Factors that inhibit the amplification of nucleic acids by PCR are present with target DNAs from many sources. The inhibitors generally act at one or more of three essential points in the reaction in the following ways: they interfere with the cell lysis necessary for extraction of DNA, they interfere by nucleic acid degradation or capture, and they inhibit polymerase activity for amplification of target DNA. Although a wide range of inhibitors is reported, the identities and modes of action of many remain unclear. These effects may have important implications for clinical and public health investigations, especially if the investigations involve food and environmental screening. Common inhibitors include various components of body fluids and reagents encountered in clinical and forensic science (e.g., hemoglobin, urea, and heparin), food constituents (e.g., organic and phenolic compounds, glycogen, fats, and Ca 21 ), and environmental compounds (e.g., phenolic compounds, humic acids, and heavy metals). Other, more widespread inhibitors include constituents of bacterial cells, nontarget DNA and contaminants, and laboratory items such as pollen, glove powder, laboratory plasticware, and cellulose. This review discusses the findings of many studies related to clinical, food, and environmental microbiology, including approaches that have been used to overcome inhibition and facilitate amplification for detection and typing. Few areas of biological science remain untouched by the invention of PCR (34, 81, 99). Other methods for amplifying nucleic acids (72, 123), such as Qb replicase (18), ligase chain reaction (13, 128), single-stranded sequence replication (17, 47), strand displacement amplification (126, 127), and nucleic acid sequence-based amplification (23, 122), have been described, but these methods have received less attention. Problems sometimes occur with PCR, however (124). Despite early indications of great sensitivity, the sensitivity of PCR may be a negative aspect of the procedure, since the most commonly reported problem is false-positive results due to cross-contamination (98, 124). This problem can be overcome by UV irradiation (100), with sodium hypochlorite (92), and by photochemical or enzymic methods (25, 36, 40, 78). One problem that is less discussed is reaction inhibition. This may be total or partial and can manifest itself as complete reaction failure or as reduced sensitivity of detection. In some cases, inhibition may be the cause of false-negative reactions, since few workers incorporate internal controls in each reaction tube. Early evidence of exquisite sensitivity with mammalian cells (53) involving detection of a single molecule of DNA from a hair was not reproduced when PCR was applied to many microbial (and some mammalian) situations, where poor sensitivity, specificity, and reproducibility have been reported (16, 82, 86, 129, 132, 134). There may also be potentially important effects in PCR typing reactions (121), and difficulties can occur in post-PCR manipulation (61). Although systematic study of inhibition has seldom been the focus of published investigations, many workers have reported these effects in the course of other studies (12, 19, 21, 124, 129, 132, 133). Considering the prevalence of this problem, it is surprising that few systematic and mechanistic studies of PCR inhibition have been reported. Rossen et al. (97) contributed the most comprehensive study of PCR inhibition, identifying inhibitory factors in foods, bacterial culture media, and various chemical compounds. These inhibitory factors included organic and inorganic chemicals, detergents, antibiotics, buffers, enzymes, polysaccharides, fats, and proteins. This review lists and discusses inhibitors and methods that can overcome the attenuation of amplification in clinical, food, and environmental microbiology. It is beyond the scope of this paper to discuss in detail the various physical, enzymic, and chemical methods used in the extraction, purification, and quantitation of nucleic acids. Those methods are presented and discussed in commercial literature and elsewhere (14, 95, 96, 106, 134).

https://aem.asm.org/content/aem/63/10/3741.full.pdf

Inhibition and facilitation of nucleic acid amplification.

