Protein misfolding cyclic amplification (PMCA) is an amplification technique (conceptually like PCR but not involving

nucleotide Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules wi ...

s) to multiply misfolded

prion Prions are misfolded proteins that have the ability to transmit their misfolded shape onto normal variants of the same protein. They characterize several fatal and transmissible neurodegenerative diseases in humans and many other animals. It ...

s originally developed by Soto and colleagues.Saborio, G.P., Permanne, B. and Soto, C. (2001) Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature, 411, 810-813. It is a test for spongiform encephalopathies like CWD or

BSE BSE may refer to: Medicine * Bovine spongiform encephalopathy, also known as mad cow disease, a neurodegenerative disease of cattle * Breast self-examination Stock exchanges * Bahrain Stock Exchange, Bahrain * Baku Stock Exchange, Azerbaijan * B ...

Technique

The technique initially incubates a small amount of abnormal prion with an excess of normal protein, so that some conversion takes place. The growing chain of misfolded protein is then blasted with

ultrasound Ultrasound is sound waves with frequency, frequencies higher than the upper audible limit of human hearing range, hearing. Ultrasound is not different from "normal" (audible) sound in its physical properties, except that humans cannot hea ...

, breaking it down into smaller chains and so rapidly increasing the amount of abnormal protein available to cause conversions. By repeating the cycle, the mass of normal protein is rapidly changed into the prion being tested for.

Development

PMCA was originally developed to,

in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...

, mimic prion replication with a similar efficiency to the

in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...

process, but with accelerated kinetics. PMCA is conceptually analogous to the

polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...

- in both systems a template grows at the expense of a substrate in a cyclic reaction, combining growing and multiplication of the template units.

Replication

PMCA has been applied to replicate the misfolded protein from diverse species. The newly generated protein exhibits the same biochemical, biological, and structural properties as brain-derived PrP^Sc and strikingly it is infectious to

wild type The wild type (WT) is the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, "m ...

animals, producing a disease with similar characteristics as the illness produced by brain-isolated prions.

Automation

The technology has been automated, leading to a dramatic increase in the efficiency of amplification. Now, a single cycle results in a 2500-fold increase in sensitivity of detection over western blotting, whereas 2 and 7 consecutive cycles result in 6 million and 3 billion-fold increases in sensitivity of detection over

western blotting The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecti ...

, a technique widely used in BSE surveillance in several countries.Saa, P., Castilla, J. and Soto, C. (2006) Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification. J.Biol.Chem., 281, 35245-35252.

Sensitivity

It has been shown that PMCA is capable of detecting as little as a single molecule of

oligomer In chemistry and biochemistry, an oligomer () is a molecule that consists of a few repeating units which could be derived, actually or conceptually, from smaller molecules, monomers.Quote: ''Oligomer molecule: A molecule of intermediate relativ ...

ic infectious PrP^Sc. PMCA possesses the ability to generate millions infectious units, starting with the equivalent to one PrP^Sc oligomer; well below the

infectivity In epidemiology, infectivity is the ability of a pathogen to establish an infection. More specifically, infectivity is a pathogen's capacity for horizontal transmission — that is, how frequently it spreads among hosts that are not in a parent� ...

threshold. This data demonstrates that PMCA has a similar power of amplification as PCR techniques used to amplify DNA. It opens a great promise for development of a highly sensitive detection of PrP^Sc, and for understanding the molecular basis of prion replication. Indeed, PMCA has been used by various groups to PrP^Sc in blood of animals experimentally infected with prions during both the symptomatic and pre-symptomatic phases as well as in urine.

Uses

The PMCA technology has been used by several groups to understand the molecular mechanism of prion replication, the nature of the infectious agent, the phenomenon of prion strains and species barrier, the effect of cellular components, to detect PrP^Sc in tissues and biological fluids and to screen for inhibitors against prion replication. Recent studies by the groups of Supattapone and Ma were able to produce prion replication in vitro by PMCA using purified PrP^C and recombinant PrP^C with the sole addition of synthetic

polyanion Polyelectrolytes are polymers whose repeating units bear an electrolyte group. Polycations and polyanions are polyelectrolytes. These groups dissociate in aqueous solutions (water), making the polymers charged. Polyelectrolyte properties are t ...

s and

lipid Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids include ...

s. These studies have shown that infectious prions can be produced in the absence of any other cellular component and constitute some of the strongest evidence in favor of the prion hypothesis. Research in 2020 concluded that protein misfolding cyclic amplification could be used to distinguish between two progressive neurodegenerative diseases,

Parkinson’s disease Parkinson's disease (PD), or simply Parkinson's, is a long-term degenerative disorder of the central nervous system that mainly affects the motor system. The symptoms usually emerge slowly, and as the disease worsens, non-motor symptoms becom ...

and

multiple system atrophy Multiple system atrophy (MSA) is a rare neurodegenerative disorder characterized by autonomic dysfunction, tremors, slow movement, muscle rigidity, and postural instability (collectively known as parkinsonism) and ataxia. This is caused by progr ...

, being the first process to give an objective diagnosis of Multiple System Atrophy instead of just a differential diagnosis.

References

{{reflist Biochemistry detection methods Molecular biology Laboratory techniques Prions