The Experts below are selected from a list of 1068 Experts worldwide ranked by ideXlab platform
Finn C. Nielsen - One of the best experts on this subject based on the ideXlab platform.
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The silent mutation nucleotide 744 G → A, Lys172Lys, in Exon 6 of BRCA2 results in Exon skipping
Breast Cancer Research and Treatment, 2010Co-Authors: Thomas V. O. Hansen, Ane Y. Steffensen, Lars Jønson, Mette K. Andersen, Bent Ejlertsen, Finn C. NielsenAbstract:Germ-line mutations in BRCA2 predispose to breast and ovarian cancer. Mutations are widespread throughout the gene and include disease-causing mutations as frameshift, nonsense, splicing mutations and large genomic rearrangements. However a large number of mutations, including missense, silent and intron variants are of unknown significance. Here, we describe the functional characterization of a silent mutation (nucleotide 744 G → A/c.516 G → A, Lys172Lys) in Exon 6 of BRCA2 in a Danish family with breast and ovarian cancer. Exon Trapping analysis showed that the mutation results in skipping of Exon 6 and/or both Exon 5 and 6, which was verified by RT-PCR analysis on RNA isolated from whole blood of the affected patient. We therefore conclude that the BRCA2 silent mutation Lys172Lys is a disease-causing mutation.
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The silent mutation nucleotide 744 G → A, Lys172Lys, in Exon 6 of results in Exon skipping
Breast Cancer Research and Treatment, 2009Co-Authors: Thomas V. O. Hansen, Ane Y. Steffensen, Lars Jønson, Mette K. Andersen, Bent Ejlertsen, Finn C. NielsenAbstract:Germ-line mutations in predispose to breast and ovarian cancer. Mutations are widespread throughout the gene and include disease-causing mutations as frameshift, nonsense, splicing mutations and large genomic rearrangements. However a large number of mutations, including missense, silent and intron variants are of unknown significance. Here, we describe the functional characterization of a silent mutation (nucleotide 744 G → A/c.516 G → A, Lys172Lys) in Exon 6 of in a Danish family with breast and ovarian cancer. Exon Trapping analysis showed that the mutation results in skipping of Exon 6 and/or both Exon 5 and 6, which was verified by RT-PCR analysis on RNA isolated from whole blood of the affected patient. We therefore conclude that the silent mutation Lys172Lys is a disease-causing mutation.
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The silent mutation nucleotide 744 G --> A, Lys172Lys, in Exon 6 of BRCA2 results in Exon skipping.
Breast cancer research and treatment, 2009Co-Authors: Thomas V. O. Hansen, Ane Y. Steffensen, Lars Jønson, Mette K. Andersen, Bent Ejlertsen, Finn C. NielsenAbstract:Germ-line mutations in BRCA2 predispose to breast and ovarian cancer. Mutations are widespread throughout the gene and include disease-causing mutations as frameshift, nonsense, splicing mutations and large genomic rearrangements. However a large number of mutations, including missense, silent and intron variants are of unknown significance. Here, we describe the functional characterization of a silent mutation (nucleotide 744 G → A/c.516 G → A, Lys172Lys) in Exon 6 of BRCA2 in a Danish family with breast and ovarian cancer. Exon Trapping analysis showed that the mutation results in skipping of Exon 6 and/or both Exon 5 and 6, which was verified by RT-PCR analysis on RNA isolated from whole blood of the affected patient. We therefore conclude that the BRCA2 silent mutation Lys172Lys is a disease-causing mutation.
D B Krizman - One of the best experts on this subject based on the ideXlab platform.
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Isolation of Exons from cloned DNA by Exon Trapping.
Current protocols in human genetics, 2001Co-Authors: P E Nisson, P C Watkins, D B KrizmanAbstract:Exon Trapping is an RNA polymerase chain reaction (PCR) method to clone expressed sequences or Exons directly from mammalian genomic DNA. The basic protocol in this unit describes the method for Trapping internal Exons from cosmid clones and the second basic protocol describes Trapping of 3 terminal Exons. An describes 3 terminal Exon Trapping, which avoids subcloning of target DNA by ligating it to the vector for direct transfection. A describes a rapid cloning procedure using uracil DNA glycosylase.
