Mode of action and application of Scorpion primers to mutation detection

Thelwell, Nicola; Millington, Stephen J.; Solinas, A; Booth, James A.; Brown, Tom

doi:10.1093/nar/28.19.3752

Cited by 303 publications

(181 citation statements)

References 22 publications

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“…Unimolecular hybridisation is kinetically more favourable as it does not depend on a chance meeting between the probe, present at relatively low concentration, and the amplicon. This allows the introduction of more rapid cycling conditions together with a significantly stronger signal strength compared with both Taqman and molecular beacons (Thelwell et al 2000). Another advantage over Taqman assays is that the PCR reaction is carried out at the optimal temperature for the polymerase, rather than at the reduced temperature required for the 5 -nuclease assay to displace and cleave the probe.…”

Section: New Technologiesmentioning

confidence: 99%

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Bustin

2002

Journal of Molecular Endocrinology

2,233

1,674

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The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

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Section: New Technologiesmentioning

confidence: 99%

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Bustin

2002

Journal of Molecular Endocrinology

2,233

1,674

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“…However, the only method we had previously to detect AIB1-⌬4 transcript was by semi-quantitative hybridization of a radioactive DNA probe to total RNA isolated from cells, thus making accurate quantification difficult. We therefore designed a quantitative assay to measure AIB1-⌬4 mRNA levels through the use of Scorpion primer technology (40,41). We designed a Scorpion primer with a primer sequence complementary to a sequence in exon 3 and a probe sequence complementary to the unique sequence generated by the splicing of exon 3 and exon 5 (Fig.…”

Section: Aib1-⌬4 Expression Is Correlated With Metastatic Potential-mentioning

confidence: 99%

Role of the Nuclear Receptor Coactivator AIB1-Δ4 Splice Variant in the Control of Gene Transcription

Chien

Kirilyuk

et al. 2011

Journal of Biological Chemistry

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The oncogene amplified in breast cancer 1 (AIB1) is a nuclear receptor coactivator that plays a major role in the progression of various cancers. We previously identified a splice variant of AIB1 called AIB1-⌬4 that is overexpressed in breast cancer. Using mass spectrometry, we define the translation initiation of AIB1-⌬4 at Met 224 of the full-length AIB1 sequence and have raised an antibody to a peptide representing the acetylated N terminus. We show that AIB1-⌬4 is predominantly localized in the cytoplasm, although leptomycin B nuclear export inhibition demonstrates that AIB1-⌬4 can enter and traffic through the nucleus. Our data indicate an import mechanism enhanced by other coactivators such as p300/CBP. We report that the endogenously and exogenously expressed AIB1-⌬4 is recruited as efficiently as full-length AIB1 to estrogen-response elements of genes, and it enhances estrogen-dependent transcription more effectively than AIB1. Expression of an N-terminal AIB1 protein fragment, which is lost in the AIB1-⌬4 isoform, potentiates AIB1 as a coactivator. This suggests a model whereby the transcriptional activity of AIB1 is squelched by a repressive mechanism utilizing the N-terminal domain and that the increased coactivator function of AIB1-⌬4 is due to the loss of this inhibitory domain. Finally, we show, using Scorpion primer technology, that AIB1-⌬4 expression is correlated with metastatic capability of human cancer cell lines.Gene transcription in eukaryotes is a complex and highly regulated process. One of the major controls of gene transcription is exerted by the coregulator family of proteins. These include both corepressors, which dampen transcription, and coactivators, which potentiate transcription. A subgroup of coactivators has been shown to be critical for the malignant progression of cancer and is known as the p160 steroid receptor coactivators (1). One member in particular was identified to be amplified in breast cancer. Amplified in breast cancer 1 (AIB1, SRC-3, NCOA3, ACTR, TRAM-1, pCIP, and RAC3) has been shown to be a gene amplified in breast cancer (2) and is also overexpressed at the mRNA and protein level in various cancers (1, 3, 4). Its role in tumorigenesis is attributed to its ability to coactivate both steroid hormone-and growth factor-dependent transcription (3, 5-7). In several oncogene-driven mouse models (8 -11), reduction of AIB1 levels leads to a decrease in tumorigenesis, and overexpression of AIB1 leads to the formation of various tumors (12). Clinically, AIB1 expression in breast cancer cases is correlated with high HER2 levels, larger tumor size, higher tumor grade, and shorter disease-free survival (13-15). Also, high levels of AIB1 in conjunction with high HER2 levels coincide with reduced disease-free survival in patients treated with tamoxifen, suggesting a role for AIB1 in tamoxifen resistance (16).We had previously identified a splice variant of AIB1, where exon 3 was spliced from the mature mRNA and the resulting protein named AIB1-⌬3 (17). More recently, an additio...

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“…In this assay, DNA melting profiles must be analyzed visually and alleles appointed manually for each individual sample, a time-consuming step introducing observer variability. In theory, MGB probes can compete with molecular beacons or scorpion primers that also have increased sequence-specificity due to the presence of a stem sequence that destabilizes mismatch hybridization [Giesendorf et al, 1998;Tyagi et al, 1998;Thelwell et al, 2000]. A disadvantage of molecular beacons is that their design can be difficult and there is no software available (yet).…”

Section: Discussionmentioning

confidence: 99%

Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes)

et al. 2002

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Novel fluorescent oligonucleotides that contain a 3¢ minor groove binding group (MGB) hybridize to single-stranded targets with increased sequence-specificity compared to ordinary DNA probes. This reduces non-specific probe hybridization and results in low background fluorescence during the 5¢ nuclease PCR assay (TaqMan, Applied Biosystems, Foster City, CA). We developed a method for closed-tube genotyping using two allele-specific MGB probes labeled with different fluorophores in one reaction. After PCR, tubes were transported to a fluorescence plate-reader for analysis of fluorescence. Common spreadsheet software was used for automated genotype assignment. As an example, DNA samples from 172 hemochromatosis patients were selected and tested for molecular defects in the HFE gene, i.e., mutations in codon 63 and 282. Tight genotype clusters were observed for both codons and results with MGB probes were identical to conventional genotyping (PCR + restriction-fragment-length-polymorphism). We show that this fast and easy method can be used for large-scale (high-throughput) genetic studies but also for routine molecular diagnostics without post-PCR manipulation of amplicons or the need for real-time quantitative PCR machines. Hum Mutat 19:554 559, 2002.

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Mode of action and application of Scorpion primers to mutation detection

Cited by 303 publications

References 22 publications

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Role of the Nuclear Receptor Coactivator AIB1-Δ4 Splice Variant in the Control of Gene Transcription

Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes)

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