scholarly journals Cytoplasmic DNA can be detected by RNA fluorescence in situ hybridization

2019 ◽  
Vol 47 (18) ◽  
pp. e109-e109
Author(s):  
Eliraz Greenberg ◽  
Hodaya Hochberg-Laufer ◽  
Shalev Blanga ◽  
Noa Kinor ◽  
Yaron Shav-Tal

Abstract Fluorescence in situ hybridization (FISH) can be used for the intracellular detection of DNA or RNA molecules. The detection of DNA sequences by DNA FISH requires the denaturation of the DNA double helix to allow the hybridization of the fluorescent probe with DNA in a single stranded form. These hybridization conditions require high temperature and low pH that can damage RNA, and therefore RNA is not typically detectable by DNA FISH. In contrast, RNA FISH does not require a denaturation step since RNA is single stranded, and therefore DNA molecules are not detectable by RNA FISH. Hence, DNA FISH and RNA FISH are mutually exclusive. In this study, we show that plasmid DNA transiently transfected into cells is readily detectable in the cytoplasm by RNA FISH without need for denaturation, shortly after transfection and for several hours. The plasmids, however, are usually not detectable in the nucleus except when the plasmids are efficiently directed into the nucleus, which may imply a more open packaging state for DNA after transfection. This detection of plasmid DNA in the cytoplasm has implications for RNA FISH experiments and opens a window to study conditions when DNA is present in the cytoplasm.

2011 ◽  
Vol 30 (9) ◽  
pp. 1779-1786 ◽  
Author(s):  
Kun Yang ◽  
Hecui Zhang ◽  
Richard Converse ◽  
Yong Wang ◽  
Xiaoying Rong ◽  
...  

Genome ◽  
1997 ◽  
Vol 40 (5) ◽  
pp. 589-593 ◽  
Author(s):  
C. Pedersen ◽  
P. Langridge

Using the Aegilops tauschii clone pAs1 together with the barley clone pHvG38 for two-colour fluorescence in situ hybridization (FISH) the entire chromosome complement of hexaploid wheat was identified. The combination of the two probes allowed easy discrimination of the three genomes of wheat. The banding pattern obtained with the pHvG38 probe containing the GAA-satellite sequence was identical to the N-banding pattern of wheat. A detailed idiogram was constructed, including 73 GAA bands and 48 pAs1 bands. Identification of the wheat chromosomes by FISH will be particularly useful in connection with the physical mapping of other DNA sequences to chromosomes, or for chromosome identification in general, as an alternative to C-banding.Key words: Triticum aestivum, chromosome identification, fluorescence in situ hybridization, repetitive DNA sequences.


Genome ◽  
1995 ◽  
Vol 38 (6) ◽  
pp. 1061-1069 ◽  
Author(s):  
A. Cuadrado ◽  
N. Jouve ◽  
C. Ceoloni

The molecular characterization of heterochromatin in six lines of rye has been performed using fluorescence in situ hybridization (FISH). The highly repetitive rye DNA sequences pSc 119.2, pSc74, and pSc34, and the probes pTa71 and pSc794 containing the 25S–5.8S–18S rDNA (NOR) and the 5S rDNA multigene families, respectively, were used. This allowed the individual identification of all seven rye chromosomes and most chromosome arms in all lines. All varieties showed similar but not identical patterns. A standard in situ hybridization map was constructed following the nomenclature system recommended for C-bands. All FISH sites observed appeared to correspond well with C-band locations, but not all C-banding sites coincided with hybridization sites of the repetitive DNA probes used. Quantitative and qualitative differences between different varieties were found for in situ hybridization response at corresponding sites. Variation between plants and even between homologous chromosomes of the same plant was found in open-pollinated lines. In inbred lines, the in situ pattern of the homologues was practically identical and no variation between plants was detected. The observed quantitative and qualitative differences are consistent with a corresponding variation for C-bands detected both within and between cultivars.Key words: fluorescence in situ hybridization, repetitive DNA, rye, Secale cereale, polymorphism.


2021 ◽  
Vol 7 (3) ◽  
pp. 215-219
Author(s):  
Mohammed Abdulazeez ◽  
◽  
Stefanie Kankel ◽  
Thomas Liehr ◽  
◽  
...  

