) and progressing to higher stringency (lower salt, higher temp)
  - Salt Concentration (e.g., SSC buffer - Saline Sodium Citrate): Salt ions (Na+) shield the negative charges on the phosphate backbones of the probe and target. High salt concentration stabilizes the duplex (lower stringency). Low salt concentration increases charge repulsion, destabilizing mismatched duplexes (higher stringency)
  - Temperature: Higher temperatures provide more kinetic energy, making it easier for hydrogen bonds to break. Thus, higher temperatures increase stringency, melting off less stable (mismatched) hybrids
  - Denaturants (e.g., Formamide): Sometimes included in hybridization/wash buffers. Formamide lowers the melting temperature of nucleic acid duplexes, effectively increasing stringency at a given temperature
- Finding the Balance: You adjust the wash conditions (time, temp, salt concentration) to get the strongest signal for your specific target with the lowest background noise
Step 4: Detection
- Visualize where the probe has bound on the membrane
- Radioactive Probes: Expose the membrane to X-ray film (autoradiography) or a phosphorimager screen. Bands appear where the probe bound
- Non-radioactive Probes: Add detection reagents
  - Chemiluminescence: If using enzyme-labeled probes (AP or HRP), add a chemiluminescent substrate. The enzyme reacts with the substrate to produce light, which is detected by exposing film or using a CCD camera imager. Very sensitive
  - Colorimetric: Add a chromogenic substrate. The enzyme converts the substrate into a colored precipitate that deposits directly onto the membrane at the location of the probe. Simpler, but less sensitive
  - Fluorescence: If using fluorescently labeled probes, detect using an appropriate fluorescence imager

Specific Blot Types

Southern Blot: DNA target separated on gel -> Transferred to membrane -> Probed with labeled DNA/RNA probe. Used to detect specific DNA sequences, gene copy number, RFLPs, structural rearrangements. (Named after Edwin Southern)
Northern Blot: RNA target separated on gel (under denaturing conditions) -> Transferred to membrane -> Probed with labeled DNA/RNA probe. Used to detect specific RNA molecules, determine transcript size, study gene expression levels
Western Blot: Protein target separated by PAGE (usually SDS-PAGE) -> Transferred to membrane -> Probed with specific antibodies. Used to detect specific proteins. (Focus of this course is nucleic acids, but good to know the analogy)

Clinical Relevance & Summary

While time-consuming and labor-intensive compared to PCR-based methods, blotting techniques (especially Southern) are still valuable for:

Detecting large deletions, insertions, or rearrangements that might be missed by PCR
Analyzing complex regions with repetitive sequences (e.g., Fragile X triplet repeats)
Confirming results from other methods
Northern blotting remains a gold standard for analyzing RNA transcript size and splice variants, though qPCR and RNA-Seq are often used for expression quantification

Understanding blotting, hybridization, washing, and especially stringency provides fundamental insight into how we achieve specificity in nucleic acid detection, principles that also apply to techniques like microarrays and in situ hybridization. It’s all about getting that labeled probe to stick specifically to its target and then washing away everything else!

Key Terms

Blotting: The process of transferring separated macromolecules (DNA, RNA, or protein) from a gel onto a solid support membrane (e.g., nitrocellulose, nylon) while maintaining the separation pattern
Probe: A labeled molecule (typically a single-stranded nucleic acid sequence - DNA, RNA, or oligonucleotide) that is complementary to a specific target sequence of interest. It is used to detect the presence of the target sequence through hybridization
Hybridization (Nucleic Acid): The process by which two complementary single-stranded nucleic acid molecules (e.g., the probe and the target sequence on the membrane) anneal (bind) to each other through specific hydrogen bonding between base pairs (A-T/U, G-C)
Target Sequence: The specific DNA or RNA sequence on the membrane that the probe is designed to detect
Stringency: A measure of the reaction conditions (primarily temperature, salt concentration, and presence of denaturants like formamide) during hybridization and washing steps that determine the degree of complementarity required between the probe and target sequence for a stable duplex to form
High Stringency Conditions: Experimental conditions (high temperature, low salt concentration) that require a high degree of base pairing complementarity for the probe to remain bound to the target. These conditions wash off non-specifically or partially matched probes, increasing specificity
Low Stringency Conditions: Experimental conditions (lower temperature, higher salt concentration) that allow probes to bind to targets even with some degree of base pair mismatch. Used when searching for related but not identical sequences
Washing (in Blotting): Post-hybridization steps involving rinsing the membrane with buffers of defined stringency to remove unbound and non-specifically bound probe, thereby reducing background noise and enhancing the specific signal from the probe bound to the target
Membrane: The solid support (e.g., nitrocellulose, nylon) onto which nucleic acids are transferred from the gel during blotting. It provides a durable surface for hybridization and detection
Fixation (Blotting): The process of permanently attaching the transferred nucleic acids to the membrane, typically by baking or UV crosslinking, to prevent them from washing off during subsequent probing steps
Blocking (Prehybridization): Treating the membrane with blocking agents (e.g., Denhardt’s solution, salmon sperm DNA) before adding the probe to saturate non-specific binding sites on the membrane surface, thus preventing the probe from sticking randomly and reducing background signal
Detection (Blotting): The method used to visualize where the labeled probe has bound to the target sequence on the membrane (e.g., autoradiography for radioactive probes, chemiluminescence or colorimetric methods for enzyme-labeled probes, fluorescence imaging for fluorescent probes)
Southern Blot: A technique used to detect specific DNA sequences, involving separation of DNA fragments by electrophoresis, transfer to a membrane, and hybridization with a labeled probe
Northern Blot: A technique used to detect specific RNA sequences (and analyze their size and abundance), involving separation of RNA fragments by electrophoresis under denaturing conditions, transfer to a membrane, and hybridization with a labeled probe

Capillary

Probe Hybridization