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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.
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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

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Amplification:

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Microarray:

Article Title: Major pathologic response and RAD51 predict survival in lung cancer patients receiving neoadjuvant chemotherapy
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Incubation:

Article Title: Nanoliter high throughput quantitative PCR
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Article Title: Behavioural and Genetic Characteristics of a New Species of Nasonia
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Modification:

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Countercurrent Chromatography:

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Cell Culture:

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Generated:

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DNA Sequencing:

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Polymerase Chain Reaction:

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Article Title: Complete mitochondrial DNA sequence of the endangered fish (Bahabataipingensis): Mitogenome characterization and phylogenetic implications
Article Snippet: .. PCR products were cleaned by adding 0.45 µl of Shrimp Alkaline Phosphatase (Biotech Pharmacon), 0.9 µl of Exonuclease I (GE Healthcare) and 1.65 µl of sterile distilled H2 O to 9 µL of PCR product and incubating at 37 °C for 30 min and 80 °C for 20 min. .. The purified product was then sequenced on ABI Prism 3730 (Applied Biosystems) from both strands with the same primers as those used for PCRs.

Article Title: Clinical importance of FANCD2, BRIP1, BRCA1, BRCA2 and FANCF expression in ovarian carcinomas
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Article Title: Deregulated expression of NKL homeobox genes in T-cell lymphomas
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Article Title: Specific variants in WDR35 cause a distinctive form of Ellis-van Creveld syndrome by disrupting the recruitment of the EvC complex and SMO into the cilium
Article Snippet: .. PCR products were enzymatically treated with alkaline phosphatase and exonuclease I (ExoSapit; GE Healthcare) prior to be sequenced using a dye terminator cycle sequencing kit and run on an ABI 3730 sequencer (Applied Biosystems). .. Sequencing of IFT 122 was conducted using an in-house next generation sequencing (NGS) panel for detection of pathogenic variants in skeletal dysplasias comprising 315 genes.

Article Title: Consuming viscous prey: a novel protein-secreting delivery system in neotropical snail-eating snakes
Article Snippet: Primers and PCR protocols for partial amplification of genes 12S, 16S, cytb, bdnf, and c-mos were those described in Grazziotin et al. [ ]. .. PCRs were purified with shrimp alkaline phosphatase and exonuclease I (GE Healthcare, Piscataway, NJ, USA) and sequences were processed using the DYEnamic ET Dye Terminator Cycle Sequencing Kit in a MegaBACE 1000 automated sequencer (GE Healthcare) following the manufacturer’s protocols.

Article Title: Root-associated fungal communities in three Pyroleae species and their mycobiont sharing with surrounding trees in subalpine coniferous forests on Mount Fuji, Japan
Article Snippet: .. Amplification products were purified using a PCR clean-up kit containing exonuclease I and shrimp alkaline phosphatase (GE Healthcare, Hertfordshire, UK). .. Sanger sequencing was performed using the Big Dye Terminator version 3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and ITS1 or ITS4.

Article Title: Major pathologic response and RAD51 predict survival in lung cancer patients receiving neoadjuvant chemotherapy
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Article Title: Ancient female philopatry, asymmetric male gene flow, and synchronous population expansion support the influence of climatic oscillations on the evolution of South American sea lion (Otaria flavescens)
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Recombinant:

Article Title: Nanoliter high throughput quantitative PCR
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DNA Extraction:

Article Title: Root-associated fungal communities in three Pyroleae species and their mycobiont sharing with surrounding trees in subalpine coniferous forests on Mount Fuji, Japan
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Fluorescence:

Article Title: A Microsphere-Based Assay for Multiplexed Single Nucleotide Polymorphism Analysis Using Single Base Chain Extension
Article Snippet: Shrimp alkaline phosphatase (SAP) and Exonuclease I (Exo I) were obtained from Amersham Pharmacia. .. Fluorescence labeled dideoxynucleotide triphosphates (ddNTPs) were obtained from NEN Life Science Products, Inc. (Boston, MA).

Mutagenesis:

Article Title: Major pathologic response and RAD51 predict survival in lung cancer patients receiving neoadjuvant chemotherapy
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Isolation:

Article Title: Mutations in FOXC2 (MFH-1), a Forkhead Family Transcription Factor, Are Responsible for the Hereditary Lymphedema-Distichiasis Syndrome
Article Snippet: To amplify the single coding exon of FOXC2, genomic DNA isolated from peripheral blood or cultured fibroblasts of probands and family members was used as template in PCR reactions. .. Before sequencing, PCR products were purified by treatment with Exonuclease I (Amersham Pharmacia Biotech and shrimp alkaline phosphatase (Amersham Pharmacia Biotech) for 15 min at 37°C.

