The origin of in situ hybridization – A personal history

Gall, Joseph G.

doi:10.1016/j.ymeth.2015.11.026

Cited by 36 publications

(18 citation statements)

References 25 publications

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“…B-allele frequency plot ratio of 0: 0.4: 0.6: 1) [9]. Mosaic deletion for whole chromosomes 1, 2, 3, 4, 5,6,7,9,11,12,13,14,15,16,17,18,19, 20 and 22 were observed, consistent with monosomy for these chromosomes. Chromosomes 8, 10 and 21 showed a normal heterozygous B-allele frequency distribution (0: 0.5: 1) consistent with disomy for these chromosomes.…”

Section: Resultssupporting

confidence: 58%

“…Microarrays and next generation sequencing are advanced, lengthy and costly techniques, which are incorporated into routine testing to detect genome aberrations. FISH is a relatively old technique developed in the early 1980s [12][13][14], but remains an essential tool for the detection of specific biomarkers such as BCR-ABL1 gene fusion, HER2 and ALK gene rearrangements, and has become crucial in the development of personalised medicine [13]. Nevertheless, it remains a rapid, economical and reliable technique in the detection of balanced rearrangement and more importantly in the detection of minimal residual disease (MRD).…”

Section: Discussionmentioning

confidence: 99%

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Fluorescence in Situ Hybridisation (FISH) is the First Tool to Identify Hypodiploidy in Paediatric Acute Lymphoblastic Leukaemia

Ghaoui¹,

Hung²,

Padhye³

2018

obm genet

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Hypodiploidy has a low incidence in childhood acute lymphoblastic leukaemia (ALL). Patients are usually stratified into three subgroups, to allocate the correct treatment according to their ploidy level: high hypodiploidy (40-45 chromosomes), low hypodiploidy (33-39 chromosomes) and near haploidy (23-29 chromosomes). In this paper, a case is presented of near-haploid childhood ALL where fluorescence in situ hybridisation (FISH) provided an insight into the near-haploidy chromosomal aberration initially missed on routine karyotyping due to culture effect and analysis software constraints. FISH primary results followed by further confirmatory studies, inclusive of karyotyping and single nucleotide polymorphism (SNP) microarray, clinically placed the patient in the correct treatment stratification. The patient remained in morphological and cytogenetic remission 12 months after commencing the high risk arm of an international collaborative (*AIEOP-BFM-ALL [Associazione Italiana Ematologia Oncologia Pediatrica-Berlin-Franklin-Munste]) 2009 treatment protocol for children and adolescents with ALL.

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Section: Resultssupporting

confidence: 58%

Section: Discussionmentioning

confidence: 99%

Fluorescence in Situ Hybridisation (FISH) is the First Tool to Identify Hypodiploidy in Paediatric Acute Lymphoblastic Leukaemia

Ghaoui¹,

Hung²,

Padhye³

2018

obm genet

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“…Several strategies were employed to study genome 3D structure 3–5 : One is genome mapping techniques, including ligation-based and ligation-free methods; another is in situ imaging of DNA, RNA, and protein in the nucleus; and the last one is computational methods to predict either chromatin interactions or 3D chromatin organization 6 . Among these strategies, 3C-derivative methods rely on digestion and proximity ligation to capture pair-wise 5,7 or multiple chromatin contacts 8 , while ligation-free methods, such as Genome Architecture Mapping (GAM) 9 , Split-Pool Recognition of Interactions by Tag Extension (SPRITE) 10 , and ChIA-Drop 11 can detect multiple chromatin interactions genome-wide, by demonstrating that multiple enhancers and highly transcribed regions are associated simultaneously.…”

