Big dye terminator version 3.1 kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Big Dye Terminator version 3.1 kit is a reagent system used for DNA sequencing. The kit contains the necessary components, including fluorescently labeled dideoxy terminators, to perform Sanger sequencing reactions.

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11 protocols using big dye terminator version 3.1 kit

Genotyping of IL-10 SNPs in Blood

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Genomic DNA was isolated from 300 μL EDTA-treated whole blood using a Commercial kit (Wizard genomic DNA purification kit, Promega, WI, USA). The procedure was carried according to the kit manufacturer's recommendation.
Genotyping analysis for detection of 3 SNPs of IL-10s was performed for all patients by using specific PCR primers. Table 1 describes primers used and PCR conditions. PCR was performed in a total volume of 25 μL using 100 ng of genomic DNA with 1.5 μL of 10 μmol/L of each primer and 12.5 μL of 2X KAPA2G Fast ReadyMix PCR Kit (Kappa Biosystems, USA). PCR amplifications were performed in PTC-100 Peltier Thermal Cycler (MJ Research, MA, USA).
PCR reaction products were sequenced using Big Dye Terminator version 3.1 kit (Applied Biosystems, Waltham, MA, USA). Samples were run on an ABI Prism Genetic Analyzer system 3130xl (Applied Biosystems, Waltham, MA, USA).

Khaleel B., Yousef A.M., Al-Zoubi M.S., Al-Ulemat M., Masadeh A.A., Abuhaliema A., Al-Batayneh K.M, & Al-Trad B. (2022). Impact of genetic polymorphisms at the promoter area of IL-10 gene on tacrolimus level in Jordanian renal transplantation recipients. Journal of Medical Biochemistry, 41(3), 327-334.

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Verification of PCR Amplicons by Sequencing

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RT-PCR was carried out in a Thermal Cycler (Applied Biosystems, Foster City, CA) in order to verify the three PCR amplicons (SsrA, RsaC and RNAIII) by agarose gel and DNA sequencing. The PCR was performed in a 20 μL reaction, containing gene-specific primers mentioned above and DreamTaq Green PCR Master Mix (Thermo Fischer Scientific, USA) according to the manufacturer's instruction. One μL of cDNA was used as the template. The cycling conditions were performed as follows: after an initial denaturation step of 2 min at 95 °C, 40 cycles were performed for 30 s at 95 °C, 60 s at 60 °C, and 1 min at 72 °C. A final extension step for 10 min at 72 °C was used. PCR products were further separated on a 1% agarose gel, stained with GelRed and visualized using Syngen Gel Imaging (Bio-Rad Laboratories Inc, USA). The PCR product was cleaned using PCR Clean-Up Kit (Promega, Norway). Sequencing reactions were performed in using a BigDye Terminator version 3.1 kit (Applied Biosystems) according to the manufacturer's instructions with the same primers as for the real-time PCR assay. Sequencing was performed on an Applied Biosystems 3,130 × l genetic analyzer.

Joshi B., Singh B., Nadeem A., Askarian F., Wai S.N., Johannessen M, & Hegstad K. (2021). Transcriptome Profiling of Staphylococcus aureus Associated Extracellular Vesicles Reveals Presence of Small RNA-Cargo. Frontiers in Molecular Biosciences, 7, 566207.

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Genetic Screening for Neuromuscular Disorders

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Genomic DNA was extracted from leukocytes by standard methods. At the beginning of the investigations, 13 patients were tested for congenital myotonia (CLCN1, at least the 3 most common mutations in Finland), 14 for DM2 (CNBP repeat expansion mutation), and 2 for DM1 (DMPK repeat expansion mutation). There was a heterozygous known recessive mutation in CLCN1 gene in 2 patients, whereas other results were negative. For all patients but one (P13), the whole coding sequence of exon 19 of SCN4A was screened. The region studied was amplified by PCR and directly sequenced with the Big-Dye Terminator version 3.1 kit on an ABI3130xl automatic DNA sequencer system (Applied Biosystems, Foster City, CA). Sequences were analyzed with Sequencher 5.1 software (Gene Codes Corporation, Ann Arbor, MI). The primers used are available on request. For P13, the genetic analysis was performed using targeted next-generation sequencing as previously described^{12 (link)} with version 2 of the MYOcap gene panel that is targeted to the exons of 236 genes known or predicted to cause muscular dystrophy or myopathy.

