Abi bigdye terminator cycle sequencing kit v3

Manufactured by Thermo Fisher Scientific

Sourced in United States

The ABI BigDye Terminator cycle sequencing kit v3.1 is a laboratory reagent used for DNA sequencing. It contains the necessary components to perform automated DNA sequencing using the Sanger sequencing method.

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9 protocols using abi bigdye terminator cycle sequencing kit v3

Molecular Cloning of TMPRSS2-ERG Variants

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TMPRSS2-ERG translocation variants were amplified with Taq DNA polymerase, purified with a QIAquick PCR Purification Kit (Qiagen, Düsseldorf, Germany), and cloned into the pCR2.1-TOPO vector using a TOPO TA Invitrogen Cloning kit. The resulting plasmids were used for the transformation of Escherichia coli XL1-blue cells; positive clones were grown in LB medium overnight. The recombinant plasmids were isolated by the DM miniprep method [50 (link)] and sequenced in both directions using M13 forward and reverse primers (Invitrogen) and ABI BigDye Terminator Cycle Sequencing Kit v3.1 (Thermo Fisher, Waltham, MA, USA) with a Gene Amp 9700 PCR System (Thermo Fisher, Carlsbad, CA, USA). The sequences were detected with an ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA USA). The sequences of TMPRSS2 (NM_005656) and ERG (NM_004449) were analyzed with the BLAST software (National Center for Biotechnology Information, NIH, GOV, USA).

Ryabov V.M., Baryshev M.M., Voskresenskiy M.A, & Popov B.V. (2023). Early Cell Cultures from Prostate Cancer Tissue Express Tissue Specific Epithelial and Cancer Markers. International Journal of Molecular Sciences, 24(3), 2830.

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DNA Sequencing Using ABI BigDye Terminator

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Restriction endonucleases and T4 DNA ligase were obtained from Thermo Fisher Scientific Inc. (Carlsbad, USA), and used with the buffer stocks recommended and provided by the company.
DNA sequencing was performed using the ABI BigDye Terminator Cycle Sequencing Kit v3.1 ThermoFisher Scientific Inc. (Carlsbad, USA) according to the manufacturer’s instructions on a Gene Amp 9700 PCR machine (Carlsbad, USA), and the sequences were detected on an ABI 3130XL Genetic Analyzer, Applied Biosystems (Carlsbad, USA).

Mikhail B., Dmitrijs M, & Ivan M. (2022). A new device-mediated miniprep method. AMB Express, 12, 21.

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APOE Exon 4 Variant Analysis

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The variants in exon four in the APOE gene (GenBank: NM_000041)—that is, c.388T > C and c.526C > T—were analyzed by polymerase chain reaction and Sanger sequencing in DNA extracted from peripheral leukocytes. Primers were designed using Primer3Plus (Bioin-formatics, Arlington, VA, USA) (34 (link)) and the polymerase chain reaction products were purified and Sanger sequenced using an ABI 3730xl DNA analyzer and an ABI BigDye terminator cycle-sequencing kit v3.1 (Thermo Fisher Scientific, Waltham, MA, USA). Sequences were analyzed with the DNA Sequencing Analysis software program (version 5.1; Applied Biosystems, Foster City, CA, USA) and the SeqScape software program (version 2.7; Thermo Fisher Scientific).

Hellstrøm T., Andelic N., Holthe Ø.Ø., Helseth E., Server A., Eiklid K, & Sigurdardottir S. (2022). APOE-ε4 Is Associated With Reduced Verbal Memory Performance and Higher Emotional, Cognitive, and Everyday Executive Function Symptoms Two Months After Mild Traumatic Brain Injury. Frontiers in Neurology, 13, 735206.

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IFT57 Gene Sequencing Protocol

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IFT57 primers were designed on the RefSeq NM_018010 (Table S1). PCR fragments were purified with the multiscreen Vacuum Manifold system (Millipore). Sequencing was performed with the ABI BigDye Terminator Cycle Sequencing kit (v.3.1) (Applied Biosystems) in ABI 3130 sequencer 7 (Applied Biosystems) according to the manufacturer’s instructions. Sequence data were analyzed with SeqScape v.2.7 (Applied Biosystems).

Thevenon J., Duplomb L., Phadke S., Eguether T., Saunier A., Avila M., Carmignac V., Bruel A.L., St-Onge J., Duffourd Y., Pazour G.J., Franco B., Attie-Bitach T., Masurel-Paulet A., Rivière J.B., Cormier-Daire V., Philippe C., Faivre L, & Thauvin-Robinet C. (2016). Autosomal Recessive IFT57 hypomorphic mutation cause ciliary transport defect in unclassified oral-facial-digital syndrome with short stature and brachymesophalangia. Clinical genetics, 90(6), 509-517.

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Genetic Analysis of Hormonal Disorders

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Genomic DNA from saliva samples of the subjects and their relatives was extracted according to manufacturer’s instructions (Oragene DNA-kit OG-500, DNA Genotek Inc., Ottawa, Ontario, Canada). The coding exons and exon-intron boundaries of GNRHR, FGFR1, TAC3, and TACR3, were PCR-amplified, the PCR products were purified with Illustra ExoProStar treatment (GE Healthcare, Chicago, Illinois, USA), and bi-directionally sequenced using the ABI BigDyeTerminator Cycle Sequencing Kit (v3.1) and ABI Prism 3730xl DNA Analyzer automated sequencer (Applied Biosystems, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The sequences were aligned and read with Sequencher 5.0 software (Gene Codes Corporation, Ann Arbor, Michigan, USA). All primer sequences are listed in S1 Table. The PCR conditions are available upon request. Monozygosity of the twin brothers was analyzed by DNA profiling (DNA Diagnostics Centre Finland).

