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Molecular Biology

Probe Structure

Understanding Probe Structures and mechanisms is crucial for choosing the right probe for a specific molecular assay, optimizing reaction conditions, and interpreting the results accurately in the clinical laboratory

The way a probe is built is directly tied to how it works and how we detect its binding to a target sequence. While the fundamental principle is always hybridization (the probe binding to its complementary target), the clever structural designs allow for different ways of generating a detectable signal, especially in real-time PCR

Probe Hybridization: The Foundation

Remember, a probe is a piece of nucleic acid (DNA, RNA, or synthetic oligonucleotide) with a known sequence, designed to specifically bind (hybridize) to a complementary target sequence. To make this binding event detectable, the probe carries a label

Simple Probes (Linear, Labeled Strands)

These are the workhorses for techniques like blotting (Southern, Northern) and traditional in situ hybridization (ISH)

  • Structure: Essentially a linear, single strand of nucleic acid (DNA, RNA, or oligo) with a label attached. The label can be:
    • Radioactive Isotope: (e.g., ³²P) - Historically common, highly sensitive
    • Hapten: (e.g., Biotin, Digoxigenin - DIG) - Small molecules incorporated into the probe. Detected indirectly using enzyme-conjugated antibodies or streptavidin
    • Enzyme: (e.g., Alkaline Phosphatase - AP, Horseradish Peroxidase - HRP) - Directly conjugated to the probe. Detected using chromogenic or chemiluminescent substrates
    • Fluorophore: (e.g., Fluorescein, Cy3, Cy5) - Directly conjugated. Detected by fluorescence microscopy (FISH) or imagers
  • Mechanism of Signal Generation: Signal is generated by the label itself after unbound probe has been washed away. The intensity of the signal (radioactivity, color, light, fluorescence) at a specific location (on a blot, in a cell) indicates the presence and relative amount of the target sequence
  • Key Feature: The signal doesn’t inherently change upon hybridization; specificity relies entirely on stringent washing to remove non-specifically bound and unbound probe before detection

Real-Time PCR Probes: Dynamic Signal Generation

These probes are designed for use during PCR amplification, allowing us to monitor product accumulation in real time. They almost always rely on fluorescence and clever use of quenching mechanisms, often involving Förster Resonance Energy Transfer (FRET)

Core Concepts

  • Fluorophore (Reporter Dye): A molecule that absorbs light energy at one wavelength and emits it at a longer wavelength (fluorescence)
  • Quencher: A molecule that can absorb the energy emitted by a fluorophore when they are in close proximity, preventing the fluorophore from emitting light. Quenchers can be:
    • Dark Quenchers: Absorb energy and release it as heat (no fluorescence). Preferred as they reduce background
    • Fluorescent Quenchers: Another fluorophore that accepts energy via FRET and may emit its own light at a different wavelength
  • FRET (Förster Resonance Energy Transfer): A distance-dependent physical process where energy is transferred non-radiatively from an excited donor fluorophore to a suitable acceptor molecule (another fluorophore or a quencher) when they are very close (typically 1-10 nm)

TaqMan® Probes (Hydrolysis Probes)

  • Structure: A linear oligonucleotide probe designed to bind to a specific target sequence between the forward and reverse PCR primers. It has:
    • A Reporter Fluorophore covalently attached to the 5’ end
    • A Quencher covalently attached to the 3’ end
    • (Often includes a minor groove binder (MGB) at the 3’ end to increase Tm, allowing for shorter, more specific probes)
  • Mechanism of Action (Hydrolysis)
    1. In the intact probe, the Quencher is close enough to the Reporter to absorb its energy (via FRET or other quenching mechanisms), keeping fluorescence low
    2. During PCR annealing, the TaqMan probe hybridizes to its target sequence on the template DNA
    3. During the extension phase, Taq polymerase synthesizes the new strand. When it encounters the bound probe, its 5’ → 3’ exonuclease activity cleaves the probe
    4. This cleavage separates the Reporter dye from the Quencher dye
    5. Freed from the Quencher’s influence, the Reporter dye fluoresces when excited by the instrument’s light source
    6. The increase in fluorescence signal is directly proportional to the amount of PCR product generated
  • Key Feature: Signal generation is dependent on enzymatic cleavage of the hybridized probe

Molecular Beacons

  • Structure: A single-stranded oligonucleotide probe designed with complementary sequences at its 5’ and 3’ ends, forcing it into a stem-loop (hairpin) structure in its free state
    • A Reporter Fluorophore is attached to one end (e.g., 5’)
    • A Quencher is attached to the other end (e.g., 3’)
    • The loop portion contains the sequence complementary to the target DNA
    • The stem is formed by the annealing of the complementary arm sequences at the ends
  • Mechanism of Action (Conformational Change)
    1. In the free state (no target present), the probe exists in the hairpin conformation. The stem keeps the Reporter and Quencher close together, resulting in efficient quenching and low fluorescence
    2. During the PCR annealing step, if the target sequence is present, the loop sequence of the Molecular Beacon hybridizes to it
    3. This probe-target binding is more stable than the stem hybrid. The binding forces the hairpin structure to open, physically separating the Reporter from the Quencher
    4. With the Quencher no longer in close proximity, the Reporter fluoresces upon excitation
    5. The fluorescence signal increases as more probe binds to the accumulating PCR product. Binding is reversible
  • Key Feature: Signal generation relies on a conformational change upon hybridization, separating Reporter and Quencher. No enzymatic cleavage is involved

FRET Probes (Dual Hybridization Probes)

