The easiest way of detecting specific nucleic acid sequences or genes of interest is through direct hybridization of a probe to microbial nucleic acid extracts. Whole-cell DNA or RNA is extracted from the environmental sample and fixed to a positively charged membrane, e.g. nylon or nitrocellulose. Bacterial colonies can also be replica-plated from agar plates to membranes and their nucleic acids exposed in situ following lysis for subsequent hybridization. Probes may be used to detect genes in the bacterial genome (Southern blots) or to detect mRNA or rRNA (Northern blots). For the in situ identification of individual whole cells it is necessary to make the cells permeable to oligonucleotide probes hybridizing with rRNA. These hybridization techniques rely on the specific binding of nucleic acid probes to complementary DNA or RNA (target nucleic acid). The probes are single strands of nucleic acid with the potential of carrying detectable marker molecules highly specifically to complementary target sequences, even if these sequences account for only a small fraction of the target nucleic acid. Either DNA or RNA can serve as a nucleic acid probe, but for a number of reasons (e.g. ease of synthesis and stability), most studies have employed DNA probes (Holben and Hedje 1988). Two general types of probes that have been developed are DNA probes complementary to a single gene or a small region of a gene and DNA probes complementary to genus- or species-specific regions of 16S rRNA for use in whole cell in situ hybridization (FISH).

Author(s):  
B.A. Hamkalo ◽  
S. Narayanswami ◽  
A.P. Kausch

The availability of nonradioactive methods to label nucleic acids an the resultant rapid and greater sensitivity of detection has catapulted the technique of in situ hybridization to become the method of choice to locate of specific DNA and RNA sequences on chromosomes and in whole cells in cytological preparations in many areas of biology. It is being applied to problems of fundamental interest to basic cell and molecular biologists such as the organization of the interphase nucleus in the context of putative functional domains; it is making major contributions to genome mapping efforts; and it is being applied to the analysis of clinical specimens. Although fluorescence detection of nucleic acid hybrids is routinely used, certain questions require greater resolution. For example, very closely linked sequences may not be separable using fluorescence; the precise location of sequences with respect to chromosome structures may be below the resolution of light microscopy(LM); and the relative positions of sequences on very small chromosomes may not be feasible.


2006 ◽  
Vol 72 (8) ◽  
pp. 5311-5317 ◽  
Author(s):  
Kengo Kubota ◽  
Akiyoshi Ohashi ◽  
Hiroyuki Imachi ◽  
Hideki Harada

ABSTRACT Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.


2008 ◽  
Vol 25 (3) ◽  
pp. 283-287
Author(s):  
CHRISTINA PETTAN-BREWER ◽  
LI FU ◽  
SAMIR S. DEEB

Many attempts have been made over the years to distinguish human and primate L (long-wavelength sensitive) from M (middle-wavelength sensitive) cone photoreceptors using either immunohistochemistry or in situ hybridization. These attempts have been unsuccessful due to the very high degree of identity between the sequences of the L and M proteins and encoding mRNAs. The recent development of chemically modified oligonucleotide probes, referred to as locked nucleic acid (LNA) probes, has shown that they hybridize with much greater affinity and specificity to the target nucleic acid. This has greatly increased the potential for differentiating L from M cones by in situ hybridization. We have designed LNA oligonucleotide probes that are complementary to either the L or M coding sequences located in exon 5 of the Macaca nemestrina L and M pigment genes. We have shown that the LNA-M and LNA-L probes hybridize specifically to their respective target nucleic acid sequences in vitro. This result strongly suggests that these probes would be instrumental in rapidly distinguishing L from M cone in the entire retina, and in defining the cone mosaic during development and in adults.


2020 ◽  
Vol 11 ◽  
Author(s):  
Andrew J. Kinloch ◽  
Yuta Asano ◽  
Azam Mohsin ◽  
Carole Henry ◽  
Rebecca Abraham ◽  
...  

In human lupus nephritis, tubulointerstitial inflammation (TII) is associated with in situ expansion of B cells expressing anti-vimentin antibodies (AVAs). The mechanism by which AVAs are selected is unclear. Herein, we demonstrate that AVA somatic hypermutation (SHM) and selection increase affinity for vimentin. Indeed, germline reversion of several antibodies demonstrated that higher affinity AVAs can be selected from both low affinity B cell germline clones and even those that are strongly reactive with other autoantigens. While we demonstrated affinity maturation, enzyme-linked immunosorbent assays (ELISAs) suggested that affinity maturation might be a consequence of increasing polyreactivity or even non-specific binding. Therefore, it was unclear if there was also selection for increased specificity. Subsequent multi-color confocal microscopy studies indicated that while TII AVAs often appeared polyreactive by ELISA, they bound selectively to vimentin fibrils in whole cells or inflamed renal tissue. Using a novel machine learning pipeline (CytoSkaler) to quantify the cellular distribution of antibody staining, we demonstrated that TII AVAs were selected for both enhanced binding and specificity in situ. Furthermore, reversion of single predicted amino acids in antibody variable regions indicated that we could use CytoSkaler to capture both negative and positive selection events. More broadly, our data suggest a new approach to assess and define antibody polyreactivity based on quantifying the distribution of binding to native and contextually relevant antigens.


