The Experts below are selected from a list of 64440 Experts worldwide ranked by ideXlab platform
D B Krizman - One of the best experts on this subject based on the ideXlab platform.
-
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.
-
Current Protocols in Human Genetics - Isolation of exons from cloned DNA by exon trapping.
Current protocols in human genetics, 1994Co-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 1 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 Alternate Protocol describes 3 terminal exon trapping, which avoids Subcloning of target DNA by ligating it to the vector for direct transfection. A Support Protocol describes a rapid cloning procedure using uracil DNA glycosylase.
Richard Daniellou - One of the best experts on this subject based on the ideXlab platform.
-
galactofuranosidase from jha 19 streptomyces sp Subcloning and biochemical characterization
Carbohydrate Research, 2019Co-Authors: Mateja Senicar, Laurent Legentil, Vincent Ferrières, Svetlana Eliseeva, Stéphane Petoud, Kaoru Takegawa, Pierre Lafite, Richard DaniellouAbstract:Abstract Despite the crucial role of the rare galactofuranose (Galf) in many pathogenic micro-organisms and our increased knowledge of its metabolism, there is still a lack of recombinant and efficient galactofuranoside hydrolase available for chemo-enzymatic synthetic purposes of specific galactofuranosyl-conjugates. Subcloning of the Galf-ase from JHA 19 Streptomyces sp. and its further overexpression lead us to the production of this enzyme with a yield of 0.5 mg/L of culture. It exhibits substrate specificity exclusively towards pNP β- d -Galf, giving a KM value of 250 μM, and the highest enzymatic efficiency ever observed of 14 mM−1 s−1. It proved to be stable to temperature up to 60 °C and to at least 4 freeze-thaw's cycles. Thus, Galf-ase demonstrated to be an efficient and stable biocatalyst with greatly improved specificity toward the galactofuranosyl entity, thus paving the way to the further development of transglycosylation and thioligation reactions.
-
Galactofuranosidase from JHA 19 Streptomyces sp:. Subcloning and biochemical characterization
Carbohydrate Research, 2019Co-Authors: Mateja Senicar, Laurent Legentil, Vincent Ferrières, Svetlana Eliseeva, Stéphane Petoud, Kaoru Takegawa, Pierre Lafite, Richard DaniellouAbstract:Despite the crucial role of the rare galactofuranose (Galf) in many pathogenic micro-organisms and our increased knowledge of its metabolism, there is still a lack of recombinant and efficient galactofuranoside hydrolase available for chemo-enzymatic synthetic purposes of specific galactofuranosyl-conjugates. Subcloning of the Galf-ase from JHA 19 Streptomyces sp. and its further overexpression lead us to the production of this enzyme with a yield of 0.5 mg/L of culture. It exhibits substrate specificity exclusively towards pNP beta-D-Galf, giving a K-M value of 250 mu M, and the highest enzymatic efficiency ever observed of 14 mM(-1) s(-1). It proved to be stable to temperature up to 60 degrees C and to at least 4 freeze-thaw's cycles. Thus, Galf-ase demonstrated to be an efficient and stable biocatalyst with greatly improved specificity toward the galactofuranosyl entity, thus paving the way to the further development of transglycosylation and thioligation reactions.
Kazuhiko Nakagawa - One of the best experts on this subject based on the ideXlab platform.
-
A Novel Mass Spectrometry-Based Assay for Diagnosis of EML4-ALK-Positive Non-Small-Cell Lung Cancer
Annals of Oncology, 2012Co-Authors: Kazuko Sakai, Ken Takezawa, Isamu Okamoto, Masayuki Takeda, Tomonori Hirashima, Hiroyasu Kaneda, Kazuko Matsumoto, Hideharu Kimura, Yoshihiko Fujita, Kazuhiko NakagawaAbstract:ABSTRACT Introduction The presence of the transforming fusion gene EML4-ALK in non-small-cell lung cancer (NSCLC) is a predictive marker for the efficacy of ALK kinase inhibitors. Currently available assays for detection of the different variants of EML4-ALK have limitations, however. Methods We developed an assay system for the detection of EML4-ALK variant 1, 2, 3a, 3b, 4, 5a, 5b, 6, or 7 transcripts in total RNA obtained from formalin-fixed, paraffin-embedded (FFPE) specimens of NSCLC tissue. The assay is based on region-specific polymerase chain reaction (PCR) amplification of EML4-ALK cDNA followed by specific single-base primer extension and analysis of the extension products by MALDI-TOF mass spectrometry. The assay was validated by fluorescence in situ hybridization (FISH) and the results confirmed by Subcloning and sequencing of PCR products. Results Evaluation of the analytic sensitivity of the assay with serial dilutions of plasmids containing EML4-ALK cDNA sequences revealed it to be capable of the reliable detection of one copy of each plasmid per reaction. The assay also detected EML4-ALK variants 1 or 3 in three FFPE samples of surgically resected NSCLC shown to be positive for ALK rearrangement by FISH. Furthermore, the assay identified variant 1 of EML4-ALK in 3 of 20 FFPE biopsy samples from patients with advanced NSCLC. All positive samples were confirmed by Subcloning and sequencing. Conclusions Our novel assay is highly sensitive and effective for the detection of EML4-ALK in FFPE specimens.