Palmio J., Sandell S., Hanna M.G., Männikkö R., Penttilä S, & Udd B. (2017). Predominantly myalgic phenotype caused by the c.3466G>A p.A1156T mutation in SCN4A gene. Neurology, 88(16), 1520-1527.

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Amplification and Sequencing of XRCC Genes

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The PCR amplifications targeting the XRCC1-exon-10, XRCC3-exon-7 and XRCC3-5’-UTR regions were performed using specific primers based on the XRCC1 and XRCC3 sequences obtained from the National Center for Biotechnology Information (NCBI) (Table 1). The PCR amplification was performed in 30 µl reaction volume that contained (75 mM Tris-HCl, 1.5 mM MgCls, 50 mM KCl, 20 mM (NH4) 2SO₄, 0.2 mM of each primer and 1 U of Taq DNA polymerase). Polymerase chain reactions were conducted under the following cycling conditions: an initial 7 minutes of denaturation at 95°C followed by 45 cycles for 45 seconds each at 94°C, 59°C, 72°C for 1 minute, and a single final extension step for 10 minutes at 72°C. Direct DNA sequencing was performed using Big Dye Terminator version 3.1 kit (Applied Biosystems, Waltham, MA, USA). Samples were run on an ABI Prism Genetic Analyzer system 3130xl (Applied Biosystems, Waltham, MA, USA).

, & Zoubi M.S. (2015). X-ray repair cross-complementing protein 1 and 3 polymorphisms and susceptibility of breast cancer in a Jordanian population. Saudi Medical Journal, 36(10), 1163-1167.

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Bisulfite PCR and Sequencing of miR31 Promoter

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A GpC site in the promoter locus of the miR31 host gene (LOC554202), which was previously demonstrated [21] (link), was PCR-amplified using bisulfate-treated genomic DNA as a template, according to the method described elsewhere [21] (link). The PCR product was sub-cloned into the pT7 blue plasmid vector (Novagen, Darmstadt, Germany), and was then subjected to a cycle dye-terminator reaction with the universal T7 promoter primer using the Big Dye Terminator Version 3.1 kit (Applied Biosystems, Foster City, CA).

Okudela K., Tateishi Y., Umeda S., Mitsui H., Suzuki T., Saito Y., Woo T., Tajiri M., Masuda M., Miyagi Y, & Ohashi K. (2014). Allelic Imbalance in the miR-31 Host Gene Locus in Lung Cancer - Its Potential Role in Carcinogenesis. PLoS ONE, 9(6), e100581.

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Pestivirus Detection in Chamois

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Viral RNA was extracted from chamois spleen tissues using a commercial RNA extraction kit (Nucleospin Viral RNA Isolation, Macherey Nagel, Düren, Germany). Reverse transcription and PCR assays targeting 5’UTR region of pestivirus were performed using 324 and 326 pan-pestivirus primers [37 (link)] following previously described protocols [32 (link)]. For each sample, amplicons of the expected size were purified and sequenced using forward and reverse primers by cycle sequencing using a Big Dye Terminator version 3.1 kit and an ABI PRISM 3130xl sequencing device (Applied Biosystems, CA, USA).
All sequences from chamois and domestic hosts were aligned with BDV reference strains, retrieved from GenBank and representative of BDV phylogenetic groups according to [12 (link)], using CLUSTALW (integrated within the Bio-Edit sequence editor, freely available at http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Phylogeny was preliminary estimated by the neighbor-joining algorithm (NJ) and the maximum likelihood (ML) method.

Luzzago C., Ebranati E., Cabezón O., Fernández-Sirera L., Lavín S., Rosell R., Veo C., Rossi L., Cavallero S., Lanfranchi P., Marco I, & Zehender G. (2016). Spatial and Temporal Phylogeny of Border Disease Virus in Pyrenean Chamois (Rupicapra p. pyrenaica). PLoS ONE, 11(12), e0168232.

