Solid-phase amplification

Most detection systems require a high number of DNA template copies to get sufficient signal threshold for detection. Therefore, it is necessary to amplify every template in several orders of magnitude from the library. This clonal amplification is performed using either emulsion PCR (emPCR) or solid-phase amplification. 

The Illumina sequencing platform, which currently is the most widely used NGS platform, was commercialized in 2006, with Illumina acquiring Solexa in early 2007 (34). It is based on sequencing-by-synthesis chemistry, with reversible terminator nucleotides for the four bases, each labeled with a different fluorescent dye, and a DNA polymerase enzyme to incorporate them (35). This reaction mixture for the sequencing reactions is supplied onto the polonies created on a surface by solid-phase amplification. After each terminator nucleotide is incorporated into the DNA strand, a fluorescent signal is... 

Regression correlation coefficient Regression coefficient of determination Rolling circle amplification Water solubility Sodium dodecyl sulfate Supercritical fluid extraction Standard operating procedure Solid-phase extraction Surface plasmon resonance Thymine... 

Synthetic oligonucleotides are very important tools in the study and manipulation of DNA, including such techniques as site-directed mutagenesis and DNA amplification by the polymerase chain reaction. The techniques for chemical synthesis of oligonucleotides are highly developed. Very efficient automated methodologies based on solid phase synthesis are used extensively in fields that depend on the availability of defined DNA sequences.52... 

The process known as SPREAD (Surface Promoted Replication and Exponential Amplification of DNA Analogues) attempts to reach the target, striven for by many researchers, of an exponential proliferation of biomolecules in model systems. As already mentioned, product inhibition (e.g., by dimerisation of the new matrices to give C2) only allowed parabolic growth. In the SPREAD process, both solid phase chemistry and feeding have a positive effect on the synthesis. Thus, no separation processes are required, as excess reagents can be removed just by washing. The synthetic process consists of four steps ... 

Adessi, C., Matton, G., Ayala, G., Turcatd, G., Mermod, J.-J., Mayer, P., and Kawashima, E. (2000) Solid phase DNA amplification Characterisation of primer attachment and amplification mechanisms. Nucleic Acids Res. 28, e87. 

Fig. 31 (A) Principle of a sandwich immunoassay using FDA particulate labels. The analyte is first immobilized by the capture antibody preadsorbed on the solid phase (a) and then exposed to antibody-coated microparticle labels (b). Every microparticle contains 108 FDA molecules. High signal amplification is achieved after solubilisation, release, and conversion of the precursor FDA into fluorescein molecules by the addition of DMSO and NaOH (c). (B) Calibration curves of IgG-FDA microcrystal labels with increasing surface coverage of detector antibody (a-d) compared with direct FITC-labeled detector antibody (e). The fluorescence signals increase with increasing IgG concentration. FDA microcrystals with a high IgG surface coverage (c,d) perform better than those with lower surface coverage (a,b). (Reprinted with permission from [189]. Copyright 2002 American Chemical Society)...

The addition of hapten-labeled primers or nucleotides allowed for the synthesis of modified PCR amplificate during PCR amplification. For immobilization of the functionalized amplificate, either hybridization to complementary solid-phase bound capture oligonucleotides ELOSA [30, 65-67] or coupling of twofold-labeled amplificate to solid-phase-bound STY... 

Immunoassays make use of the high specificity of antibody-antigen interactions. Also, coupled reactions are often used to introduce an element of amplification in the detection signal, giving the assays highly desirable properties of specificity and sensitivity. The antibody-antigen interactions can be carried out either in solution or in the solid phase in the latter, the immobilised species can be either the antigen or antibody. 

Lindsay K, et al. Quantitative detection of hepatitis C vims RNA with a solid-phase signal amplification method Definition of optimal conditions for specimen collection and clinical application in interferon-treated patients. Hepatology 1994 19 1337-41. 

Fig. 2.1. Design of solid-phase EIA. One of the immunoreactants (R or Rj, generally antibody or antigen) is immobilized on the solid phase and serves to capture the complementary reactant from the sample ( immunoextraction ). In EIA, the substance to be assayed is thus added in the second layer. Amplification of the signal of reaction between R and R is achieved with Rj which may consist of several layers.

Based on the principle of enzyme amplification for enzyme immunoassays adopted in the 1980s, Bobrow and associates developed a catalyzed reporter deposition technique (CARD) to achieve amplified signal for solid-phase immunoassay system and membrane immunoassays. Subsequently, this CARD technique was introduced to IHC in 1992. ... 

Another use for drug conjugation is for signal labeling. For example, the drug will be conjugated to an enzyme in EIA to biotin, avidin, or streptavidin in biotin amplification to a protein for adsorption onto a solid-phase support or to polystyrene beads by direct covalent linkage. 

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