Many processes requiring analysis or manipulation of nucleic acids (NAs, e.g., DNA and RNA), such as detection and identification of viruses and bacteria, diagnosis of infections, sequencing of genomes, forensics, and plasmid preparation require the extraction of NAs from complex samples and media. A currently familiar example is the use of polymerase chain reaction (PCR) for the diagnosis of COVID-19 infections. A critical step in such diagnoses is the preparation of samples for PCR analysis by isolation of NA components from nasal swabs, saliva or other bodily specimens. A typical NA extraction process uses a kit with proprietary chemicals and specialized equipment in a well-equipped lab that require technical expertise as well as extended processing time. As the COVID-19 pandemic has shown, there is significant room for improvement in terms of time, price and availability of tests, for which the extraction process can be limiting. Extraction of NAs represents a bottleneck in other processes such as those noted above.
Researchers have developed simple, rapid, and efficient methods for extraction of NAs from complex samples based on the tunable, temperature-sensitive and reversible liquid-liquid phase separation (LLPS) property of cationic elastin-like polypeptides (ELPs). Cationic ELPs can form electrostatic complexes with NAs in aqueous solutions and then can also concentrate those NAs into a complex coacervate (i.e., protein and NA rich aqueous phase) upon triggered LLPS by application of a mild temperature stimulus. The resultant protein/NA-poor phase can be decanted to easily remove cell-debris, viral-debris and other biomacromolecules (e.g., other proteins and polysaccharides) present in the original specimen. The ELP/NA-rich phase can then be resuspended by adding an aqueous solvent at a lower (e.g., room) temperature. The solution conditions of the aqueous electrolyte can be chosen to disrupt ELP-NA bonding to liberate the NA and subsequent thermally triggered LLPS of the unbound ELP can be used to remove it, and recover a solution of aqueous NA for further (e.g., PCR) analysis or processing.
- Simplicity and cost of NA isolation extraction methodology (no specialized equipment or training required) for point of need applications.
- The extraction methodology is adaptable and scalable to multiple nonlaboratory or laboratory settings.
- ELPs can be produced recombinantly in E. coli in standard bioreactors that are amenable to scale up.
- The properties of ELPs allows their simple and efficient purification from cell lysate by repeated cycles of LLPS.
- ELPs are intrinsically stable. Upon purification, they can be stored at room temperature and are very stable in lyophilized form, or in buffer solution.
- ELPs are potentially recyclable for use in multiple extraction cycles.
- Detection and identification of viruses, bacteria and other pathogens
- Diagnosis of infections
- Sequencing of genomes
- Plasmid preparation
- Identification of genetic biomarkers (e.g., single nucleotide polymorphisms)
- Measurement of viral load during infection
Ime: Gregg Banninger