Single-copy Gene Detection Using Branched DNA (bDNA) In Situ Hybridization

Player, Audrey; Shen, Lu-Ping; Kenny, Daryn A.; Antao, Vincent; Kolberg, Janice A.

doi:10.1177/002215540104900507

Cited by 162 publications

(134 citation statements)

References 42 publications

(45 reference statements)

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“…Functionalities can be attached directly to primers and incorporated into the probe during amplification, or they can be brought in by the hybridization of a secondary oligo (48,50,51) that is homologous to the primer sequence; either way, significant cost savings can be achieved by bulk orders of modified oligos that can then be applied to any number of libraries. The availability of primer sequences further opens opportunities for bringing in functionalities via DNA binding factors or assembling DNA structures, such as those used in branched signal amplification (11,27). Thus, we believe that Oligopaints has the potential to become a reagent not only for visualization, but also a broader spectrum of methods that require the targeting of biochemical modifications and functional chemistries to nucleic acids in a sequence-specific fashion.…”

Section: Discussionmentioning

confidence: 99%

“…Also, as these probes are typically short (∼20-50 bases) (24)(25)(26) and single stranded, they diffuse efficiently into fixed cells and tissues and are unhindered by competitive hybridization with complementary probe fragments. Oligo probes have allowed the visualization of single-copy viral DNA as well as individual mRNA molecules using branched DNA signal amplification (27) or a handful to a few dozen short oligo probes (26,28), and, by targeting blocks of repetitive sequences as a strategy to amplify signal, enabled the first FISHbased genome-wide RNAi screen (29). Oligo probes have also been generated directly from genomic DNA using parallel PCR reactions (30,31).…”

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confidence: 99%

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Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes

Beliveau

Joyce

Apostolopoulos

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

396

472

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A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide-and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.T he role of chromosome positioning in gene regulation and chromosome stability is fueling a growing interest in technologies that reveal the in situ organization of the genome. Among these technologies are chromosome conformation capture (3C) (1) and its several iterations, such as Hi-C (2), which are applied to populations of nuclei to identify chromosomal regions that are in close proximity to each other (3, 4). Another technology is fluorescence in situ hybridization (FISH), wherein nucleic acids are targeted by fluorescently labeled probes and then visualized via microscopy; this technology is an extension of methods that once used radioactive probes and autoradiography but have since been adapted to use nonradioactive labels (5-11). FISH is a single-cell assay, making it especially powerful for the detection of rare events that might otherwise be lost in mixed or asynchronous populations of cells. In addition, because FISH is applied to fixed cells, it can reveal the positioning of chromosomes relative to nuclear, cytoplasmic, and even tissue structures. FISH can also be used to visualize RNA, permitting the simultaneous assessment of gene expression, chromosome position, and protein localization.FISH probes are typically derived from cloned genomic regions or flow-sorted chromosomes, which are labeled directly via nick translation or PCR in the presence of fluorophore-conjugated nucleotides or labeled indirectly with nucleotide-conjugated haptens, such as biotin and digoxigenin, and then visualized with secondary detection reagents. Probe DNA is often fragmented into ∼150-to 250-bp pieces to facilitate its penetration into fixed cells (12) and, as many genomic clones contain repetitive sequences that occur abundantly in the genome, hybridization is typically performed in the presence of unlabeled repetitive DNA (13). Another limitation to clone-based probes is that the genomic regions that can be visualized with them are restricted by the availability of clones and the size of ...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes

Beliveau

Joyce

Apostolopoulos

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

396

472

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“…19 These early ISH methods were based on moderately sensitive approaches but, in the last decade, numerous more sensitive (F)ISH assays have been developed that allow the detection of a single-copy of HPV. 17,[20][21][22][23][24] Applying these sensitive methods to CIN II/III lesions results, however, in an increase in the number of FISH signals within individual nuclei. The inherent problem of this approach is that signals originating from integrated HPV can be hidden in a background of episomal copies.…”

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confidence: 99%

HPV in situ hybridization: Impact of different protocols on the detection of integrated HPV

