High-resolution nucleic acid sequence mapping via in situ hybridization at the Electron Microscope level

1995 ◽  
Vol 53 ◽  
pp. 752-753
Author(s):  
B.A. Hamkalo ◽  
S. Narayanswami ◽  
A.P. Kausch
Keyword(s):  
Nucleic Acid ◽  
Interphase Nucleus ◽  
Genome Mapping ◽  
Precise Location ◽  
Electron Microscope Level ◽  
Rna Sequences ◽  
Fundamental Interest ◽  
Whole Cells ◽  
Dna And Rna

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.

scholarly journals Detection of viral DNA and RNA by in situ hybridization.

1986 ◽  
Vol 34 (1) ◽  
pp. 33-38 ◽  
Author(s):  
J K McDougall ◽  
D Myerson ◽  
A M Beckmann
Keyword(s):  
Dna Sequences ◽  
Viral Dna ◽  
Rna Sequences ◽  
Hpv Dna ◽  
Dna And Rna ◽  
Common Warts ◽  
Anogenital Region ◽  
Simplex Virus ◽  
Human Viruses

Using cloned restriction endonuclease fragments of Herpes simplex virus (HSV), human papillomavirus (HPV), and cytomegalovirus (CMV) DNA as probes, viral DNA and RNA sequences have been detected in human tissues. The probes were labeled either with a radioactive isotope, for subsequent detection by autoradiography, or with biotin. This latter technique has been successfully used to visualize HPV DNA in tissues that have been fixed in formalin and embedded in paraffin, and is therefore of value in retrospective studies of histological specimens. HPV DNA was detected under non-stringent conditions (Tm = -42 degrees C) with heterologous probes in plantar and common warts, laryngeal papillomas, and anogenital condylomas. The specific type of HPV was established using stringent hybridization conditions (Tm = - 17 degrees C). Results from these and from malignant tissues show the distribution and localization of HSV and HPV RNA and DNA sequences in malignancies of squamous cell origin in the anogenital region. Both HSV and HPV DNA sequences have occasionally been detected in the same tumor, providing a further impetus to test the hypothesis that an initiator-promoter relationship might involve these common human viruses in the development of some tumors.

scholarly journals Use of DNA and Peptide Nucleic Acid Molecular Beacons for Detection and Quantification of rRNA in Solution and in Whole Cells

2003 ◽  
Vol 69 (9) ◽  
pp. 5673-5678 ◽  
Author(s):  
Chuanwu Xi ◽  
Michal Balberg ◽  
Stephen A. Boppart ◽  
Lutgarde Raskin
Keyword(s):  
Nucleic Acid ◽  
Peptide Nucleic Acid ◽  
Molecular Beacon ◽  
Signal To Noise Ratio ◽  
Microfluidic Devices ◽  
Molecular Beacons ◽  
Target Cells ◽  
Signal To Noise ◽  
Whole Cells

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.

scholarly journals The Application of the CRISPR/Cas9 System in the Treatment of Hepatitis B Liver Cancer

2021 ◽  
Vol 20 ◽  
pp. 153303382110452
Author(s):  
Wei Lv ◽  
Tao Li ◽  
Shanshan Wang ◽  
Huihui Wang ◽  
Xuemei Li ◽  
...  
Keyword(s):  
Liver Cancer ◽  
Hepatitis B ◽  
Adaptive Immune System ◽  
Cancer Genes ◽  
Rna Sequences ◽  
Entire Genome ◽  
Dna And Rna ◽  
B Virus ◽  
In Situ Repair

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system was originally discovered in prokaryotes and functions as part of the adaptive immune system. The experimental research of many scholars, as well as scientific and technological advancements, has allowed prokaryote-derived CRISPR/Cas genome-editing systems to transform our ability to manipulate, detect, image, and annotate specific DNA and RNA sequences in the living cells of diverse species. Through modern genetic engineering editing technology and high-throughput gene sequencing, we can edit and splice covalently closed circular DNA to silence it, and correct the mutation and deletion of liver cancer genes to achieve precise in situ repair of defective genes and prohibit viral infection or replication. Such manipulations do not destroy the structure of the entire genome and facilitate the cure of diseases. In this review, we discussed the possibility that CRISPR/Cas could be used as a treatment for patients with liver cancer caused by hepatitis B virus infection, and reviewed the challenges incurred by this effective gene-editing technology.

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).

2003 ◽  
pp. 224-224
Keyword(s):  
Nucleic Acid ◽  
Specific Binding ◽  
Bacterial Genome ◽  
Dna Probes ◽  
Whole Cell ◽  
Whole Cells ◽  
Hybridization Probes ◽  
Target Nucleic Acid ◽  
Species Specific

Fluorescence in situ hybridisation

Biologia ◽  
2007 ◽  
Vol 62 (3) ◽  
Author(s):  
Martina Lakatošová ◽  
Beáta Holečková
Keyword(s):  
In Situ Hybridisation ◽  
Molecular Cytogenetics ◽  
Fluorescence In Situ Hybridisation ◽  
Rna Sequences ◽  
Reliable Technique ◽  
Molecular Cytogenetic ◽  
Dna And Rna ◽  
Hybridisation Probe ◽  
Cytogenetic Method

AbstractFluorescence in situ hybridisation (FISH) is a rapid and reliable technique for chromosomal investigations that is used for a wide variety of cytogenetic purposes at present. This molecular-cytogenetic method has been developed continuously for many years. As a consequence, various modifications with different kinds of fluorescently labelled probes have been introduced to optimise the detection of DNA and RNA sequences. This review articlepaper presents the general principles of in situ hybridisation, probe labelling and examples of proper use of different kinds of probes. In addition, some newer FISH methods and their usefulness in human molecular cytogenetics are described.

scholarly journals Sulfur Modification in Natural RNA and Therapeutic Oligonucleotides

2021 ◽  
Author(s):  
Ya Ying Zheng ◽  
Ying Wu ◽  
Thomas Begley ◽  
Jia Sheng
Keyword(s):  
Nucleic Acid ◽  
Dna And Rna ◽  
Sulfur Modification ◽  
Oxygen Atoms

Sulfur modifications have been discovered on both DNA and RNA. Sulfur substitution of oxygen atoms at nucleobase or backbone locations in the nucleic acid framework led to a wide variety...

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