Bigdye

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

BigDye is a DNA sequencing technology developed by Thermo Fisher Scientific. It is a reagent system used for DNA sequencing applications in molecular biology laboratories. The core function of BigDye is to facilitate the determination of the nucleotide sequence of DNA molecules.

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70 protocols using bigdye

Genetic Etiology of Deafness in Family PKDF063

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Family PKDF063 includes 153 individuals of which 39 individuals were consented for participation in our study. Hearing loss segregating in members of family PKDF063 was noted during their first year after birth by their mothers. Bilateral severe to profound deafness was confirmed by an audiologist. Although not an objective evaluation, parents said that their deaf children, including the PS proband, have normal intelligence and motor development.
Genomic DNA was extracted from peripheral blood leukocytes. One affected individual and the proband were initially screened by di-deoxy sequencing using BigDye (Applied Biosystems, Foster City, CA, USA) for pathogenic variants in CLPP, one of the five reported PS genes. We also screened for variants of GJB2 (DFNB1A, MIM 220290) and HGF (DFNB39, MIM 608265) since in Pakistan mutant alleles of these two genes are common causes of deafness. WES was performed with genomic DNA from five individuals (four affected; one unaffected) of family PKDF063 (Fig. 1) using a Nextera Rapid Capture Exome kit and a HiSeq1500 instrument (Illumina San Diego, CA, USA). Computational analyses used a GATK (Genome Analysis Toolkit) pipeline followed by variant calls that were annotated with Annovar v2014_07_14. Short-listed variants were verified by di-deoxy sequencing using an ABI3730XL genetic analyzer (Applied Biosystems).

Faridi R., Rehman A.U., Morell R.J., Friedman P.L., Demain L., Zahra S., Khan A.A., Tohlob D., Assir M.Z., Beaman G., Khan S.N., Newman W.G., Riazuddin S, & Friedman T.B. (2016). Mutations of SGO2 and CLDN14 collectively cause coincidental Perrault syndrome. Clinical genetics, 91(2), 328-332.

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BigDye Cycle Sequencing Reaction Protocol

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The sequencing reactions were carried out in a mixture containing 3.25 µL water, 1.75 µL 5× BigDye buffer (Applied Biosystems), 1 µL of each primer (5 pmol/µL), 2 µL of the PCR product, and 2 µL BigDye (Applied Biosystems). The cycle sequencing reaction was initiated at 96 °C for 1 min, followed by 40 cycles of denaturation at 96 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 4 min. The sequencing reaction product was precipitated with 1 µL 125 mM EDTA, 1 µL 3 M sodium acetate, and 25 µL 100 % ethanol. After homogenization, the solution was left to stand for 15 min and then centrifuged at 3000g for 15 min at 4 °C. The supernatant was removed by inverting the tube and 35 µL 70 % ethanol was added. The solution was centrifuged at 1650g for 15 min at 4 °C. After removal of the supernatant by inversion, 10 µL HiDi formamide (Applied Biosystems) was added and the mixture was left to stand for 5 min at 95 °C and for 2 min on ice. The product was run on an 8-capillary ABI 3500 sequencer (50 cm) using POP7 polymer (Applied Biosystems).

Monteiro A.C., Fortaleza C.M., Ferreira A.M., Cavalcante R.D., Mondelli A.L., Bagagli E, & da Cunha M.D. (2016). Comparison of methods for the identification of microorganisms isolated from blood cultures. Annals of Clinical Microbiology and Antimicrobials, 15, 45.

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Site-Directed Mutagenesis Protocol for S. oneidensis prpF

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Amplification conditions for site-directed mutagenesis were as follows: 95°C for 1 min, followed by 19 cycles of 95°C for 50 s, 60°C for 50 s, 68°C for 6 min 15 s, ending with 68°C for 7 min. All plasmids carrying mutant S. oneidensis prpF alleles were sequenced using two described primers [19 (link)]. DNA sequencing reactions were performed using BigDye® (Applied Biosystems), were purified using CleanSEQ protocols (Agentcourt Biotechnology), and were resolved at the University of Wisconsin-Madison Biotechnology Center.

Rocco C.J., Wetterhorn K.M., Garvey G.S., Rayment I, & Escalante-Semerena J.C. (2017). The PrpF protein of Shewanella oneidensis MR-1 catalyzes the isomerization of 2-methyl-cis-aconitate during the catabolism of propionate via the AcnD-dependent 2-methylcitric acid cycle. PLoS ONE, 12(11), e0188130.

