Antibodies for use in the methods and devices of this disclosure can be monoclonal or polyclonal. Merely by way of example, monoclonal antibodies первый житель россии официально заразился коронавирусом can be prepared from murine hybridomas according to the classical method of Kohler and Milstein ( Nature первый житель россии официально заразился коронавирусом 256:495–97, 1975) or derivative methods thereof. Detailed procedures for monoclonal antibody production are described in Harlow and Lane, Using Antibodies: A Laboratory Manual , CSHL, New York, 1999. Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. In one embodiment, an antigen is a coronavirus antigen. Binding or Stable Binding: An oligonucleotide binds or stably binds to a target nucleic acid if a sufficient amount of the oligonucleotide forms base pairs or is hybridized первый житель россии официально заразился коронавирусом to its target nucleic acid, to permit detection первый житель россии официально заразился коронавирусом of that binding. Binding can be detected by either physical or functional properties of the target:oligonucleotide complex. Binding between a target and an oligonucleotide can be detected by any procedure known to one skilled in the art, including functional or physical binding assays. Binding can be detected первый житель россии официально заразился коронавирусом первый житель россии официально заразился коронавирусом functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation, and the like. Physical methods of detecting the binding of complementary strands of DNA or RNA are well known in the art, and include such первый житель россии официально заразился коронавирусом methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, Northern blotting, Southern blotting, dot blotting, and light absorption detection procedures.
For example, a method which is widely used, because it is so simple and reliable, involves observing a change in light absorption of a solution containing an oligonucleotide (or an analog) and a target nucleic acid at 220 to 300 nm as the temperature is slowly increased. If the oligonucleotide or analog has bound to its target, there is a sudden increase in absorption at a characteristic temperature as the oligonucleotide (or analog) первый житель россии официально заразился коронавирусом and target dissociate or melt. The binding between an oligomer and its target nucleic acid is frequently characterized by the temperature (T m ) at which 50% of the oligomer is melted from первый житель россии официально заразился коронавирусом its target. A higher T m means a stronger or more stable complex relative to a complex with a lower T m . cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells. Electrophoresis: Electrophoresis refers to the migration of charged solutes or particles in a liquid medium under the influence of an electric field. Electrophoretic separations are widely первый житель россии официально заразился коронавирусом used for analysis of macromolecules. Of particular importance is the identification of proteins and nucleic acid sequences. Such separations can be based on differences in size and/or charge.
Nucleotide sequences have a uniform charge and are therefore separated based on differences in size. Electrophoresis can be performed in an unsupported liquid medium (for example, capillary electrophoresis), but more commonly the liquid medium travels through a solid supporting medium.
The most widely used supporting media are gels, for example, polyacrylamide and agarose gels. Sieving gels (for example, agarose) impede the flow of molecules.
The pore size of the gel determines the size of a molecule that первый житель россии официально заразился коронавирусом can flow freely through the gel. The amount of time to travel through the gel increases as the size of the molecule increases. As первый житель россии официально заразился коронавирусом a result, small molecules travel through the gel more quickly than large molecules and thus progress первый житель россии официально заразился коронавирусом further from the sample application area than larger molecules, in a given time period. Such gels are used for size-based separations of nucleotide sequences. Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log 10 of their molecular weight.
By using gels with different concentrations of agarose, different sizes of DNA fragments can be resolved.
Higher concentrations of agarose facilitate separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs. Hybridization: Oligonucleotides and their analogs hybridize by hydrogen bonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary bases. Generally, nucleic acid consists of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and the bonding of the pyrimidine to the purine is referred to as “base pairing.” More specifically, A will hydrogen bond to T or U, and G will bond to C. “Complementary” refers to the base pairing that occurs between to distinct nucleic acid sequences or первый житель россии официально заразился коронавирусом two distinct regions of the same nucleic acid sequence.
