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GE Healthcare exonuclease i
Schematic presentation of the microsphere-based single base chain extension assays. DNA fragments containing the polymorphic site to be typed were amplified either individually or by multiplexed PCR (step 1 ). The PCR products containing a SNP site were pooled and treated with SAP and <t>exonuclease</t> I (step 2 ). After heat inactivation of the enzymes, the PCR products were used in the SBCE reactions (step 3 ) as described in Methods. For every SNP, one capture oligonucleotide probe with a unique ZipCode sequence was designed and used to assay the two alleles in each of two separate wells with a different labeled ddNTP per well. Multiplexed SNP analysis could be achieved by the employment of different ZipCode sequences for different SNPs in the presence of pooled PCR products. After the completion of the SBCE reaction, ∼1200 of each type of microsphere [with an attached oligonucleotide encoding the complement to the ZipCode sequences and a common luciferase sequence (SeqLUC)] were added to the completed SBCE reactions. The hybridization reactions were carried out at 40°C in the presence of NaCl for > 2 hr (step 4 ). The microspheres were then subjected to flow cytometric analysis (step 5 ). Minimums of 100 of each type of microsphere were read and the mean value of MESF was used for determining the genotypes. The fluorescence signal of the corresponding microsphere without SBCE reactions (microsphere alone) or SBCE reactions without AmpliTaq FS were subtracted from the MESF values.
Exonuclease I, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 191 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "A Microsphere-Based Assay for Multiplexed Single Nucleotide Polymorphism Analysis Using Single Base Chain Extension"

Article Title: A Microsphere-Based Assay for Multiplexed Single Nucleotide Polymorphism Analysis Using Single Base Chain Extension

Journal: Genome Research

doi:

Schematic presentation of the microsphere-based single base chain extension assays. DNA fragments containing the polymorphic site to be typed were amplified either individually or by multiplexed PCR (step 1 ). The PCR products containing a SNP site were pooled and treated with SAP and exonuclease I (step 2 ). After heat inactivation of the enzymes, the PCR products were used in the SBCE reactions (step 3 ) as described in Methods. For every SNP, one capture oligonucleotide probe with a unique ZipCode sequence was designed and used to assay the two alleles in each of two separate wells with a different labeled ddNTP per well. Multiplexed SNP analysis could be achieved by the employment of different ZipCode sequences for different SNPs in the presence of pooled PCR products. After the completion of the SBCE reaction, ∼1200 of each type of microsphere [with an attached oligonucleotide encoding the complement to the ZipCode sequences and a common luciferase sequence (SeqLUC)] were added to the completed SBCE reactions. The hybridization reactions were carried out at 40°C in the presence of NaCl for > 2 hr (step 4 ). The microspheres were then subjected to flow cytometric analysis (step 5 ). Minimums of 100 of each type of microsphere were read and the mean value of MESF was used for determining the genotypes. The fluorescence signal of the corresponding microsphere without SBCE reactions (microsphere alone) or SBCE reactions without AmpliTaq FS were subtracted from the MESF values.
Figure Legend Snippet: Schematic presentation of the microsphere-based single base chain extension assays. DNA fragments containing the polymorphic site to be typed were amplified either individually or by multiplexed PCR (step 1 ). The PCR products containing a SNP site were pooled and treated with SAP and exonuclease I (step 2 ). After heat inactivation of the enzymes, the PCR products were used in the SBCE reactions (step 3 ) as described in Methods. For every SNP, one capture oligonucleotide probe with a unique ZipCode sequence was designed and used to assay the two alleles in each of two separate wells with a different labeled ddNTP per well. Multiplexed SNP analysis could be achieved by the employment of different ZipCode sequences for different SNPs in the presence of pooled PCR products. After the completion of the SBCE reaction, ∼1200 of each type of microsphere [with an attached oligonucleotide encoding the complement to the ZipCode sequences and a common luciferase sequence (SeqLUC)] were added to the completed SBCE reactions. The hybridization reactions were carried out at 40°C in the presence of NaCl for > 2 hr (step 4 ). The microspheres were then subjected to flow cytometric analysis (step 5 ). Minimums of 100 of each type of microsphere were read and the mean value of MESF was used for determining the genotypes. The fluorescence signal of the corresponding microsphere without SBCE reactions (microsphere alone) or SBCE reactions without AmpliTaq FS were subtracted from the MESF values.

Techniques Used: Amplification, Polymerase Chain Reaction, Sequencing, Labeling, Luciferase, Hybridization, Flow Cytometry, Fluorescence

Related Articles

Polymerase Chain Reaction:

Article Title: Mutation screening of GRIN2B in schizophrenia and autism spectrum disorder in a Japanese population
Article Snippet: .. After PCR amplification, amplicons were processed with Illustra Exonuclease I and Alkaline Phosphatase (GE Healthcare and Life Science, Little Chalfont, United Kingdom) to digest excess primers. .. The cycle sequencing reaction was then performed with the Big Dye Terminator v3.1 Cycle sequencing kit (Applied Biosystems, Foster City, CA, USA).

