Cycling probe technology (CPT) is a

molecular biological Molecular biology is the branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interact ...

technique for detecting specific DNA sequences. CPT operates under

isothermal In thermodynamics, an isothermal process is a type of thermodynamic process in which the temperature ''T'' of a system remains constant: Δ''T'' = 0. This typically occurs when a system is in contact with an outside thermal reservoir, and ...

conditions. In some applications, CPT offers an alternative to

PCR PCR or pcr may refer to: Science * Phosphocreatine, a phosphorylated creatine molecule * Principal component regression, a statistical technique Medicine * Polymerase chain reaction ** COVID-19 testing, often performed using the polymerase chain r ...

. However, unlike PCR, CPT does not generate multiple copies of the target DNA itself, and the amplification of the signal is linear, in contrast to the exponential amplification of the target DNA in PCR. CPT uses a sequence specific chimeric probe which hybridizes to a complementary target DNA sequence and becomes a substrate for

RNase H Ribonuclease H (abbreviated RNase H or RNH) is a family of non-sequence-specific endonuclease enzymes that catalyze the cleavage of RNA in an RNA/ DNA substrate via a hydrolytic mechanism. Members of the RNase H family can be found in nearly ...

. Cleavage occurs at the RNA internucleotide linkages and results in dissociation of the probe from the target, thereby making it available for the next probe molecule. Integrated electrokinetic systems have been developed for use in CPT.

Probe

Cycling probe technology makes use of a chimeric nucleic acid probe to detect the presence of a particular DNA sequence. The chimeric probe consists of an

RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...

segment sandwiched between two DNA segments. The RNA segment contains 4 contiguous

purine Purine is a heterocyclic aromatic organic compound that consists of two rings ( pyrimidine and imidazole) fused together. It is water-soluble. Purine also gives its name to the wider class of molecules, purines, which include substituted purin ...

nucleotides. The probes should be less than 30 nucleotides in length and designed to minimize intra-probe and inter-probe interactions.

Process

Cycling probe technology utilizes a cyclic, isothermal process that begins with the

hybridization Hybridization (or hybridisation) may refer to: *Hybridization (biology), the process of combining different varieties of organisms to create a hybrid *Orbital hybridization, in chemistry, the mixing of atomic orbitals into new hybrid orbitals *Nu ...

of the chimeric probe with the target DNA. Once hybridized, the probe becomes a suitable substrate for

. RNase H, an

endonuclease Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonuclease ...

, cleaves the RNA portion of the probe, resulting in two chimeric fragments. The melting temperature (T_m) of the newly cleaved fragments is lower than the melting temperature of original probe. Because the CPT reaction is isothermally kept just above the melting point of the original probe, the cleaved fragments dissociate from the target DNA. Once dissociated, the target DNA is free to hybridize with a new probe, beginning the cycle again. After the fragments have been cleaved and dissociated, they become detectable. A common strategy for detecting the fragments involves

fluorescence Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, ...

. With this method, a fluorescent marker is attached to the 5’ end of the probe and a

quencher In materials science, quenching is the rapid cooling of a workpiece in water, oil, polymer, air, or other fluids to obtain certain material properties. A type of heat treating, quenching prevents undesired low-temperature processes, such as pha ...

is attached to the 3’ end of the probe. When RNase H cleaves the probe, the quencher and fluorescent marker separate, increasing the intensity of the fluorescent marker. Cleaved fragments can alternatively be detected via amplification (e.g.,

) or further modification to allow for other chemical means of detection. When working with small concentrations of target DNA, the CPT protocol can be modified to increase specificity and efficiency. Increasing allotted time has been shown to improve probe cleavage efficiency. Both increasing RNase H concentrations and use of a probe that isn’t prone to inter-probe and intra-probe interactions has been show to increase specificity.

Advantages

Because cycling probe technology does not involve the amplification of target DNA, CPT has a lower risk of

cross contamination Contamination is the presence of a constituent, impurity, or some other undesirable element that spoils, corrupts, infects, makes unfit, or makes inferior a material, physical body, natural environment, workplace, etc. Types of contamination Wi ...

than PCR. In addition, CPT is faster than PCR and doesn’t require a specialized

thermocycler The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus most commonly used to amplify segments of DNA via the polymerase chain reaction (PCR). Thermal cyclers may also be used in laboratories to ...

. CPT also does not require running CPT products on a

gel A gel is a semi-solid that can have properties ranging from soft and weak to hard and tough. Gels are defined as a substantially dilute cross-linked system, which exhibits no flow when in the steady-state, although the liquid phase may still di ...

Disadvantages

CPT requires specialized chimeric probes, making CPT assays more expensive than PCR. Because CPT probes are so specific, a new probe must be designed for each unique assay, further increasing cost. Clinical implementation is hampered financially, but it is also limited by the possibility of samples containing nonspecific RNases other than RNase H.

Applications

CPT can be used to detect specific DNA sequences and by extension specific

genotype The genotype of an organism is its complete set of genetic material. Genotype can also be used to refer to the alleles or variants an individual carries in a particular gene or genetic location. The number of alleles an individual can have in a ...

s. For example CPT can be used to distinguish

GMO A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques. The exact definition of a genetically modified organism and what constitutes genetic engineering varies, with ...

produce from non-GMO produce. Clinically, CPT can be used as an alternative to cell culturing in order to detect

antibacterial resistance Antimicrobial resistance (AMR) occurs when microbes evolve mechanisms that protect them from the effects of antimicrobials. All classes of microbes can evolve resistance. Fungi evolve antifungal resistance. Viruses evolve antiviral resistance. P ...

of a pathogen. CPT, at its core, detects whether a specific sequence is present in a sample. But because cleaved probes accumulate following linear rate kinetics, the amount of target DNA can be quantified. Consequently, CPT has been used to quantify the number of non-coding repeats in organisms. CPT can be used in conjunction with other technologies, like

molecular beacon Molecular beacons, or molecular beacon probes, are oligonucleotide hybridization probes that can report the presence of specific nucleic acids in homogenous solutions. Molecular beacons are hairpin-shaped molecules with an internally quenched fluor ...

s and

qPCR A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...

References

{{reflist Molecular biology Laboratory techniques Amplifiers