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Transcript Mapping in a 46-kb Sequenced Region at the Core of 12q13.3 Amplification in Human Cancers
Genomics, 1997Co-Authors: Abdel G. Elkahloun, D B Krizman, Zhili Wang, Tiffany A. Hofmann, Bruce A. Roe, Paul S. MeltzerAbstract:We used a combination of sequence analysis and Exon Trapping in an effort to determine the complete transcript map for a cosmid (6E5) derived from 12q13.3, a region of DNA sequence amplification in human cancers. This cosmid, previously known to contain three genes (CDK4, SAS, and OS9), was sequenced, and that information was used for computer-assisted analysis. In addition, 6E5 was subjected to both internal and 3'-terminal Exon-Trapping protocols, and the results of these studies were used to guide cDNA cloning experiments. These studies demonstrate that this cosmid is derived from a remarkably gene-dense region and add two new transcripts (KIAA0167 and 6E5.2) to the list of sequences that are expressed in tumors bearing amplification of this region.
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Gene Identification by 3′ Terminal Exon Trapping
Genetic engineering, 1996Co-Authors: D B KrizmanAbstract:The fact that most eukaryotic genes are split into Exons and introns accounts for the difficulty in identifying those regions of the eukaryotic genome that code for protein. To compound the problem, introns in higher eukaryotes are generally quite long while the Exons are usually very short. In order to translate correct peptides from split genes, the cell utilizes the mechanism of mRNA splicing to bring the coding regions together while removing the non-coding intervening sequences. The technology of Exon Trapping, sometimes called Exon amplification, strives to exploit the phenomenon of mRNA splicing to discover genes directly from genomic DNA.
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gene identification by 3 terminal Exon Trapping
Genetic engineering, 1996Co-Authors: D B KrizmanAbstract:The fact that most eukaryotic genes are split into Exons and introns accounts for the difficulty in identifying those regions of the eukaryotic genome that code for protein. To compound the problem, introns in higher eukaryotes are generally quite long while the Exons are usually very short. In order to translate correct peptides from split genes, the cell utilizes the mechanism of mRNA splicing to bring the coding regions together while removing the non-coding intervening sequences. The technology of Exon Trapping, sometimes called Exon amplification, strives to exploit the phenomenon of mRNA splicing to discover genes directly from genomic DNA.
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Identification of 3'-terminal Exons from yeast artificial chromosomes.
PCR methods and applications, 1995Co-Authors: D B Krizman, Tiffany A. Hofmann, Paul S. Meltzer, Udaya Desilva, Eric D. Green, Jeffrey M. TrentAbstract:We report an extension of 3'-terminal Exon Trapping technology to the identification of transcribed sequences from yeast artificial chromosomes (YACs). A 350-kb YAC containing mouse genomic DNA was gel-purified and used as the target DNA for the 3'-terminal Exon Trapping strategy. A novel direct ligation/transfection approach was employed to increase the efficiency of Trapping 3'-terminal Exons from recombinant vector-derived chimeric mRNA. The resulting RT-PCR product was then used to generate a plasmid library. Randomly chosen individual subclones from this library were sequenced, and the results indicate that 86% met sequence criteria characteristic of 3'-terminal Exons, whereas 14% were background from identified sources. PCR mapping efforts suggest eight putative last Exons present within this YAC, whereas RT-PCR studies demonstrate that three reside within valid expressed sequences.
Johan T. Den Dunnen - One of the best experts on this subject based on the ideXlab platform.
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Cosmid-based Exon Trapping.
Methods in Enzymology, 1999Co-Authors: Johan T. Den DunnenAbstract:Publisher Summary Exon Trapping is a widely used gene identification method. In contrast to RNA-based methods, such as screening of cDNA libraries and cDNA selection, Exon Trapping is independent of gene expression. Exon Trapping provides a functional assay in which cloned DNA sequences are selectively isolated based on the presence of functional splice sites. Consequently, sequences are isolated directly from the clone under analysis without knowledge or availability of tissues expressing the unknown gene. This chapter discusses cosmid based Exon Trapping that was developed to bypass problems related to plasmid-based Exon Trapping. It discusses the principle of cosmid-based Exon Trapping. The chapter also discusses reverse transcription (RT) and amplification by polymerase chain reaction (PCR), which is the most critical step of the entire procedure.