The complexity of the regulatory mechanisms in biological systems has become increasingly apparent. This has stimulated a demand for efficient physical separation methods of cells to enable investigation of functionally homogeneous cell populations. Ideally the separation according to physical parameters should also be a separation according to functional characteristics of the cells. It has long been known that there is a close association between cell function and membrane specificity. Thus separation according to specific membrane properties as reflected, for example, in changes in cell surface charge is likely to offer a more promising approach than separation on the basis of volume or density, as in the classical centrifugation techniques. Changes in the surface charge of cells or membranes in most cases reflects processes of differentiation or transformation or effects resulting from the influence of stimulating factors. This type of change in surface charge, which is associated with functional changes of cells or membranes, can be recognized and used in electrophoretic separation techniques. This has been known effectively for twenty years, which makes it all the more surprising that electrophoretic cell separation methods have not been more widely applied.

New aspects in preparative and analytical continuous free‐flow cell electrophoresis

Free flow electrophoresis can separate mouse spleen and lymph node lymphocytes into two fractions: a high mobility (more negatively charged) and a low mobility (less negatively charged) fraction. The high mobility lymphocytes are sensitive to anti-Θ serum and complement but resistant to an anti-serum against mouse specific B lymphocyte antigen (MBLA). They produce rosettes with sheep red cells and induce a graft-vs-host reaction when injected into F1-hybrid recipients. The high mobility lymphocytes also respond to phytohemagglutinin in vitro and are cytotoxic to allogeneic target cells after appropriate immunization. The low mobility lymphocytes are sensitive to anti-MBLA serum and complement but resistant to anti-Θ serum, form rosettes and hemolytic plaques against sheep red cells after immunization, but neither respond to phytohemagglutinin in vitro nor induce a graft-vs-host reaction. In CBA mice the percentages of high and low mobility lymphocytes in the spleen and lymph nodes correspond to the known distributions of T and B cells in these organs. The separated cells are viable since they are able to perform biological functions. Thus free flow cell electrophoresis seems to produce viable populations of mouse T and B lymphocytes.

Separation of T and B lymphocytes by preparative cell electrophoresis.

Isoelectric focusing (IEF) is an excellent tool at analytical scales but has some drawbacks at preparative and process scales. Alternative electrofocusing methods have been around for over a decade but have only recently reached the point where they can begin to compete head-to-head with IEF. This paper describes some of the advances made in this field since the mid-1980s and shows how they are related to IEF by a common mathematical expression. In addition, one new technique is described which allows real-time computer-control of the focusing gradient.

A Brief Review of Alternative Electrofocusing Techniques

Thymus-dependent (T) lymphocytes are responsible for a wide range of immunological phenomena. Evidence is accumulating that this general Tlymphocyte class includes distinct sub-sets, with differing physiological properties and a degree of functional specialisation. These subpopulations may represent differentiation steps in a single developmental pathway. Alternatively, they may refiect the existence of either branched pathways leading to different specialised end-points, or of entirely separate and specialised lymphocyte lineages. To understand their function and interrelationships, we need to be able to recognise these subpopulations on the basis of some reproducible characteristic, and to experimentally separate them from each other. Cell separation techniques which depend on the overall physical properties of the cells can fulfil many of these requirements.

Subpopulations of T‐Lymphocytes

Publisher Summary This chapter describes the principle of free-flow electrophoresis. “Continuous deflection electrophoresis” is a method of separation, based on a different principle and also suitable for preparative problems. In this electrophoretic procedure the mixture to be separated is injected continuously in a fine stream into the medium—that is flowing uniformly at right angles to the electric field. Molecules and particles with differing charge are then deflected from the direction of flow at an angle that depends on the rate of flow of the medium and the electrophoretic mobility of the particle. As it is possible to apply this principle to a buffer film that flows vertically, continuous free-flow electrophoresis can be applied both for analysis and preparation to a whole spectrum of materials from low-molecular substances to coarse particles. The method thus permits not only the separation of cells and subcellular particles or organelles, but also the separation and preparative isolation of their enzyme systems without loss of activity.

Chapter VIII Free-flow Electrophorisis: (A Technique for Continuous Preparative and Analytical Separation)

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Chapter VIII Free-flow Electrophorisis: (A Technique for Continuous Preparative and Analytical Separation)