  • Structure: This system uses two separate oligonucleotide probes designed to bind adjacent to each other on the target sequence
    • Probe 1 (Donor Probe): Labeled at its 3’ end with a Donor Fluorophore
    • Probe 2 (Acceptor Probe): Labeled at its 5’ end with an Acceptor Fluorophore (which can also act as a quencher for the donor, or emit its own light)
  • Mechanism of Action (FRET upon adjacent hybridization)
    1. When the probes are free in solution or bound far apart, exciting the Donor fluorophore results only in its characteristic emission; little or no signal is detected from the Acceptor
    2. During PCR annealing, both probes hybridize to their respective adjacent sites on the target DNA
    3. This brings the Donor and Acceptor fluorophores into very close proximity (within FRET distance)
    4. Now, when the instrument’s light source excites the Donor fluorophore, the energy is efficiently transferred via FRET to the Acceptor fluorophore
    5. The Acceptor fluorophore then emits light at its characteristic longer wavelength, and this is the signal that is detected
    6. The intensity of the Acceptor fluorescence increases as more PCR product accumulates, allowing more probe pairs to bind adjacently
  • Key Feature: Signal generation (Acceptor emission) depends on the simultaneous hybridization of two adjacent probes, bringing the Donor and Acceptor dyes close enough for FRET. Excellent for melt curve analysis after PCR

Scorpion® Probes

  • Structure: A complex unimolecular probe that contains both the probe sequence and a PCR primer element, linked together
    • A PCR Primer at the 5’ end
    • A Stem-Loop structure (similar to a Molecular Beacon) attached to the 3’ end of the primer sequence, containing:
      • A Fluorophore (often within the loop or on one arm)
      • A Quencher (on the opposing arm)
      • A probe sequence within the loop/arms complementary to a target site downstream of the primer binding site on the same strand
    • A PCR Blocker element between the primer and the probe stem-loop. This prevents the polymerase from reading through and copying the probe sequence itself during PCR extension
  • Mechanism of Action (Intramolecular Hybridization)
    1. In the initial PCR cycles, the Scorpion primer anneals and is extended by the polymerase. The probe portion remains in its quenched hairpin form
    2. After denaturation and during the subsequent annealing step, the probe sequence (which is now covalently tethered to the 5’ end of the newly synthesized strand via the primer) hybridizes intramolecularly to the complementary target sequence that was just synthesized further down the same strand
    3. This intramolecular binding opens the hairpin structure, separating the Fluorophore from the Quencher
    4. Fluorescence signal increases as more product containing the incorporated Scorpion element is generated
  • Key Feature: Signal generation involves an intramolecular hybridization event following primer incorporation. This is kinetically very fast

Summary Table

Probe Type Structure Mechanism of Signal Generation Signal Change upon Binding Key Application(s)
Simple Probe Linear strand + Label (Radio, Hapten, Fluor) Label detection after washing unbound probe None (signal is inherent) Blotting (Southern, Northern), ISH/FISH
TaqMan® Linear oligo; 5’-Fluor, 3’-Quencher Enzymatic hydrolysis separates Fluor from Quencher Fluorescence Increase Real-time qPCR (Quantification, SNP)
Molecular Beacon Stem-loop; Fluor on one end, Quencher on other Conformational change separates Fluor from Quencher Fluorescence Increase Real-time qPCR (SNP), ISH
FRET Probes Two adjacent probes; 3’-Donor, 5’-Acceptor FRET occurs upon adjacent binding Acceptor Fluorescence Incr. Real-time qPCR (SNP, Melt Curve)
Scorpion® Primer linked to stem-loop probe (F/Q) Intramolecular hybridization opens stem-loop Fluorescence Increase Real-time qPCR (Quantification, SNP)

Key Terms

  • Probe: A labeled nucleic acid strand used to detect a complementary target sequence via hybridization
  • Hybridization: The process of a probe binding to its complementary target sequence through base pairing
  • Label: A detectable molecule (radioisotope, hapten, enzyme, fluorophore) attached to a probe
  • Fluorophore: A molecule that emits light (fluorescence) after absorbing light at a specific wavelength
  • Quencher: A molecule that absorbs energy from a nearby fluorophore, preventing it from emitting light
  • FRET (Förster Resonance Energy Transfer): Non-radiative energy transfer between a donor fluorophore and an acceptor molecule (quencher or another fluorophore) when in close proximity (1-10 nm)
  • TaqMan® Probe (Hydrolysis Probe): A linear probe with 5’-Fluorophore and 3’-Quencher, cleaved by Taq polymerase’s 5’-exonuclease activity during PCR extension, separating Fluorophore from Quencher and generating signal
  • Molecular Beacon: A stem-loop (hairpin) structured probe with Fluorophore and Quencher on opposite ends of the stem. Hybridization to target opens the hairpin, separating Fluorophore from Quencher and generating signal
  • FRET Probes (Hybridization Probes): A pair of probes binding adjacently; one carries a donor fluorophore, the other an acceptor. FRET occurs upon binding, generating signal (acceptor emission)
  • Scorpion® Probe: A unimolecular probe covalently linked to a PCR primer. Contains a stem-loop structure with Fluorophore and Quencher. Intramolecular hybridization after primer extension opens the hairpin, generating signal
  • Stem-Loop Structure (Hairpin): A secondary structure formed in single-stranded nucleic acids when complementary sequences within the strand anneal, forming a double-stranded stem and a single-stranded loop
  • 5’ → 3’ Exonuclease Activity: An enzymatic activity possessed by some DNA polymerases (like Taq) that allows them to degrade nucleic acid strands encountered ahead of the direction of synthesis. Crucial for TaqMan probes
  • Intramolecular: Occurring within the same molecule
  • Intermolecular: Occurring between different molecules