-
A Novel Mass Spectrometry-Based Assay for Diagnosis of EML4-ALK-Positive Non-Small Cell Lung Cancer
Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer, 2012Co-Authors: Kazuko Sakai, Ken Takezawa, Isamu Okamoto, Masayuki Takeda, Tomonori Hirashima, Hiroyasu Kaneda, Kazuko Matsumoto, Hideharu Kimura, Yoshihiko Fujita, Kazuhiko NakagawaAbstract:Introduction: The presence of the transforming fusion gene echinoderm microtubule-associated protein–like 4 ( EML4 ) – anaplastic lymphoma kinase ( ALK ) in non–small-cell lung cancer (NSCLC) is a predictive marker for the efficacy of anaplastic lymphoma kinase inhibitors. However, the currently available assays for the detection of the different variants of EML4-ALK have limitations. Methods: We developed an assay system for the detection of EML4-ALK variants 1, 2, 3a, 3b, 4, 5a, 5b, 6, or 7 transcripts in total RNA obtained from formalin-fixed, paraffin-embedded (FFPE) specimens of NSCLC tissue. The assay is based on region-specific polymerase chain reaction amplification of EML4-ALK complementary DNA followed by specific single-base primer extension and analysis of the extension products by matrix-assisted laser desorption/ionization–time of flight mass spectrometry. The assay was validated by fluorescence in situ hybridization and the results confirmed by Subcloning and sequencing of polymerase chain reaction products. Results: Evaluation of the analytic sensitivity of the assay with serial dilutions of plasmids containing EML4-ALK complementary DNA sequences revealed it to be capable of the reliable detection of one copy of each plasmid per reaction. The assay also detected EML4-ALK variants 1 or 3 in three FFPE samples of surgically resected NSCLC shown to be positive for anaplastic lymphoma kinase rearrangement by fluorescence in situ hybridization. Furthermore, the assay identified variant 1 of EML4-ALK in 3 of 20 FFPE biopsy samples from patients with advanced NSCLC. All positive samples were confirmed by Subcloning and sequencing. Conclusions: Our novel assay is highly sensitive and effective for the detection of EML4-ALK in FFPE specimens.
P E Nisson - One of the best experts on this subject based on the ideXlab platform.
-
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.
-
Current Protocols in Human Genetics - Isolation of exons from cloned DNA by exon trapping.
Current protocols in human genetics, 1994Co-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 1 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 Alternate Protocol describes 3 terminal exon trapping, which avoids Subcloning of target DNA by ligating it to the vector for direct transfection. A Support Protocol describes a rapid cloning procedure using uracil DNA glycosylase.
Mateja Senicar - One of the best experts on this subject based on the ideXlab platform.
-
galactofuranosidase from jha 19 streptomyces sp Subcloning and biochemical characterization
Carbohydrate Research, 2019Co-Authors: Mateja Senicar, Laurent Legentil, Vincent Ferrières, Svetlana Eliseeva, Stéphane Petoud, Kaoru Takegawa, Pierre Lafite, Richard DaniellouAbstract:Abstract Despite the crucial role of the rare galactofuranose (Galf) in many pathogenic micro-organisms and our increased knowledge of its metabolism, there is still a lack of recombinant and efficient galactofuranoside hydrolase available for chemo-enzymatic synthetic purposes of specific galactofuranosyl-conjugates. Subcloning of the Galf-ase from JHA 19 Streptomyces sp. and its further overexpression lead us to the production of this enzyme with a yield of 0.5 mg/L of culture. It exhibits substrate specificity exclusively towards pNP β- d -Galf, giving a KM value of 250 μM, and the highest enzymatic efficiency ever observed of 14 mM−1 s−1. It proved to be stable to temperature up to 60 °C and to at least 4 freeze-thaw's cycles. Thus, Galf-ase demonstrated to be an efficient and stable biocatalyst with greatly improved specificity toward the galactofuranosyl entity, thus paving the way to the further development of transglycosylation and thioligation reactions.
-
Galactofuranosidase from JHA 19 Streptomyces sp:. Subcloning and biochemical characterization
Carbohydrate Research, 2019Co-Authors: Mateja Senicar, Laurent Legentil, Vincent Ferrières, Svetlana Eliseeva, Stéphane Petoud, Kaoru Takegawa, Pierre Lafite, Richard DaniellouAbstract:Despite the crucial role of the rare galactofuranose (Galf) in many pathogenic micro-organisms and our increased knowledge of its metabolism, there is still a lack of recombinant and efficient galactofuranoside hydrolase available for chemo-enzymatic synthetic purposes of specific galactofuranosyl-conjugates. Subcloning of the Galf-ase from JHA 19 Streptomyces sp. and its further overexpression lead us to the production of this enzyme with a yield of 0.5 mg/L of culture. It exhibits substrate specificity exclusively towards pNP beta-D-Galf, giving a K-M value of 250 mu M, and the highest enzymatic efficiency ever observed of 14 mM(-1) s(-1). It proved to be stable to temperature up to 60 degrees C and to at least 4 freeze-thaw's cycles. Thus, Galf-ase demonstrated to be an efficient and stable biocatalyst with greatly improved specificity toward the galactofuranosyl entity, thus paving the way to the further development of transglycosylation and thioligation reactions.