Variants in size of the acrocentric short arms (acro-ps) are normally not reported and considered as chromosomal heteromorphisms (CHMs) without any influence on the carrier’s phenotype. However, if acro-ps are translocated to ends of A-chromosomes (i.e. human chromosomes 1-22 and X or Y), those rearrangements are studied in more detail. The aim of the study: Here we characterized 11 healthy carriers of a non-acrocentric satellited chromosomes der(A)t(A;acro)(pter or qter;p1?1.2) to determine the frequency of chromosome 15p and 22p in such rearrangements. Materials and methods: 11 carriers of one (10 cases) or two (1 case) der(A)t(A;acro) were identified during routine cytogenetic analyses. They were originally referred due to infertility or due to a mentally retarded child with otherwise abnormal karyotype. Here derivative chromosomes were studied by fluorescence in situ hybridization applying probes D15Z1 (specific for 15p11.2) and D22Z4 (specific for 22p11.2). As there are no DNA-sequences available for 13p11.2, 14p11.2 and 21p11.2 these regions could not be tested. Results: D15Z1 sequences were identified in 1 out of 12 derivatives der(A)t(A;acro). D22Z1 could not be detected in any of the 11 remainder derivatives. However, only 3 of the 12 der(A)t(A;acro) had acro-ps large enough to potentially comprise sub-band p11.2. Conclusion: In contrast to der(Y)t(Y;acro)(q12;p1?1.2), where in at least 65% of the cases the acro-p part contains D15Z1 sequences, here it could be shown that in der(A)t(A;acro) 15p involvement can be substantiated much less frequently. Also, in none of the two groups D22Z4-sequences were detected in acro-p-parts yet. Besides, breakpoint of acro-pparts in der(A)t(A;acro) seem to be in ~75% of the cases distal from p11.2.


2020 ◽  
Vol 17 (3) ◽  
pp. 393-410
Author(s):  
Hoang Thi Nhu Phuong ◽  
Huynh Thi Thu Hue ◽  
Cao Xuan Hieu

Fluorescence in situ hybridization (FISH) technique enables the direct detection of DNA sequences inintact cellular materials (e.g. individual chromosomes in metaphase spreads). This review article focuses on theapplications of FISH in genome research, including validation and correction of the genome assembly from thenext-generation sequencing (NGS) projects. DNA probes for specific DNA fragments of the assembly can beobtained from PCR amplicon or cloned products using different vector systems. Localization of these probeson their respective chromosomal regions can be visualized by FISH, providing useful information to crosscheckthe assembly data. Furthermore, the recent refinements in the FISH technology including using smartpooling scheme of differently colored DNA probes, together with consecutive FISH experiments (stripping andreprobing of the same slide) are described. These advances in multicolor FISH can provide crucial linkageinformation on association of linkage groups and assembly scaffolds, resulting in so-called cytogenetic maps.Integration of the cytogenetic maps and assembly sequences assists to resolve the chromosome-level genomeassembly and to reveal new insights in genome architecture and genome evolution. Especially, comparativechromosome painting with pooled DNA probes from one reference species can be used to investigate ancestralrelationships (chromosome homeology and rearrangements) among other not-yet-sequenced species. Inaddition, FISH using DNA probes for certain specific classes of repetitive DNA elements as well as for basicchromosome structures (e.g. centromere or telomere DNA repeats, ribosomal DNA loci) can be used to studythe genome organization and karyotype differentiation. We also discussed about limitations and futureperspectives of the FISH technology.


Genome ◽  
2006 ◽  
Vol 49 (9) ◽  
pp. 1057-1068 ◽  
Author(s):  
Jiming Jiang ◽  
Bikram S. Gill

Fluorescence in situ hybridization (FISH), which allows direct mapping of DNA sequences on chromosomes, has become the most important technique in plant molecular cytogenetics research. Repetitive DNA sequence can generate unique FISH patterns on individual chromosomes for karyotyping and phylogenetic analysis. FISH on meiotic pachytene chromosomes coupled with digital imaging systems has become an efficient method to develop physical maps in plant species. FISH on extended DNA fibers provides a high-resolution mapping approach to analyze large DNA molecules and to characterize large genomic loci. FISH-based physical mapping provides a valuable complementary approach in genome sequencing and map-based cloning research. We expect that FISH will continue to play an important role in relating DNA sequence information to chromosome biology. FISH coupled with immunoassays will be increasingly used to study features of chromatin at the cytological level that control expression and regulation of genes.


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