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scanning for genes in large genomic regions cosmid based Exon Trapping of multiple Exons in a single product
Nucleic Acids Research, 1996Co-Authors: Nicole A. Datson, Esther Van De Vosse, Hans G Dauwerse, Mattie Bout, Gertjan B Van Ommen, Johan T. Den DunnenAbstract:To facilitate the scanning of large genomic regions for the presence of Exonic gene segments we have constructed a cosmid-based Exon trap vector. The vector serves a dual purpose since it is also suitable for contig construction and physical mapping. The Exon trap cassette of vector sCOGH1 consists of the human growth hormone gene driven by the mouse mettallothionein-1 promoter. Inserts are cloned in the multicloning site located in intron 2 of the hGH gene. The efficiency of the system is demonstrated with cosmids containing multiple Exons of the Duchenne Muscular Dystrophy gene. All Exons present in the inserts were successfully retrieved and no cryptic products were detected. Up to seven Exons were isolated simultaneously in a single spliced product. The system has been extended by a transcription-translation-test protocol to determine the presence of large open reading frames in the trapped products, using a combination of tailed PCR primers directing protein synthesis in three different reading frames, followed by in vitro transcription-translation. Having larger stretches of coding sequence in a single Exon trap product rather than small single Exons greatly facilitates further analysis of potential genes and offers new possibilities for direct mutation analysis of Exon trap material.
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Specific isolation of 3′-terminal Exons of human genes by Exon Trapping
Nucleic Acids Research, 1994Co-Authors: Nicole A. Datson, Geoffrey M. Duyk, Gert-jan B. Van Ommen, Johan T. Den DunnenAbstract:Exon Trapping is a method to functionally clone expressed sequences from genomic DNA. We have previously developed the vector system pETV-SD2, which contains only a splice donor site (SD) followed by a LacZ gene, allowing Trapping of internal Exons of human genes by blue-white selection. We now describe the adaptation of the same system for the efficient Trapping of 3'-terminal Exons, by using different RT-PCR primers in a 3' RACE reaction. The addition of a T7 promoter to the RT-PCR products derived from pETV-SD2 allows their amplification in an isothermic amplification reaction called NASBA (nucleic acid sequence-based amplification reaction) and results in a strong signal from amplified 3' Exons in addition to a great reduction of non-specific background. As a test for the system, 3' Exon Trapping was performed using a cosmid containing the alpha-globin gene cluster on chromosome 16. The 3'-terminal Exons of the human alpha 1-, zeta 2-, and theta-globin genes were trapped, as well as a correctly spliced and polyadenylated sequence in the 3' flanking region of the alpha 1-globin gene. This Exon appears to belong to a previously unidentified gene within the alpha-globin gene cluster. This 3' Exon Trapping strategy should facilitate the cloning of genes from large genomic regions.
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specific isolation of 3 terminal Exons of human genes by Exon Trapping
Nucleic Acids Research, 1994Co-Authors: Nicole A. Datson, Geoffrey M. Duyk, Gert-jan B. Van Ommen, Johan T. Den DunnenAbstract:Exon Trapping is a method to functionally clone expressed sequences from genomic DNA. We have previously developed the vector system pETV-SD2, which contains only a splice donor site (SD) followed by a LacZ gene, allowing Trapping of internal Exons of human genes by blue-white selection. We now describe the adaptation of the same system for the efficient Trapping of 3'-terminal Exons, by using different RT-PCR primers in a 3' RACE reaction. The addition of a T7 promoter to the RT-PCR products derived from pETV-SD2 allows their amplification in an isothermic amplification reaction called NASBA (nucleic acid sequence-based amplification reaction) and results in a strong signal from amplified 3' Exons in addition to a great reduction of non-specific background. As a test for the system, 3' Exon Trapping was performed using a cosmid containing the alpha-globin gene cluster on chromosome 16. The 3'-terminal Exons of the human alpha 1-, zeta 2-, and theta-globin genes were trapped, as well as a correctly spliced and polyadenylated sequence in the 3' flanking region of the alpha 1-globin gene. This Exon appears to belong to a previously unidentified gene within the alpha-globin gene cluster. This 3' Exon Trapping strategy should facilitate the cloning of genes from large genomic regions.
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An Exon Trapping System Providing Size Selection of Spliced Clones and Facilitating Direct Cloning
Identification of Transcribed Sequences, 1994Co-Authors: Nicole A. Datson, Geoffrey M. Duyk, Gert-jan B. Van Ommen, L. A. J. Blonden, Johan T. Den DunnenAbstract:Exon Trapping is a method to functionally clone expressed sequences from genomic DNA. We have developed an Exon Trapping procedure based on the use of vector pETVSD2. Cosmid DNA is partially digested and cloned in pETV-SD2. DNA of an entire library of subclones is introduced into COS-1 cells and transiently expressed. RNA is isolated and vector-derived transcripts are amplified by RT-PCR. Cloning of the RT-PCR products, which contain a ColEI-origin of replication and a supF marker, is established by NotI digestion and intramolecular circularisation. Due to their shorter length, spliced clones are preferentially amplified and cloned.