Hopman

Kamps

Smedts³

et al. 2005

Intl Journal of Cancer

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Although there is consensus that HPV integration is common in invasive cervical carcinomas and uncommon or absent in lowgrade uterine cervical intraepithelial neoplasia (CIN I), estimates for HPV integration in CIN II/III range from 5 to 100% using different PCR-based and in situ hybridization (ISH) approaches. It has been suggested that HPV integration can be identified using ISH by scoring of punctate signals. The increased sensitivity of fluorescence ISH (FISH) methods, allowing the detection of single copies of HPV, complicates the distinction between integrated and episomal HPV. Recently it has been suggested that, in such assays, the signals originating from integrated virus can be hidden in a background of episomal HPV. We therefore compared 2 different FISH protocols for the detection of integrated HPV in a series of CIN II/III lesions: 1) a mild protocol in which episomal HPV and RNA is retained and 2) a harsh protocol that extensively extracts proteins and RNA, and which promotes the partial loss of episomal HPV but not integrated HPV. A series of 28 HPV 16/18 positive CIN II/III lesions (17 solitary lesions and 11 lesions adjacent to microinvasive carcinoma) were studied. A punctate signal pattern was identified in 7 of these lesions with both protocols. Punctate signal was also present in control samples from lesions that are known to be associated with HPV integration (invasive squamous cell carcinoma (n ¼ 3), adenocarcinoma in situ (n ¼ 3), and invasive adenocarcinoma (n ¼ 1). HPV RNA contributed significantly to the intensity of punctate FISH signal, especially when applying the mild protocol, as shown by omitting DNA denaturation, including RNase pretreatment steps and measuring the fluorescence signal intensity. Also, HPV RNA was frequently detected in addition to episomal/integrated HPV DNA in the majority of the other 21 CIN II/III lesions; this resulted in intense granular/ diffuse FISH signals throughout the epithelium. However, in 7 of these lesions, the harsh protocol gave a more consistent punctate pattern in cells throughout the full thickness of the epithelium. This supports the hypothesis that the harsh protocol unmasks integrated HPV more efficiently by extracting RNA and episomal HPV. Overall, with this harsh protocol, a clonally expanded population of cells containing punctate HPV signals was found in 5 of 17 (29%) solitary CIN II/III lesions and in 9 of 11 (88%) CIN II/ III lesions associated with microinvasive carcinoma. Combining these data with the results from our previous study, with the harsh protocol in 7 of 40 (18%) Several premalignant stages can be distinguished in the development of carcinoma of the uterine cervix. These include cervical intraepithelial neoplasia grades I, II and III (CIN I-III), also designated low-grade squamous intraepithelial lesions (LSIL, comprising CIN I) and high grade SIL (HSIL, comprising CIN II-III). 1-4Nearly all invasive cervical carcinomas (ICCs) and CIN grade II/ III lesions contain human papillomavirus (HPV) DNA. Epidemiological studies ...

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“…The application of nucleic acid target and signal amplification technologies to ISH has enabled re-searchers to detect as few as one or two copies of specific DNA molecules in cell preparations (Femino et al 1998;Player et al 2001). However, the sensitive detection of nucleic acid sequences in tissue biopsy specimens has proven more challenging.…”

Section: Detection Of Viral Infection and Gene Expression In Clinicalmentioning

confidence: 99%

“…We recently reported the development of a branched DNA (bDNA) ISH method for detection of DNA and mRNA in whole cells (Player et al 2001). The bDNA ISH method is a signal amplification system in which target nucleic acid sequences are hybridized to a series of synthetic oligonucleotide probes and visualized through generation of chromogenic or fluorescent signals in an alkaline phosphatase (AP)-catalyzed reaction.…”

Section: Detection Of Viral Infection and Gene Expression In Clinicalmentioning

confidence: 99%

Detection of Viral Infection and Gene Expression in Clinical Tissue Specimens Using Branched DNA (bDNA) In Situ Hybridization

Kenny

Shen

Kolberg

2002

J Histochem Cytochem.

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S U M M A R YIn situ hybridization (ISH) methods for detection of nucleic acid sequences have proved especially powerful for revealing genetic markers and gene expression in a morphological context. Although target and signal amplification technologies have enabled researchers to detect relatively low-abundance molecules in cell extracts, the sensitive detection of nucleic acid sequences in tissue specimens has proved more challenging. We recently reported the development of a branched DNA (bDNA) ISH method for detection of DNA and mRNA in whole cells. Based on bDNA signal amplification technology, bDNA ISH is highly sensitive and can detect one or two copies of DNA per cell. In this study we evaluated bDNA ISH for detection of nucleic acid sequences in tissue specimens. Using normal and human papillomavirus (HPV)-infected cervical biopsy specimens, we explored the cell type-specific distribution of HPV DNA and mRNA by bDNA ISH. We found that bDNA ISH allowed rapid, sensitive detection of nucleic acids with high specificity while preserving tissue morphology. As an adjunct to conventional histopathology, bDNA ISH may improve diagnostic accuracy and prognosis for viral and neoplastic diseases.

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Single-copy Gene Detection Using Branched DNA (bDNA) In Situ Hybridization

Cited by 162 publications

References 42 publications

Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes

Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes

HPV in situ hybridization: Impact of different protocols on the detection of integrated HPV

Detection of Viral Infection and Gene Expression in Clinical Tissue Specimens Using Branched DNA (bDNA) In Situ Hybridization

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