Article Title: Clinical importance of the EMSY gene expression and polymorphisms in ovarian cancer
Article Snippet: .. PCR products were purified with exonuclease I and alkaline phosphatase (Illustra ExoProStar, GE Healthcare Life Sciences, Little Chalfont, UK) for 18 min. at 37°C followed by 18 min. at 80°C to inactivate the enzymes. .. Then, the purified PCR products were sequenced with the use of BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) on ABI PRISM 3100 DNA sequencer (Life Technologies) according to the manufacturer’s recommendations.

Article Title: Clinical importance of FANCD2, BRIP1, BRCA1, BRCA2 and FANCF expression in ovarian carcinomas
Article Snippet: .. Prior to sequencing, the PCR products were purified enzymatically with exonuclease I and alkaline phosphatase (Illustra ExoProStar, GE Healthcare Life Sciences). .. TP53 protein accumulation status Analysis of TP53 accumulation in the nuclei of tumor cells population was described previously by our team., Briefly, TP53 accumulation was visualized by an immunohistochemical method, with the use of PAb1801 monoclonal antibody (1/3000, Sigma-Genosys) on paraffin-embedded material, after heat-induced epitope retrieval (HIER).

Article Title: Identification of Rare, Single-Nucleotide Mutations in NDE1 and Their Contributions to Schizophrenia Susceptibility
Article Snippet: .. After PCR amplification, aliquots of PCR products were purified using Illustra Exonuclease I and Alkaline Phosphatase (GE Healthcare and Life Science). .. These were then sequenced using the Sanger method and a 3130XL Genetic Analyzer (Applied Biosystems).

Article Title: Deregulated expression of NKL homeobox genes in T-cell lymphomas
Article Snippet: .. PCR products were treated using exonuclease 1 and alkaline phosphatase Illustra ExoProStar according to the recommended protocol for a 5 μl aliquot (GE Healthcare Life Sciences, Freiburg, Germany). .. The sequencing reactions were performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher) for 25 cycles in a Veriti Thermal Cycler (Thermo Fisher).

Article Title: Systematics of South American snail-eating snakes ( Serpentes, Dipsadini), with the description of five new species from Ecuador and Peru
Article Snippet: .. PCR products were cleaned with either ExoSAP-IT (Affymetrix, Cleveland, OH), or Exonuclease I and Alkaline Phosphatase (Illustra ExoProStar by GE Healthcare) before they were sent to Macrogen Inc (Korea) for sequencing. ..

Article Title: Resequencing and Association Analysis of PTPRA, a Possible Susceptibility Gene for Schizophrenia and Autism Spectrum Disorders
Article Snippet: .. The Takara LA taq Kit (Takara Bio Inc. Shiga, Japan) was used for PCR amplification, and products were cleaned up with Illustra Exonuclease I and Alkaline Phosphatase (GE Healthcare & Life Science, Little Chalfont, United Kingdom). .. After that, Sanger sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California, United States).

Sequencing:

Article Title: Clinical importance of FANCD2, BRIP1, BRCA1, BRCA2 and FANCF expression in ovarian carcinomas
Article Snippet: .. Prior to sequencing, the PCR products were purified enzymatically with exonuclease I and alkaline phosphatase (Illustra ExoProStar, GE Healthcare Life Sciences). .. TP53 protein accumulation status Analysis of TP53 accumulation in the nuclei of tumor cells population was described previously by our team., Briefly, TP53 accumulation was visualized by an immunohistochemical method, with the use of PAb1801 monoclonal antibody (1/3000, Sigma-Genosys) on paraffin-embedded material, after heat-induced epitope retrieval (HIER).

Article Title: Combining GWAS and RNA-Seq Approaches for Detection of the Causal Mutation for Hereditary Junctional Epidermolysis Bullosa in Sheep
Article Snippet: .. The amplicons were purified using Illustra Exonuclease I and alkaline phosphatase (Illustra ExoProStar 1-Step, GE Healthcare Life Sciences) treatment and were dideoxy-sequenced in both directions using the Big Dye Terminator Cycle Sequencing Kit, v3.1 (Applied Biosystems) using the same primers used for the fragment amplification. .. After sequencing on an ABI Prism 3130 automatic sequencer (Applied Biosystems), the sequence data were analyzed using SeqScape v2.5 (Applied Biosystems).