Section: Introductionmentioning

confidence: 99%

Imaging chromatin interactions at sub-kilobase resolution Via Tn5-FISH

Zhang

Niu

et al. 2019

Preprint

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There is increasing interest in understanding how the three-dimensional organization of the genome is regulated. Different strategies have been employed to identify chromatin interactions genome wide. However, due to the current limitations in resolving genomic contacts, visualization and validation of these genomic loci with sub-kilobase resolution remain the bottleneck for many years. Here, we describe Tn5 transposase-based Fluorescence in situ Hybridization (Tn5-FISH), a Polymerase Chain Reaction (PCR)-based, cost-effective imaging method, which achieved the co-localization of genomic loci with sub-kilobase resolution, to fine dissect genome architecture at sub-kilobase resolution and to verify chromatin interactions detected by Chromatin Configuration Capture (3C)-derivative methods. Especially, Tn5-FISH is very useful to verify short-range chromatin interactions inside of contact domain and Topologically Associated Domain (TAD). It also offers one powerful molecular diagnosis tool for clinical detection of cytogenetic changes in cancers.the genomic resolution of traditional FISH based on BAC clones is too low to precisely label and to image the interactions within the genomic distance less than 500 Kb, such as inside of a TAD or contact domain, whereas most of the chromatin interactions identified by 3C-based techniques fall into this range [25][26][27] .There are advanced FISH techniques with higher genomic resolution, such as oligopaint 28 , HD-FISH 29 , CasFISH 30 and MB-FISH 31 , etc. But they are either very expensive 28,31 or require complex preparation 29,30 , which is difficult for laboratories that do not routinely use various FISH techniques to adopt and to study genomic loci. Molecular beacon-based FISH 31 can offer a resolution of 2.5 kb, but is also technically complex and lacks cost-effectiveness.Here we report a novel and cost-effective method named Tn5-FISH (Tn5 transposase based fluorescence in situ hybridization) that offers more than one order of magnitude higher resolution than traditional FISH. Tn5-FISH utilized the hyperactive Tn5 transposase for probe library construction, and PCR for probe library amplification and labeling. Foreign DNA sequences can be inserted into host genome with the help of Tn5 transposase through "cut and paste" mechanism 32 , which granted Tn5 transposase the ability to induce the DNA double breaks at insertion site. Efficient segmentation of targeted DNA sequence is favored in probe library construction 33,34 . With the aid of bioinformatic tools, we can obtain the repeat-free DNA sequence suitable for probing via genomic PCR.Then Tn5-FISH was employed to verify the chromatin interactions, for example, the ones inside of type II KRT locus in chromosome 12 of K562 cells measured by Hi-C contact map from published data. Triple-color Tn5-FISH can verify these interactions well, with triple-color traditional BAC FISH as positive control, indicating that Tn5-FISH is suitable to visualize specific genomic loci and chromatin interactions inside a typical TAD, in ei...

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“…Initial reports of in situ hybridization achieved denaturation either with high temperatures or chemically, with NaOH or salts (John et al, 1969;Buongiorno-Nardelli and Amaldi, 1970;Barsacchi and Gall, 1972;Gall, 2016). Subsequent research favored the use of formamide at a concentration of 50-70 % in the hybridization buffer, in order to lower hybridization temperature and efficiently denature DNA/RNA (see for example (Barbera et al, 1979;Bauman et al, 1980;Gerhard et al, 1981;Hafen et al, 1983;Levine et al, 1983;Braissant and Wahli, 1998;Brown, 1998).…”

Section: Introductionmentioning

confidence: 99%

A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species

Sinigaglia

Thiel

Hejnol

et al. 2018

Developmental Biology

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In situ hybridization is a widely employed technique allowing spatial visualization of gene expression in fixed specimens. It has proven to be essential to our understanding of biological processes, including developmental regulation. In situ protocols are today routine in numerous laboratories, and although details might change, they all include a hybridization step, where specific antisense RNA or DNA probes anneal to the target nucleic acids strand. This step, in general, is carried out at high temperatures and in a denaturing solution, the hybridization buffer, commonly containing 50% (v/v) formamide. An important drawback is that hot formamide poses a significant health risk and so must be handled with great care.We were prompted to test alternative hybridization solutions for in situ detection of gene expression in the medusa of the hydrozoan Clytia hemisphaerica, where traditional protocols caused extensive deterioration of the morphology and texture during hybridization, hindering observation and interpretation of results. Inspired by optimized protocols for Northern and Southern blot analysis, we substituted the 50% formamide with an equal volume of 8 M urea solution in the hybridization buffer. The new protocol yielded better morphologies and consistency of tissues, and also notably improved the resolution of the signal, allowing more precise localization of gene expression, as well as reduced staining at non-specific sites. Given the improved results using a less toxic hybridization solution, we tested the urea protocol on a number of other metazoans: two brachiopod species (Novocrania anomala and Terebratalia transversa) and the worm Priapulus caudatus, obtaining a similar reduction of aspecific probe binding. Overall, substitution of formamide by urea in in situ hybridization offers safer alternative protocols, potentially useful in research, medical and teaching contexts. We encourage other workers to test this approach on their study organisms, and hope that they will also obtain better sample preservation, more precise expression patterns and fewer problems due to aspecific staining, as we report here for Clytia medusae and Novocrania and Terebratalia developing larvae.

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The origin of in situ hybridization – A personal history

Cited by 36 publications

References 25 publications

Fluorescence in Situ Hybridisation (FISH) is the First Tool to Identify Hypodiploidy in Paediatric Acute Lymphoblastic Leukaemia

Fluorescence in Situ Hybridisation (FISH) is the First Tool to Identify Hypodiploidy in Paediatric Acute Lymphoblastic Leukaemia

Imaging chromatin interactions at sub-kilobase resolution Via Tn5-FISH

A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species

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