The 5' nuclease PCR assay detects the accumulation of specific PCR product by hybridization and cleavage of a double-labeled fluorogenic probe during the amplification reaction. The probe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. An increase in reporter fluorescence intensity indicates that the probe has hybridized to the target PCR product and has been cleaved by the 5'-->3' nucleolytic activity of Taq DNA polymerase. In this study, probes with the quencher dye attached to an internal nucleotide were compared with probes with the quencher dye attached to the 3'-end nucleotide. In all cases, the reporter dye was attached to the 5' end. All intact probes showed quenching of the reporter fluorescence. In general, probes with the quencher dye attached to the 3'-end nucleotide exhibited a larger signal in the 5' nuclease PCR assay than the internally labeled probes. It is proposed that the larger signal is caused by increased likelihood of cleavage by Taq DNA polymerase when the probe is hybridized to a template strand during PCR. Probes with the quencher dye attached to the 3'-end nucleotide also exhibited an increase in reporter fluorescence intensity when hybridized to a complementary strand. Thus, oligonucleotides with reporter and quencher dyes attached at opposite ends can be used as homogeneous hybridization probes.

/pdf/oligonucleotides-with-fluorescent-dyes-at-opposite-ends-343i3ami73.pdf

Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.

Plant DNA flow cytometry and estimation of nuclear genome size.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

Unimolecular and bimolecular hybridization probes for the detection of nucleic acid target sequences comprise a target complement sequence, an affinity pair holding the probe in a closed conformation in the absence of target sequence, and either a label pair that interacts when the probe is in the closed conformation or, for certain unimolecular probes, a non-interactive label. Hybridization of the target and target complement sequences shifts the probe to an open conformation. The shift is detectable due to reduced interaction of the label pair or by detecting a signal from a non-interactive label. Certain unimolecular probes can discriminate between target and non-target sequences differing by as little as one nucleotide. Also, universal stems and kits useful for constructing said probes. Also, assays utilizing said probes and kits for performing such assays. Also, such probes capable of allelic discrimination.

Detectably labeled dual conformation oligonucleotide probes, assays and kits

Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4′,6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species-specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.

Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

We describe a simple and rapid method for determining the linearity of a flow cytometer amplification system. The method is based on a fundamental characteristic of linear amplifiers: The difference between two amplified signals increases linearly with increasing amplifier gain. Two populations of beads or cells, differing slightly in fluorescence intensity, are analyzed by the flow cytometer at increasing photomultiplier tube high-voltage settings. The distribution of the populations' mean difference versus mean position is a straight line intersecting the origin for linear amplifiers. Although some types of nonlinearities cannot be detected with this technique, deviations from linearity indicate nonlinear components in the flow cytometer amplification system. The correlation coefficient is used to quantify degree of nonlinearity. We also describe a method for amplifier nonlinearity compensation.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/cyto.990100604

A simple and rapid method for determining the linearity of a flow cytometer amplification system.

Five commonly used fixatives (AZF, B-5, Bouin's, formalin, and Zenker's) were evaluated for their effect on the flow cytometric analysis of DNA and total nuclear protein (TNP) in solid tumors. Data were obtained with the use of colonic adenocarcinoma, squamous carcinoma of the lung, mammary adenocarcinoma, and spleen with a plasma cell leukemic infiltrate. The parameters examined were G0-G1 DNA staining intensity, %G0-G1, percent coefficient of variation (%CV), percent debris, and TNP staining intensity. The results showed that variations in the fixation of solid tumor significantly affected flow cytometric-derived parameters. In this study, paraffin-embedded tissue (PET) fixed in 10% (v/v) neutral buffered formalin (NBF) produced the best results, with a %CV below 4.7, whereas fixatives such as Zenker's and B-5 produced poor %CVs (above 6.0) or uninterpretable TNP and light scatter data. These data suggest that a portion of all tissue samples be fixed in NBF to allow for subsequent analysis by fixative-sensitive assays such as DNA in situ hybridization and flow cytometry.

Effects of several commonly used fixatives on DNA and total nuclear protein analysis by flow cytometry.

A new homogeneous assay system for specific nucleic acid sequences: poly-dA and poly-A detection

As the technology of cytometry matures, there is mounting pressure to address two major issues with data analyses. The first issue is to develop new analysis methods for high-dimensional data that can directly reveal and quantify important characteristics associated with complex cellular biology. The other issue is to replace subjective and inaccurate gating with automated methods that objectively define subpopulations and account for population overlap due to measurement uncertainty. Probability state modeling (PSM) is a technique that addresses both of these issues. The theory and important algorithms associated with PSM are presented along with simple examples and general strategies for autonomous analyses. PSM is leveraged to better understand B-cell ontogeny in bone marrow in a companion Cytometry Part B manuscript. Three short relevant videos are available in the online supporting information for both of these papers. PSM avoids the dimensionality barrier normally associated with high-dimensionality modeling by using broadened quantile functions instead of frequency functions to represent the modulation of cellular epitopes as cells differentiate. Since modeling programs ultimately minimize or maximize one or more objective functions, they are particularly amenable to automation and, therefore, represent a viable alternative to subjective and inaccurate gating approaches. © 2015 International Society for Advancement of Cytometry

Probability state modeling theory.

The amnion regulates movement of fetally derived alpha-fetoprotein into maternal blood.

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