Bigdye terminator v3.0 cycle sequencing ready reaction kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The BigDye Terminator v3.0 Cycle Sequencing Ready Reaction Kit is a reagent kit used for DNA sequencing. It contains the necessary components for performing cycle sequencing reactions, including labeled nucleotides and DNA polymerase. The kit is designed to work with automated DNA sequencing instruments.

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Lab products found in correlation

Abi 3130xl genetic analyzer, by Thermo Fisher Scientific (2 mentions) Architect instrument, by Abbott (1 mentions) Anti hbe, by Roche (1 mentions) Viral rna mini kit, by Qiagen (1 mentions) One step primescript rt pcr kit, by Takara Bio (1 mentions) Pmd18 t vector, by Takara Bio (1 mentions) Qiaex 2 gel extraction kit, by Qiagen (1 mentions) Abi 3100 genetic analyzer, by Thermo Fisher Scientific (1 mentions) Abi 3130xl genetic analyser, by Thermo Fisher Scientific (1 mentions) Revertra plus kit, by Toyobo (1 mentions) 3130 genetic analyser, by Thermo Fisher Scientific (1 mentions) In fusion hd cloning kit, by Takara Bio (1 mentions) Kod plus dna polymerase, by Toyobo (1 mentions) Instaclone pcr cloning kit, by Thermo Fisher Scientific (1 mentions) Escherichia coli top10, by Thermo Fisher Scientific (1 mentions)

6 protocols using bigdye terminator v3.0 cycle sequencing ready reaction kit

Hepatitis B Serological Profiling and Genotyping

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HBsAg and anti-HBc were run on the Architect instrument (Abbott, Germany) and values equal to or greater than 1.00 Sample/Cutoff (S/CO) were considered positive. Anti-HBs titres (Architect, Abbott, Germany) were reported as milli-international units per ml (mIU/ml) according to WHO international reference standard (Hollinger and Dreesman, 1986). Samples equal to or greater than 10mIU/ml were considered positive for anti-HBs. Samples positive for HBsAg were tested for HBeAg and anti-HBe (Architect, Abbot, Germany) with cutoff at 1.00 S/CO.
Hepatitis B viral load was performed on HBsAg positive samples using the COBAS AmpliPrep/COBAS TaqMan HBV Test, v2.0, quantitative assay (Roche Molecular Systems, New Jersey, USA). Sequencing was performed on the polymerase region (nucleotides 2624–1240), overlapping the complete S region (nucleotides 2848–2835) using BigDye Terminator v3.0 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) on the ABI 3130XL Genetic Analyzer (Applied Biosystems) [38 (link)]. Neighbour-joining phylogenetic analysis with a bootstrap of 1000 replicates was conducted using Molecular Evolutionary Genetics Analysis (MEGA 5)[39 (link)].

Prabdial-Sing N., Makhathini L., Smit S.B., Manamela M.J., Motaze N.V., Cohen C, & Suchard M.S. (2019). Hepatitis B sero-prevalence in children under 15 years of age in South Africa using residual samples from community-based febrile rash surveillance. PLoS ONE, 14(5), e0217415.

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Viral Gene Sequencing Protocol

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Subtyping of the HA and NA genes, and genotyping of the other six viral genes, was carried out by RT-PCR followed by sequencing. Briefly, RNA was extracted from the allantoic fluid using a Viral RNA Mini Kit (Qiagen, Hilden, Germany) and PCR amplification of the specific viral genes was carried out using a PrimeScript™ One-Step RT-PCR Kit (TaKaRa, Dalian, Japan). All viral genes were amplified with primers as described previously [19 (link)]. PCR products were purified using the QIAEX II Gel Extraction Kit (Qiagen) and cloned into the pMD18-T vector (TaKaRa). To ensure accuracy of the detected sequence, clones were sequenced using a Big Dye Terminator V.3.0 Cycle Sequencing Ready Reaction Kit (Applied Biosystems Inc., Foster City, CA, USA) for each cloned gene.

Xu H., Meng F., Huang D., Sheng X., Wang Y., Zhang W., Chang W., Wang L, & Qin Z. (2015). Genomic and Phylogenetic Characterization of Novel, Recombinant H5N2 Avian Influenza Virus Strains Isolated from Vaccinated Chickens with Clinical Symptoms in China. Viruses, 7(3), 887-898.