Article Title: Systematics of South American snail-eating snakes ( Serpentes, Dipsadini), with the description of five new species from Ecuador and Peru
Article Snippet: .. PCR products were cleaned with either ExoSAP-IT (Affymetrix, Cleveland, OH), or Exonuclease I and Alkaline Phosphatase (Illustra ExoProStar by GE Healthcare) before they were sent to Macrogen Inc (Korea) for sequencing. ..

Amplification:

Article Title: Mutation screening of GRIN2B in schizophrenia and autism spectrum disorder in a Japanese population
Article Snippet: .. After PCR amplification, amplicons were processed with Illustra Exonuclease I and Alkaline Phosphatase (GE Healthcare and Life Science, Little Chalfont, United Kingdom) to digest excess primers. .. The cycle sequencing reaction was then performed with the Big Dye Terminator v3.1 Cycle sequencing kit (Applied Biosystems, Foster City, CA, USA).

Article Title: Identification of Rare, Single-Nucleotide Mutations in NDE1 and Their Contributions to Schizophrenia Susceptibility
Article Snippet: .. After PCR amplification, aliquots of PCR products were purified using Illustra Exonuclease I and Alkaline Phosphatase (GE Healthcare and Life Science). .. These were then sequenced using the Sanger method and a 3130XL Genetic Analyzer (Applied Biosystems).

Article Title: Combining GWAS and RNA-Seq Approaches for Detection of the Causal Mutation for Hereditary Junctional Epidermolysis Bullosa in Sheep
Article Snippet: .. The amplicons were purified using Illustra Exonuclease I and alkaline phosphatase (Illustra ExoProStar 1-Step, GE Healthcare Life Sciences) treatment and were dideoxy-sequenced in both directions using the Big Dye Terminator Cycle Sequencing Kit, v3.1 (Applied Biosystems) using the same primers used for the fragment amplification. .. After sequencing on an ABI Prism 3130 automatic sequencer (Applied Biosystems), the sequence data were analyzed using SeqScape v2.5 (Applied Biosystems).

Article Title: Resequencing and Association Analysis of PTPRA, a Possible Susceptibility Gene for Schizophrenia and Autism Spectrum Disorders
Article Snippet: .. The Takara LA taq Kit (Takara Bio Inc. Shiga, Japan) was used for PCR amplification, and products were cleaned up with Illustra Exonuclease I and Alkaline Phosphatase (GE Healthcare & Life Science, Little Chalfont, United Kingdom). .. After that, Sanger sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California, United States).

Purification:

Article Title: Clinical importance of the EMSY gene expression and polymorphisms in ovarian cancer
Article Snippet: .. PCR products were purified with exonuclease I and alkaline phosphatase (Illustra ExoProStar, GE Healthcare Life Sciences, Little Chalfont, UK) for 18 min. at 37°C followed by 18 min. at 80°C to inactivate the enzymes. .. Then, the purified PCR products were sequenced with the use of BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) on ABI PRISM 3100 DNA sequencer (Life Technologies) according to the manufacturer’s recommendations.

Article Title: Clinical importance of FANCD2, BRIP1, BRCA1, BRCA2 and FANCF expression in ovarian carcinomas
Article Snippet: .. Prior to sequencing, the PCR products were purified enzymatically with exonuclease I and alkaline phosphatase (Illustra ExoProStar, GE Healthcare Life Sciences). .. TP53 protein accumulation status Analysis of TP53 accumulation in the nuclei of tumor cells population was described previously by our team., Briefly, TP53 accumulation was visualized by an immunohistochemical method, with the use of PAb1801 monoclonal antibody (1/3000, Sigma-Genosys) on paraffin-embedded material, after heat-induced epitope retrieval (HIER).

Article Title: Identification of Rare, Single-Nucleotide Mutations in NDE1 and Their Contributions to Schizophrenia Susceptibility
Article Snippet: .. After PCR amplification, aliquots of PCR products were purified using Illustra Exonuclease I and Alkaline Phosphatase (GE Healthcare and Life Science). .. These were then sequenced using the Sanger method and a 3130XL Genetic Analyzer (Applied Biosystems).

Article Title: Combining GWAS and RNA-Seq Approaches for Detection of the Causal Mutation for Hereditary Junctional Epidermolysis Bullosa in Sheep
Article Snippet: .. The amplicons were purified using Illustra Exonuclease I and alkaline phosphatase (Illustra ExoProStar 1-Step, GE Healthcare Life Sciences) treatment and were dideoxy-sequenced in both directions using the Big Dye Terminator Cycle Sequencing Kit, v3.1 (Applied Biosystems) using the same primers used for the fragment amplification. .. After sequencing on an ABI Prism 3130 automatic sequencer (Applied Biosystems), the sequence data were analyzed using SeqScape v2.5 (Applied Biosystems).

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