Hietamäki J., Hero M., Holopainen E., Känsäkoski J., Vaaralahti K., Iivonen A.P., Miettinen P.J, & Raivio T. (2017). GnRH receptor gene mutations in adolescents and young adults presenting with signs of partial gonadotropin deficiency. PLoS ONE, 12(11), e0188750.

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Molecular Characterization of S. aureus Isolates

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We performed molecular characterization (MLST, spa typing, and PVL testing) on all 366 isolates. MLST was performed as previously described.^{7 (link)} Briefly, internal fragments of seven housekeeping genes were amplified by PCR and sequenced using the ABI Big Dye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems, Foster City, CA). MLST STs were assigned using the online S. aureus MLST database (www.mlst.net). MLST STs were clustered into MLST CCs using eBURST, with the minimum number of common alleles set at six of seven.^{6 (link)}spa typing was performed on isolates by amplifying and sequencing the spa repeat region using primers as previously described.^{13 (link)}spa sequence analysis was performed using Ridom StaphType (VERSION 2.0; Ridom GmbH, Würzburg, Germany), and the resulting spa types were then clustered into related spa CCs using the BURP algorithm with parameters set to exclude spa types that are shorter than five repeats and if costs were less than or equal to four.
We tested for the presence of the PVL genes using PCR amplification and gel electrophoresis using primers and PCR conditions as described previously.^{7 (link)}

O'Hara F.P., Suaya J.A., Ray G.T., Baxter R., Brown M.L., Mera R.M., Close N.M., Thomas E, & Amrine-Madsen H. (2016). spa Typing and Multilocus Sequence Typing Show Comparable Performance in a Macroepidemiologic Study of Staphylococcus aureus in the United States. Microbial Drug Resistance, 22(1), 88-96.

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Genetic Screening for Mitochondrial and Optic Neuropathy

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We first screened for the three primary mtDNA mutations (m.11778G>A, m.14484T>C m.3460G>A) in all 304 patients and then screened for mutations in OPA1 and OPA3 in the patients without mtDNA mutations utilizing the ABI BigDye Terminator cycle sequencing kit v3.1 and an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). All of the primers for OPA1 and OPA3 amplification and sequencing are listed in the S2 and S3 Tables.

Li Y., Li J., Jia X., Xiao X., Li S, & Guo X. (2017). Genetic and Clinical Analyses of DOA and LHON in 304 Chinese Patients with Suspected Childhood-Onset Hereditary Optic Neuropathy. PLoS ONE, 12(1), e0170090.

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Sanger Sequencing Validation of Genetic Variants

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Sanger sequencing was used to confirm the potential pathogenic variants detected by exome sequencing, including missense, nonsense, indels, and splice site variants. Primers used to amplify the sequence with each variant were either referred to in previous studies [13] (link),[15] (link),[17] (link) or designed by online tool Primer3 (http://frodo.wi.mit.edu/primer3/) (Table S1). The nucleotide sequences of amplicons were analyzed using an ABI BigDye Terminator cycle sequencing kit v3.1 (Applied Biosystems, Foster City, CA) on an ABI 3130 Genetic Analyzer (Applied Biosystems). Sequencing results were aligned with consensus sequences from the National Center for Biotechnology Information (NCBI) human genome database (http://www.ncbi.nlm.nih.gov/), using the SeqManII program of the Lasergene package (DNAStar Inc. Madison, WI). Confirmed variants were further sequenced in the available family members and 96 unrelated control individuals. The descriptions of the variants followed the nomenclature recommended by the Human Genomic Variation Society (HGVS; http://www.hgvs.org/mutnomen/). The effects of missense variations were evaluated by the PolyPhen-2 [18] (link) and SIFT [19] (link) online tools, and the effects of intronic variants on splicing site changes were predicted by the Berkeley Drosophila Genome Project (BDGP; http://www.fruitfly.org/seq_tools/splice.html[20] (link).

Sun W., Xiao X., Li S., Guo X, & Zhang Q. (2014). Exome Sequencing of 18 Chinese Families with Congenital Cataracts: A New Sight of the NHS Gene. PLoS ONE, 9(6), e100455.

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Genotyping RSV Strains via Hemi-nested PCR

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HVR2 region of the G gene was ampli ed using a Hemi-nested PCR as previously reported (Malekshahi et al. 2020) employing the primer pairs in Sato et al.'s method (Sato et al. 2005) which detected both the RSV A and B types (table2). To determine if the genotypes of RSV strains tested positive, the PCR products were sequenced using the ABI BigDye® Terminator Cycle Sequencing Kit v3.1, on the 3130 Genetic Analyzer Automated Sequencer (Applied Biosystems Foster City, California, USA).

Tavakoli F., Izadi A., Yavarian J., Sharifi-Zarchi A., Salimi V, & Mokhtari-Azad T. (2021). Determination of genetic characterization and circulation pattern of Respiratory Syncytial Virus (RSV) in children with a respiratory infection, Tehran, Iran, during 2018-2019. Virus research, 305.

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