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Sequencing for Discordant TB Resistance

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Sanger sequencing was performed whenever results between MTBDRplus and phenotypic DST remained discordant upon repeating both tests. Isolates showing conflicting results for INH had the mabA-inhA regulatory region (positions -168 to 80, relative to codon) amplified and sequenced with primers mabA-inhAF and mabA-inhAR,¹⁶⁾ as well as the entire inhA and katG genes by using the primer pairs inhA3 and inhA4, inhA3F and inhA5R, and the forward and reverse primers katG-P4, -P5, -P6, -P7 and -P8.¹⁷ For isolates with RIF-discordant results, primers RPOB-1 and RPOB-2¹⁸⁾ were used to amplify and sequence a 350-bp fragment of rpoB encompassing the RIF resistance-determining region.
Single PCR included 12.5 µL of PrimeSTAR Max DNA Polymerase (Takara Bio, Shiga, Japan), 5 pmol of primers for mabA-inhA and katG, 10 pmol of primers for inhA and rpoB, 2 µL of DNA template and PCR-grade water for a final volume of 25 µL. Amplification comprised 30 cycles of 98 °C for 10 seconds, 55 °C for 5 seconds, and 72 °C for 20 seconds. Amplimers purified with ExoSAP-it (Affymetrix, SCL, CA, USA) were sequenced with an ABI 3130xL Genetic Analyzer and the BigDye Terminator version 3.1 Kit (Applied Biosystems, FSTC, CA, USA). Sequences were aligned and analysed using the BioEdit v7.2.5 software¹⁹ and the web-based MUBII-TB-DB²⁰ and BLAST²¹ tools.

Brandao A.P., Pinhata J.M., Oliveira R.S., Galesi V.M., Caiaffa-Filho H.H, & Ferrazoli L. (2019). Speeding up the diagnosis of multidrug-resistant tuberculosis in a high-burden region with the use of a commercial line probe assay. Jornal Brasileiro de Pneumologia, 45(2), e20180128.

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DNA Purification and Sequencing

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The PCR products were purified from the agarose gel by using Nucleospin @ Extract II kit (Macherey-Nagel, Germany) as per manufacturer's recommended protocol. DNA (200 ng of gel-purified product) was used with the Big Dye Terminator kit (version 3.1) (Applied Biosystem, Foster City, CA, USA) for the sequencing PCR. The template was purified and sequenced on a 3130xl Genetic Analyzer (Applied Biosystem, Foster City, CA, USA).

Fredrick T., Ponnaiah M., Murhekar M.V., Jayaraman Y., David J.K., Vadivoo S, & Joshua V. (2015). Cholera Outbreak Linked with Lack of Safe Water Supply Following a Tropical Cyclone in Pondicherry, India, 2012. Journal of Health, Population, and Nutrition, 33(1), 31-38.

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DNA Sequencing Protocol Optimization

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Amplified fragments were firstly purified using the illustra ExoProStar1-Step (GE Healthcare Life Sciences). Direct sequencing of amplicons was performed using Big Dye Terminator kit (version 3.1) (Applied Biosystem, Foster City, CA, USA) that includes dideoxynucleotides labelled with four fluorochromes of different colours. For each PCR product, both strands were sequenced, in independent reactions, using the mentioned above primers. The resulting chromatograms were manually edited to ensure sequence accuracy and added to the alignment component of MEGA 5 software.

Oudghiri A., Karimi H., Chetioui F., Zakham F., Bourkadi J.E., Elmessaoudi M.D., Laglaoui A., Chaoui I, & El Mzibri M. (2018). Molecular characterization of mutations associated with resistance to second-line tuberculosis drug among multidrug-resistant tuberculosis patients from high prevalence tuberculosis city in Morocco. BMC Infectious Diseases, 18, 98.

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Sanger Sequencing for Variant Validation

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Automated Sanger sequencing was carried out to confirm variants of interest and for segregation analysis when familial samples were available. Polymerase chain reaction–amplified regions (primer sequences are available on request) were purified enzymatically with Illustra ExoProStar (GE Healthcare Life Sciences) and sequenced using a BigDye Terminator, version 3.1 kit (Thermo Fisher Scientific) on an ABI 3130x1 automated DNA sequencer (Thermo Fisher).

Rocha-Braz M.G., França M.M., Fernandes A.M., Lerario A.M., Zanardo E.A., de Santana L.S., Kulikowski L.D., Martin R.M., Mendonca B.B, & Ferraz-de-Souza B. (2020). Comprehensive Genetic Analysis of 128 Candidate Genes in a Cohort With Idiopathic, Severe, or Familial Osteoporosis. Journal of the Endocrine Society, 4(12), bvaa148.

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