scholarly journals Extending the Useful Life Span of DNA Probes for Fluorescence In Situ Hybridization

BioTechniques ◽  
1999 ◽  
Vol 26 (6) ◽  
pp. 1068-1072
Author(s):  
Allen T. Christian ◽  
Holly E. Garcia ◽  
James D. Tucker
Keyword(s):  
In Situ Hybridization ◽  
Life Span ◽  
Fluorescence In Situ Hybridization ◽  
Dna Probes ◽  
Useful Life

scholarly journals Fluorescence In Situ Hybridization (FISH) on Human Chromosomes Using Photoprobe Biotin-labeled Probes

2003 ◽  
Vol 51 (4) ◽  
pp. 549-551 ◽  
Author(s):  
Anja Weise ◽  
Peter Harbarth ◽  
Uwe Claussen ◽  
Thomas Liehr
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
Dna Probes ◽  
Human Chromosomes ◽  
First Time ◽  
Established Technique ◽  
Whole Chromosome Painting

Fluorescence in situ hybridization (FISH) on human chromosomes in meta-and interphase is a well-established technique in clinical and tumor cytogenetics and for studies of evolution and interphase architecture. Many different protocols for labeling the DNA probes used for FISH have been published. Here we describe for the first time the successful use of Photoprobe biotin-labeled DNA probes in FISH experiments. Yeast artificial chromosome (YAC) and whole chromosome painting (wcp) probes were tested.

scholarly journals Fluorescence in situ hybridization in diluted and flow cytometrically sorted boar spermatozoa using specific DNA direct probes labelled by nick translation

Reproduction ◽  
2003 ◽  
pp. 317-325 ◽  
Author(s):  
I Parrilla ◽  
JM Vazquez ◽  
M Oliver-Bonet ◽  
J Navarro ◽  
J Yelamos ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Y Chromosome ◽  
Internal Control ◽  
Dna Probes ◽  
Chromosome 1 ◽  
Nick Translation ◽  
Fluorescent Signal ◽  
Sperm Separation

Successful evaluation of X- and Y-chromosome-bearing sperm separation technology using flow cytometry-cell sorter is of great importance. Fluorescence in situ hybridization (FISH), which allows for the detection of specific nucleic acid sequences on morphologically preserved spermatozoa, is an ideal method for quantitatively and qualitatively assessing the purity of sorted sperm samples. In this study specific pig DNA direct probes for small regions of chromosomes 1 and Y were used. Chromosome 1 was labelled in green and used as internal control to detect a lack of hybridization, whereas chromosome Y was labelled in red. Nick translation was used as the labelling method for the preparation of these probes. Spermatozoa, unsorted and sorted for high and low Y-chromosome purity from ejaculates of five boars, were fixed on slides and two-colour direct FISH was performed for chromosomes 1 and Y. About 500 non-sorted and 200 sorted spermatozoa per sample were scored. The proportion of Y-chromosome-bearing spermatozoa was determined by the presence of a red fluorescent signal on the sperm head and the proportion of X-chromosome-bearing spermatozoa was determined by subtraction. The efficiency of the hybridization procedure was established as near 98% on sorted and unsorted samples. The results of this study confirm that direct FISH using specific pig DNA probes labelled by nick translation provides a useful tool for laboratory validation of sperm separation by flow sorting technology. Moreover, the ease of nick translation and the quality of the fluorescent signal obtained using this method makes this procedure the most appropriate method for labelling pig DNA probes to be used for direct FISH on pig spermatozoa.

scholarly journals Combined immunocytochemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle, genomic, and phenotypic parameters of tumor cells.

1994 ◽  
Vol 42 (7) ◽  
pp. 961-966 ◽  
Author(s):  
E J Speel ◽  
J Herbergs ◽  
F C Ramaekers ◽  
A H Hopman
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Tumor Cells ◽  
Simultaneous Detection ◽  
Dna Probes ◽  
Accurate Localization ◽  
Fast Red ◽  
Pre Treatment ◽  
High Fluorescence

We describe the development and application of a sensitive high-resolution fluorescence alkaline phosphatase (APase)-Fast Red immunocytochemical (ICC) staining method in combination with fluorescence in situ hybridization (ISH) and bromodeoxyuridine (BrdU) detection. The high fluorescence intensity, accurate localization, and advantageous slow-fading properties make the APase-Fast Red reaction a valuable tool for detection of antigens or specific DNA probes in biological cell preparations. Since the enzyme precipitate proved to be resistant to enzymatic pre-treatment steps and stable during the entire ISH procedure, APase-Fast Red immunostaining could be combined with subsequent visualization of DNA target sequences by fluorescence ISH. The lung cancer cell lines NCI-H82 and EPLC 65 were used as a model system for simultaneous detection of cell proteins, such as the neural cell adhesion molecule (N-CAM), cytokeratin filaments, lamin or the Ki67 antigen (Ki67-Ag), and centromere-specific DNA probes for human chromosomes 1, 7, or 17. In addition, the combined ICC/ISH procedure could be extended with the immunodetection of BrdU incorporated by tumor cells in S-phase. As a consequence, a combined ICC/ISH/BrdU detection procedure is now available that enables analysis of relatively complex tumor populations on the basis of different ICC and genetic markers as well as proliferative activity.

scholarly journals Applications of fluorescence in situ hybridization (FISH) in genome research

2020 ◽  
Vol 17 (3) ◽  
pp. 393-410
Author(s):  
Hoang Thi Nhu Phuong ◽  
Huynh Thi Thu Hue ◽  
Cao Xuan Hieu
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Dna Sequences ◽  
Dna Probes ◽  
Dna Repeats ◽  
Genome Research ◽  
Multicolor Fish ◽  
Vector Systems ◽  
Assembly Sequences

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.

Sign in / Sign up

Export Citation Format

Share Document