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Genetic Analysis of Fabry Disease

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Patients with persistent low plasma α-Gal A activity (< 3.0 nmol/hr/mL) were recalled for further testing and DNA analysis of the GLA gene. Whole blood was collected and sent for analysis to the Medical Genetics Clinic and Laboratory, Asan Medical Center, Seoul, Korea. All patients who underwent genetic testing had previously provided informed consent, as required according to the Korean Law on Gene Technology.
Genomic DNA was isolated from peripheral blood leukocytes using a Puregene DNA isolation kit (Gentra, Minneapolis, MN, USA). Seven GLA exons and their intronic flanking sequences were amplified by polymerase chain reaction (PCR) using seven sets of previously described primers, followed by single-strand conformational polymorphism analysis and direct sequencing.16 (link) DNA sequencing used the same primers as PCR with a BigDye Terminator V3.0 Cycle Sequencing Ready reaction kit (Applied Biosystems, Foster City, CA, USA). Electrophoresis and analysis of reactions was performed on an ABI 3100 Genetic analyzer (Applied Biosystems). If any variant of GLA gene was found in sequence analysis, we could explore in the documentations of the Human Gene Mutation Database (HGMD; http://www.hgmd.org), Clin Var (https://www.ncbi.nlm.nih.gov/clinvar/), and Genome Aggregation Database (gnomAD; http://gnomad.broadinstitute.org/).

Kim W.S., Kim H.S., Shin J., Park J.C., Yoo H.W., Takenaka T, & Tei C. (2019). Prevalence of Fabry Disease in Korean Men with Left Ventricular Hypertrophy. Journal of Korean Medical Science, 34(7), e63.

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Sanger Sequencing of Viral Genomes

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The amplicons were prepared for direct sequencing using the BigDye Terminator v3.0 Cycle Sequencing Ready Reaction Kit (Applied Biosystems., Foster City, USA) using second-round forward PCR primers. Sequencing was performed by the Central Analytical Facility, Stellenbosch University, South Africa, using the ABI 3130XL Genetic analyser (Applied Biosystems, Foster City, CA). 5’UTR and NS5B region sequences were analysed in the forward directions of a single fragment.

Oyaro M., Wylie J., Chen C.Y., Ondondo R.O, & Kramvis A. (2018). Human immunodeficiency virus infection predictors and genetic diversity of hepatitis B virus and hepatitis C virus co-infections among drug users in three major Kenyan cities. Southern African Journal of HIV Medicine, 19(1), 737.

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Molecular Cloning and Sequencing Protocols

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Escherichia coli strain XL10gold (Stratagene) was used to prepare plasmids. Ligation of DNA fragments was performed using an In-Fusion HD Cloning Kit (Clontech). PCR was performed using KOD Plus DNA polymerase (Toyobo). Reverse transcription-PCR (RT-PCR) was performed using a ReverTra -Plus- kit (Toyobo). DNA sequencing was performed using a BigDye Terminator v3.0 cycle sequencing ready reaction kit (Applied Biosystems) and analysed with a 3130 genetic analyser (Applied Biosystems).

Arita M, & Iwai-Itamochi M. (2019). Evaluation of antigenic differences between wild and Sabin vaccine strains of poliovirus using the pseudovirus neutralization test. Scientific Reports, 9, 11970.

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PCR Amplicon Cloning and Sequencing

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After nested PCR, the 307 nucleotide amplicon (1653–1959 from EcoR1 site) was gel-purified and cloned into pTZ57R/T vector (55 ng/µl) using Instaclone PCR Cloning Kit (Fermentas, Waltham, MA, USA), and transformed into TOP10 Escherichia coli (Invitrogen, Carlsbad, CA, USA). The transformants were grown on Ampicillin plates. Positive clones were identified by restriction fragment length polymorphism (RFLP) assay. At least 20 clones per sample were sequenced by direct sequencing, using a BigDye Terminator v3.0 Cycle Sequencing Ready Reaction Kit (Applied Biosystems., Foster City, USA) on an ABI 3130XL Genetic Analyzer (Applied Biosystems). The sequencing primer used was M13 forward (5′-GTAAAACGACGGCCAGT-3′). A phylogenetic tree was generated as described previously [17] (link). Clone sequences have been deposited in GenBank: Genotype D: KJ496256-KJ496297, Genotype E: KJ496206-KJ496255.

Yousif M., Bell T.G., Mudawi H., Glebe D, & Kramvis A. (2014). Analysis of Ultra-Deep Pyrosequencing and Cloning Based Sequencing of the Basic Core Promoter/Precore/Core Region of Hepatitis B Virus Using Newly Developed Bioinformatics Tools. PLoS ONE, 9(4), e95377.

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