Thomas V. O. Hansen - One of the best experts on this subject based on the ideXlab platform.
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The silent mutation nucleotide 744 G → A, Lys172Lys, in Exon 6 of BRCA2 results in Exon skipping
Breast Cancer Research and Treatment, 2010Co-Authors: Thomas V. O. Hansen, Ane Y. Steffensen, Lars Jønson, Mette K. Andersen, Bent Ejlertsen, Finn C. NielsenAbstract:Germ-line mutations in BRCA2 predispose to breast and ovarian cancer. Mutations are widespread throughout the gene and include disease-causing mutations as frameshift, nonsense, splicing mutations and large genomic rearrangements. However a large number of mutations, including missense, silent and intron variants are of unknown significance. Here, we describe the functional characterization of a silent mutation (nucleotide 744 G → A/c.516 G → A, Lys172Lys) in Exon 6 of BRCA2 in a Danish family with breast and ovarian cancer. Exon Trapping analysis showed that the mutation results in skipping of Exon 6 and/or both Exon 5 and 6, which was verified by RT-PCR analysis on RNA isolated from whole blood of the affected patient. We therefore conclude that the BRCA2 silent mutation Lys172Lys is a disease-causing mutation.
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The silent mutation nucleotide 744 G → A, Lys172Lys, in Exon 6 of results in Exon skipping
Breast Cancer Research and Treatment, 2009Co-Authors: Thomas V. O. Hansen, Ane Y. Steffensen, Lars Jønson, Mette K. Andersen, Bent Ejlertsen, Finn C. NielsenAbstract:Germ-line mutations in predispose to breast and ovarian cancer. Mutations are widespread throughout the gene and include disease-causing mutations as frameshift, nonsense, splicing mutations and large genomic rearrangements. However a large number of mutations, including missense, silent and intron variants are of unknown significance. Here, we describe the functional characterization of a silent mutation (nucleotide 744 G → A/c.516 G → A, Lys172Lys) in Exon 6 of in a Danish family with breast and ovarian cancer. Exon Trapping analysis showed that the mutation results in skipping of Exon 6 and/or both Exon 5 and 6, which was verified by RT-PCR analysis on RNA isolated from whole blood of the affected patient. We therefore conclude that the silent mutation Lys172Lys is a disease-causing mutation.
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The silent mutation nucleotide 744 G --> A, Lys172Lys, in Exon 6 of BRCA2 results in Exon skipping.
Breast cancer research and treatment, 2009Co-Authors: Thomas V. O. Hansen, Ane Y. Steffensen, Lars Jønson, Mette K. Andersen, Bent Ejlertsen, Finn C. NielsenAbstract:Germ-line mutations in BRCA2 predispose to breast and ovarian cancer. Mutations are widespread throughout the gene and include disease-causing mutations as frameshift, nonsense, splicing mutations and large genomic rearrangements. However a large number of mutations, including missense, silent and intron variants are of unknown significance. Here, we describe the functional characterization of a silent mutation (nucleotide 744 G → A/c.516 G → A, Lys172Lys) in Exon 6 of BRCA2 in a Danish family with breast and ovarian cancer. Exon Trapping analysis showed that the mutation results in skipping of Exon 6 and/or both Exon 5 and 6, which was verified by RT-PCR analysis on RNA isolated from whole blood of the affected patient. We therefore conclude that the BRCA2 silent mutation Lys172Lys is a disease-causing mutation.
Nicole A. Datson - One of the best experts on this subject based on the ideXlab platform.