1994 ◽  
Vol 6 (3) ◽  
pp. 308-314 ◽  
Author(s):  
AnaPatricia Garcia ◽  
Kenneth S. Latimer ◽  
Frank D. Niagro ◽  
Branson W. Ritchie ◽  
Raymond P. Campagnoli

Liver sections from 32 psittacine birds with multifocal to coalescing hepatocellular necrosis were examined to determine the cause of disease. Avian polyomavirus (APV) infection (19 of 32 birds), bacterial hepatitis (5 of 32 birds), and chlamydiosis (3 of 32 birds) were major causes of hepatic disease. The presence of APV inclusions or nucleic acid was demonstrated using hematoxylin and eosin (HE) staining, DNA in situ hybridization, and DNA amplification with Southern blotting. Amphophilic intranuclear inclusions, suggestive of APV infection, were observed in HE-stained liver sections from 5 of 32 birds. Hepatocellular karyomegaly was present in liver tissues from 10 birds (5 birds with typical APV inclusions and 5 birds without discernable inclusions). DNA in situ hybridization recognized intranuclear APV nucleic acid in liver sections of 18 of 32 birds. DNA amplification with Southern or dot blots also identified APV nucleic acid in processed, paraffin-embedded livers of 18 of 32 birds. This study demonstrates that acute APV infection is a frequent cause of multifocal to coalescing hepatocellular necrosis in psittacine birds. Furthermore, APV infection is best diagnosed using DNA probes, especially when typical intranuclear inclusions are not observed microscopically.


2006 ◽  
Vol 72 (8) ◽  
pp. 5453-5462 ◽  
Author(s):  
Sandra A. Wilks ◽  
C. William Keevil

ABSTRACT Using fluorescence in situ hybridization to detect bacterial groups has several inherent limitations. DNA probes are generally used, targeting sites on the 16S rRNA. However, much of the 16S rRNA is highly conserved, with variable regions often located in inaccessible areas where secondary structures can restrict probe access. Here, we describe the use of peptide nucleic acid (PNA) probes as a superior alternative to DNA probes, especially when used for environmental samples. A complex bacterial genus (Legionella) was studied, and two probes were designed, one to detect all species and one targeted to Legionella pneumophila. These probes were developed from existing sequences and are targeted to low-binding-affinity sites on the 16S rRNA. In total, 47 strains of Legionella were tested. In all cases, the Legionella spp. PNA probe labeled cells strongly but did not bind to any non-Legionella species. Likewise, the specific L. pneumophila PNA probe labeled only strains of L. pneumophila. By contrast, the equivalent DNA probes performed poorly. To assess the applicability of this method for use on environmental samples, drinking-water biofilms were spiked with a known concentration of L. pneumophila bacteria. Quantifications of the L. pneumophila bacteria were compared using PNA hybridization and standard culture methods. The culture method quantified only 10% of the number of L. pneumophila bacteria found by PNA hybridization. This illustrates the value of this method for use on complex environmental samples, especially where cells may be in a viable but noncultivable state.


2003 ◽  
Vol 69 (9) ◽  
pp. 5673-5678 ◽  
Author(s):  
Chuanwu Xi ◽  
Michal Balberg ◽  
Stephen A. Boppart ◽  
Lutgarde Raskin

ABSTRACT DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution. In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA MB. DNA and PNA molecular beacons were successfully used to detect whole cells in fluorescence in situ hybridization (FISH) experiments without a wash step. The FISH results with the PNA molecular beacons were superior to those with the DNA molecular beacons: the hybridization kinetics were much faster, the signal-to-noise ratio was much higher, and the specificity was much better for the PNA molecular beacons. Finally, it was demonstrated that the combination of the use of PNA molecular beacons in FISH and flow cytometry makes it possible to rapidly collect quantitative FISH data. Thus, PNA molecular beacons might provide a solution for limitations of traditional FISH methods, such as variable target site accessibility, poor sensitivity for target cells with low rRNA content, background fluorescence, and applications of FISH in microfluidic devices.


Parasitology ◽  
1988 ◽  
Vol 97 (1) ◽  
pp. 63-73 ◽  
Author(s):  
W. C. Gibson ◽  
P. Dukes ◽  
J. K. Gashumba

SUMMARYWe have obtained 5 specific DNA probes for African trypanosomes of the subgenera Trypanozoon and Nannomonas. Each probe consists of one repeat unit of the major repetitive DNA (satellite DNA) of each species or intra-specific group. One probe hybridized with all members of subgenus Trypanozoon (except T. equiperdum which was not tested). In subgenus Nannomonas, one probe recognized T. simiae, but 3 probes were needed to identify all stocks of T. congolense available. Each of the 3 latter probes recognized trypanosomes from one of the 3 major groups of T. congolense previously defined by isoenzyme characterization, i.e. savannah, forest and Kenya coast types. As few as 100 trypanosomes could be unequivocally identified by dot blot hybridization and individual trypanosomes could be identified by in situ hybridization. We show how this simple methodology can be used in the field for the identification of immature and mature trypanosome infections in tsetse.


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