Labeling:

Article Title: A Microsphere-Based Assay for Multiplexed Single Nucleotide Polymorphism Analysis Using Single Base Chain Extension
Article Snippet: Shrimp alkaline phosphatase (SAP) and Exonuclease I (Exo I) were obtained from Amersham Pharmacia. .. Fluorescence labeled dideoxynucleotide triphosphates (ddNTPs) were obtained from NEN Life Science Products, Inc. (Boston, MA).

Purification:

Article Title: Mutations in FOXC2 (MFH-1), a Forkhead Family Transcription Factor, Are Responsible for the Hereditary Lymphedema-Distichiasis Syndrome
Article Snippet: .. Before sequencing, PCR products were purified by treatment with Exonuclease I (Amersham Pharmacia Biotech and shrimp alkaline phosphatase (Amersham Pharmacia Biotech) for 15 min at 37°C. .. Enzymes were heat inactivated by incubating the reaction at 80°C for 15 min. Sequencing was performed by the Sequencing Core Laboratory at the University of Michigan, by the dideoxy termination method and an ABI 377 automated sequencer.

Article Title: Complete mitochondrial DNA sequence of the endangered fish (Bahabataipingensis): Mitogenome characterization and phylogenetic implications
Article Snippet: PCR products were cleaned by adding 0.45 µl of Shrimp Alkaline Phosphatase (Biotech Pharmacon), 0.9 µl of Exonuclease I (GE Healthcare) and 1.65 µl of sterile distilled H2 O to 9 µL of PCR product and incubating at 37 °C for 30 min and 80 °C for 20 min. .. The purified product was then sequenced on ABI Prism 3730 (Applied Biosystems) from both strands with the same primers as those used for PCRs.

Article Title: Nanoliter high throughput quantitative PCR
Article Snippet: RNA was purified using an RNeasy Mini Kit (Qiagen Inc.) and reverse transcription was done using a High Capacity cDNA Archive Kit (Applied Biosystems). .. Samples were treated with Exonuclease I (10 U/μl) (Amersham Biosciences).

Article Title: Clinical importance of FANCD2, BRIP1, BRCA1, BRCA2 and FANCF expression in ovarian carcinomas
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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). .. For purification we used CleanSEQ reagent in combination with the Agencourt CleanSEQ magnetic plate (Beckman Coulter, Krefeld, Germany).

Article Title: Consuming viscous prey: a novel protein-secreting delivery system in neotropical snail-eating snakes
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Article Title: Root-associated fungal communities in three Pyroleae species and their mycobiont sharing with surrounding trees in subalpine coniferous forests on Mount Fuji, Japan
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Article Title: Large-scale molecular phylogeny, morphology, divergence-time estimation, and the fossil record of advanced caenophidian snakes (Squamata: Serpentes)
Article Snippet: .. PCRs were purified with shrimp alkaline phosphatase and exonuclease I (GE Healthcare, Piscataway, NJ). .. Sequences were generated in Brazil at the Laboratório de Biologia Genômica e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul (Porto Alegre, Rio Grande do Sul) using the DYEnamic ET Dye Terminator Cycle Sequencing Kit in a MegaBACE 1000 automated sequencer (GE Healthcare); and in China at Laboratory for Conservation and Utilization of Bio-resources, Yunnan University (Kunming, Yunnan) using BigDye Terminator cycle sequencing kit in an ABI 3700 sequencer (Applied Biosystems, Foster City, CA).

Article Title: Major pathologic response and RAD51 predict survival in lung cancer patients receiving neoadjuvant chemotherapy
Article Snippet: Subsequently, the biotinylated PCR products were purified and made into single‐stranded DNA to which a sequencing primer was annealed using a vacuum prep tool (Pyrosequencing, Inc., Westborough, MA). .. For direct sequencing, all PCR amplification products were incubated using exonuclease I and shrimp alkaline phosphatase (Amersham Biosciences, Piscataway, NJ) and sequenced by the MD Anderson Core Sequencing and Microarray Facility.

Article Title: Ancient female philopatry, asymmetric male gene flow, and synchronous population expansion support the influence of climatic oscillations on the evolution of South American sea lion (Otaria flavescens)
Article Snippet: .. Amplification products were purified with shrimp alkaline phosphatase and exonuclease I (Amersham Biosciences). .. The purified products were sequenced in both directions using the DYEnamic ET Dye Terminator Cycle Sequencing Kit (Amersham Biosciences) and run in a MegaBace 1000 automated sequencer (Amersham Biosciences).