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scanning for genes in large genomic regions cosmid based Exon Trapping of multiple Exons in a single product
Nucleic Acids Research, 1996Co-Authors: Nicole A. Datson, Esther Van De Vosse, Hans G Dauwerse, Mattie Bout, Gertjan B Van Ommen, Johan T. Den DunnenAbstract:To facilitate the scanning of large genomic regions for the presence of Exonic gene segments we have constructed a cosmid-based Exon trap vector. The vector serves a dual purpose since it is also suitable for contig construction and physical mapping. The Exon trap cassette of vector sCOGH1 consists of the human growth hormone gene driven by the mouse mettallothionein-1 promoter. Inserts are cloned in the multicloning site located in intron 2 of the hGH gene. The efficiency of the system is demonstrated with cosmids containing multiple Exons of the Duchenne Muscular Dystrophy gene. All Exons present in the inserts were successfully retrieved and no cryptic products were detected. Up to seven Exons were isolated simultaneously in a single spliced product. The system has been extended by a transcription-translation-test protocol to determine the presence of large open reading frames in the trapped products, using a combination of tailed PCR primers directing protein synthesis in three different reading frames, followed by in vitro transcription-translation. Having larger stretches of coding sequence in a single Exon trap product rather than small single Exons greatly facilitates further analysis of potential genes and offers new possibilities for direct mutation analysis of Exon trap material.
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Specific isolation of 3′-terminal Exons of human genes by Exon Trapping
Nucleic Acids Research, 1994Co-Authors: Nicole A. Datson, Geoffrey M. Duyk, Gert-jan B. Van Ommen, Johan T. Den DunnenAbstract:Exon Trapping is a method to functionally clone expressed sequences from genomic DNA. We have previously developed the vector system pETV-SD2, which contains only a splice donor site (SD) followed by a LacZ gene, allowing Trapping of internal Exons of human genes by blue-white selection. We now describe the adaptation of the same system for the efficient Trapping of 3'-terminal Exons, by using different RT-PCR primers in a 3' RACE reaction. The addition of a T7 promoter to the RT-PCR products derived from pETV-SD2 allows their amplification in an isothermic amplification reaction called NASBA (nucleic acid sequence-based amplification reaction) and results in a strong signal from amplified 3' Exons in addition to a great reduction of non-specific background. As a test for the system, 3' Exon Trapping was performed using a cosmid containing the alpha-globin gene cluster on chromosome 16. The 3'-terminal Exons of the human alpha 1-, zeta 2-, and theta-globin genes were trapped, as well as a correctly spliced and polyadenylated sequence in the 3' flanking region of the alpha 1-globin gene. This Exon appears to belong to a previously unidentified gene within the alpha-globin gene cluster. This 3' Exon Trapping strategy should facilitate the cloning of genes from large genomic regions.
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specific isolation of 3 terminal Exons of human genes by Exon Trapping
Nucleic Acids Research, 1994Co-Authors: Nicole A. Datson, Geoffrey M. Duyk, Gert-jan B. Van Ommen, Johan T. Den DunnenAbstract:Exon Trapping is a method to functionally clone expressed sequences from genomic DNA. We have previously developed the vector system pETV-SD2, which contains only a splice donor site (SD) followed by a LacZ gene, allowing Trapping of internal Exons of human genes by blue-white selection. We now describe the adaptation of the same system for the efficient Trapping of 3'-terminal Exons, by using different RT-PCR primers in a 3' RACE reaction. The addition of a T7 promoter to the RT-PCR products derived from pETV-SD2 allows their amplification in an isothermic amplification reaction called NASBA (nucleic acid sequence-based amplification reaction) and results in a strong signal from amplified 3' Exons in addition to a great reduction of non-specific background. As a test for the system, 3' Exon Trapping was performed using a cosmid containing the alpha-globin gene cluster on chromosome 16. The 3'-terminal Exons of the human alpha 1-, zeta 2-, and theta-globin genes were trapped, as well as a correctly spliced and polyadenylated sequence in the 3' flanking region of the alpha 1-globin gene. This Exon appears to belong to a previously unidentified gene within the alpha-globin gene cluster. This 3' Exon Trapping strategy should facilitate the cloning of genes from large genomic regions.
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An Exon Trapping System Providing Size Selection of Spliced Clones and Facilitating Direct Cloning
Identification of Transcribed Sequences, 1994Co-Authors: Nicole A. Datson, Geoffrey M. Duyk, Gert-jan B. Van Ommen, L. A. J. Blonden, Johan T. Den DunnenAbstract:Exon Trapping is a method to functionally clone expressed sequences from genomic DNA. We have developed an Exon Trapping procedure based on the use of vector pETVSD2. Cosmid DNA is partially digested and cloned in pETV-SD2. DNA of an entire library of subclones is introduced into COS-1 cells and transiently expressed. RNA is isolated and vector-derived transcripts are amplified by RT-PCR. Cloning of the RT-PCR products, which contain a ColEI-origin of replication and a supF marker, is established by NotI digestion and intramolecular circularisation. Due to their shorter length, spliced clones are preferentially amplified and cloned.