Sequencing:

Article Title: Colorectal Tumors from APC*I1307K Carriers Principally Harbor Somatic APC Mutations outside the A8 Tract
Article Snippet: .. PCR products were prepared for sequencing by treatment of 5 µl of PCR product with 2 U of shrimp alkaline phosphatase and 10 U of exonuclease I (Amersham Pharmacia, Chalfont St Giles, UK) at 37°C for 30 minutes and then 80°C for 15 minutes. .. Sequencing reactions of 10 µl were carried out using 1–3 µl of prepared PCR product, 1.6 picomoles of primer, BigDye ready reaction mix (Applied Biosystems) version 2 diluted 1∶2 with Half Big Dye reagent (Genetix, http://www.genetix.com/ ).

Article Title: Mutations in FOXC2 (MFH-1), a Forkhead Family Transcription Factor, Are Responsible for the Hereditary Lymphedema-Distichiasis Syndrome
Article Snippet: .. Before sequencing, PCR products were purified by treatment with Exonuclease I (Amersham Pharmacia Biotech and shrimp alkaline phosphatase (Amersham Pharmacia Biotech) for 15 min at 37°C. .. Enzymes were heat inactivated by incubating the reaction at 80°C for 15 min. Sequencing was performed by the Sequencing Core Laboratory at the University of Michigan, by the dideoxy termination method and an ABI 377 automated sequencer.

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: Deregulated expression of NKL homeobox genes in T-cell lymphomas
Article Snippet: Paragraph title: Sanger sequencing ... 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).

Article Title: Specific variants in WDR35 cause a distinctive form of Ellis-van Creveld syndrome by disrupting the recruitment of the EvC complex and SMO into the cilium
Article Snippet: .. PCR products were enzymatically treated with alkaline phosphatase and exonuclease I (ExoSapit; GE Healthcare) prior to be sequenced using a dye terminator cycle sequencing kit and run on an ABI 3730 sequencer (Applied Biosystems). .. Sequencing of IFT 122 was conducted using an in-house next generation sequencing (NGS) panel for detection of pathogenic variants in skeletal dysplasias comprising 315 genes.

Article Title: Consuming viscous prey: a novel protein-secreting delivery system in neotropical snail-eating snakes
Article Snippet: .. PCRs were purified with shrimp alkaline phosphatase and exonuclease I (GE Healthcare, Piscataway, NJ, USA) and sequences were processed using the DYEnamic ET Dye Terminator Cycle Sequencing Kit in a MegaBACE 1000 automated sequencer (GE Healthcare) following the manufacturer’s protocols. ..

Article Title: Root-associated fungal communities in three Pyroleae species and their mycobiont sharing with surrounding trees in subalpine coniferous forests on Mount Fuji, Japan
Article Snippet: Amplification products were purified using a PCR clean-up kit containing exonuclease I and shrimp alkaline phosphatase (GE Healthcare, Hertfordshire, UK). .. Sanger sequencing was performed using the Big Dye Terminator version 3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and ITS1 or ITS4.

Article Title: Large-scale molecular phylogeny, morphology, divergence-time estimation, and the fossil record of advanced caenophidian snakes (Squamata: Serpentes)
Article Snippet: PCRs were purified with shrimp alkaline phosphatase and exonuclease I (GE Healthcare, Piscataway, NJ). .. Sequences were generated in Brazil at the Laboratório de Biologia Genômica e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul (Porto Alegre, Rio Grande do Sul) using the DYEnamic ET Dye Terminator Cycle Sequencing Kit in a MegaBACE 1000 automated sequencer (GE Healthcare); and in China at Laboratory for Conservation and Utilization of Bio-resources, Yunnan University (Kunming, Yunnan) using BigDye Terminator cycle sequencing kit in an ABI 3700 sequencer (Applied Biosystems, Foster City, CA).

Article Title: Major pathologic response and RAD51 predict survival in lung cancer patients receiving neoadjuvant chemotherapy
Article Snippet: .. For direct sequencing, all PCR amplification products were incubated using exonuclease I and shrimp alkaline phosphatase (Amersham Biosciences, Piscataway, NJ) and sequenced by the MD Anderson Core Sequencing and Microarray Facility. .. Histopathologic evaluation Immunohistochemical staining for biomarkers was performed as described previously .

Article Title: Ancient female philopatry, asymmetric male gene flow, and synchronous population expansion support the influence of climatic oscillations on the evolution of South American sea lion (Otaria flavescens)
Article Snippet: Paragraph title: Mitochondrial DNA amplification, sequencing and analysis ... Amplification products were purified with shrimp alkaline phosphatase and exonuclease I (Amersham Biosciences).

Article Title: Behavioural and Genetic Characteristics of a New Species of Nasonia
Article Snippet: .. To clean the reactions before sequencing, amplified reactions (8uL) were incubated with 0.5 U shrimp alkaline phosphatase and 1.0 U of exonuclease I (Amersham, Piscataway, NJ) along with the supplied buffer. .. Sequencing was performed directly from the amplified products using a BigDye v3.0 terminator sequencing kit and an ABI 3700 or 3730 xl ) for the cox1 sequences as well as the concatenated data set for the nuclear markers.

Software:

Article Title: Specific variants in WDR35 cause a distinctive form of Ellis-van Creveld syndrome by disrupting the recruitment of the EvC complex and SMO into the cilium
Article Snippet: PCR products were enzymatically treated with alkaline phosphatase and exonuclease I (ExoSapit; GE Healthcare) prior to be sequenced using a dye terminator cycle sequencing kit and run on an ABI 3730 sequencer (Applied Biosystems). .. Nevertheless, to be consistent with the previous WDR35 literature, variants were named using and as reference sequences and checked with Mutalyzer software ( ) ( ).

Multiplex Assay:

Article Title: Root-associated fungal communities in three Pyroleae species and their mycobiont sharing with surrounding trees in subalpine coniferous forests on Mount Fuji, Japan
Article Snippet: The internal transcribed spacer (ITS) regions of fungal rDNA were amplified by polymerase chain reaction (PCR) using the forward primer ITS1F and the reverse primers ITS4, LR21, LR22, and LBW with a Multiplex PCR kit (QIAGEN, Hilden, Germany), following the manufacturer’s instructions. .. Amplification products were purified using a PCR clean-up kit containing exonuclease I and shrimp alkaline phosphatase (GE Healthcare, Hertfordshire, UK).

Sample Prep:

Article Title: Nanoliter high throughput quantitative PCR
Article Snippet: Paragraph title: Human umbilical vein endothelial cells (HUVEC sample preparation ... Samples were treated with Exonuclease I (10 U/μl) (Amersham Biosciences).

Ethanol Precipitation:

Article Title: Colorectal Tumors from APC*I1307K Carriers Principally Harbor Somatic APC Mutations outside the A8 Tract
Article Snippet: PCR products were prepared for sequencing by treatment of 5 µl of PCR product with 2 U of shrimp alkaline phosphatase and 10 U of exonuclease I (Amersham Pharmacia, Chalfont St Giles, UK) at 37°C for 30 minutes and then 80°C for 15 minutes. .. Unincorporated nucleotides and primers were removed by ethanol precipitation.

Next-Generation Sequencing:

Article Title: Specific variants in WDR35 cause a distinctive form of Ellis-van Creveld syndrome by disrupting the recruitment of the EvC complex and SMO into the cilium
Article Snippet: PCR products were enzymatically treated with alkaline phosphatase and exonuclease I (ExoSapit; GE Healthcare) prior to be sequenced using a dye terminator cycle sequencing kit and run on an ABI 3730 sequencer (Applied Biosystems). .. Sequencing of IFT 122 was conducted using an in-house next generation sequencing (NGS) panel for detection of pathogenic variants in skeletal dysplasias comprising 315 genes.

Sampling:

Article Title: Consuming viscous prey: a novel protein-secreting delivery system in neotropical snail-eating snakes
Article Snippet: Sixty-one Dipsadinae terminals composed the ingroup, representing an increase of 25 species in respect to the taxon sampling used by Pyron et al. [ ] and 31 species to the one used by Grazziotin et al. [ ]. .. PCRs were purified with shrimp alkaline phosphatase and exonuclease I (GE Healthcare, Piscataway, NJ, USA) and sequences were processed using the DYEnamic ET Dye Terminator Cycle Sequencing Kit in a MegaBACE 1000 automated sequencer (GE Healthcare) following the manufacturer’s protocols.

Variant Assay:

Article Title: Colorectal Tumors from APC*I1307K Carriers Principally Harbor Somatic APC Mutations outside the A8 Tract
Article Snippet: Extended DNA Sequencing Samples in which a variant was identified by FSSCP were re-amplified using unlabeled PCR primers. .. PCR products were prepared for sequencing by treatment of 5 µl of PCR product with 2 U of shrimp alkaline phosphatase and 10 U of exonuclease I (Amersham Pharmacia, Chalfont St Giles, UK) at 37°C for 30 minutes and then 80°C for 15 minutes.

Article Title: Specific variants in WDR35 cause a distinctive form of Ellis-van Creveld syndrome by disrupting the recruitment of the EvC complex and SMO into the cilium
Article Snippet: Paragraph title: Sequence variant screening and nomenclature ... PCR products were enzymatically treated with alkaline phosphatase and exonuclease I (ExoSapit; GE Healthcare) prior to be sequenced using a dye terminator cycle sequencing kit and run on an ABI 3730 sequencer (Applied Biosystems).

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    GE Healthcare exonuclease i shrimp alkaline phosphatase
    Exonuclease I Shrimp Alkaline Phosphatase, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 87/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exonuclease i shrimp alkaline phosphatase/product/GE Healthcare
    Average 87 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    exonuclease i shrimp alkaline phosphatase - by Bioz Stars, 2020-01
    87/100 stars
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