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Amplification and Sequencing of Ocular Genes

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gDNA was diluted to 20 ng/ul concentration and PCR amplification of coding regions of all four genes was performed using primers flanking exons following a protocol used earlier [34 (link)]. Primer sequences are available on request. Bidirectional sequencing of all fragments was carried out using BigDye (Applied Biosystems, Foster city, CA) chain termination chemistry. Fragments were then separated on AB 3500 genetic analyzer (Life Technologies). All sequenced fragments were analyzed using BioEdit software (http://www.mbio.ncsu.edu/bioedit/bioedit.html) and compared to the reference sequences of corresponding genes from UCSC genome browser (http://genome.ucsc.edu/cgi-bin/hgGateway).
In house controls were used and it was ensured that controls are healthy individuals without having any ocular disease(s) or previous ophthalmic surgeries.

Al-Barry M.A., Albalawi A.M., Sayf M.A., Badawi A., Afzal S., Latif M., Samman M.I, & Basit S. (2016). Sequence analysis of four vitamin D family genes (VDR, CYP24A1, CYP27B1 and CYP2R1) in Vogt-Koyanagi-Harada (VKH) patients: identification of a potentially pathogenic variant in CYP2R1. BMC Ophthalmology, 16, 172.

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Tau Protein Mutant Generation

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MARK2/Par1-myc was kindly provided by B. Lu (Stanford University School of Medicine, Stanford, CA), whereas GFP and wild-type V5-tagged tau (4R0N isoform) constructs for mammalian expression were cloned into pcDNA3.1 in our laboratory (31 (link), 36 (link)). The QuikChange mutagenesis kit (Agilent Technologies, Santa Clara, CA) was used to generate K163Q, K174Q, K180Q, K234Q, K240Q, K259Q, K274Q, K280Q, K290Q, K311Q, K321Q, K353Q, K369Q, K290Q/K321Q, K259Q/K290Q/K321Q/K353Q, K321R, S324A, S324D, and S324E mutants from the wild-type tau-V5 parent construct, following the manufacturer's protocol. For protein expression in bacteria, wild-type and mutant tau constructs were cloned into the pET30a vector (EMD Millipore, Billerica, MA). All constructs were sequence-verified using ABI3730 with Big Dye chemistry following the manufacturer's protocols (Applied Biosystems, Foster City, CA).

Carlomagno Y., Chung D.E., Yue M., Castanedes-Casey M., Madden B.J., Dunmore J., Tong J., DeTure M., Dickson D.W., Petrucelli L, & Cook C. (2017). An acetylation–phosphorylation switch that regulates tau aggregation propensity and function. The Journal of Biological Chemistry, 292(37), 15277-15286.

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Sanger Sequencing of SNV Loci

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Sanger sequencing analysis was conducted to confirm the location of specific SNVs in the detected genes. PCR was performed using the PrimeSTAR Max DNA polymerase system (Takara Bio, Kusatsu, Japan). Thereafter, PCR-amplified products were extracted using the QIAquick Gel Extraction Kit (QIAGEN). Sequencing in the reverse direction was undertaken according to the manufacturer’s instructions (BigDye; Applied Biosystems, Warrington, UK). Sequencing of the products was performed using the ABI 3500 automated DNA sequencer (Applied Biosystems).

Mihara Y., Hirasaki M., Horita Y., Fujino T., Fukushima H., Kamakura Y., Uranishi K., Hirano Y., Ryozawa S., Yasuda M., Makino Y., Shibazaki S, & Hamaguchi T. (2023). PTEN-induced kinase 1 gene single-nucleotide variants as biomarkers in adjuvant chemotherapy for colorectal cancer: a retrospective study. BMC Gastroenterology, 23, 339.

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FMR1 Promoter CGG Repeat Sequencing

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Primers covering the FMR1 promoter region (Additional file 1: Table S1) were designed to amplify the CGG repeat segment. A modified protocol containing PCR enhancer solution (Life Technology, Grand Island, NY, USA) was used to amplify this GC-rich region. The PCR products were purified (Exonuclease I, New England Biolabs, Ipswich, MA, USA) and sequenced using the standard protocol (BigDye, Applied Biosystems, Foster City, CA, USA). Raw sequences were visualized by Mutation Surveyor V3.30 (SoftGenetics, State College, PA, USA) and blasted to the human reference sequence (http://genome.ucsc.edu/, hg19) to determine the CGG repeat number.

Chen X., Wang J., Xie H., Zhou W., Wu Y., Wang J., Qin J., Guo J., Gu Q., Zhang X., Ji T., Zhang Y., Xiong Z., Wang L., Wu X., Latham G.J, & Jiang Y. (2015). Fragile X syndrome screening in Chinese children with unknown intellectual developmental disorder. BMC Pediatrics, 15, 77.

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Sanger Sequencing of PCR Amplicons

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Amplicons were characterised through DNA Sanger sequencing. The PCR amplicons were purified using 6% Sephadex G-50 gel filtration (Merck KGaA, Darmstadt, Germany). The purified products were visualized on an agarose gel using the protocol described above. For sequencing, we used a 10 µL sequencing reaction volume containing 5.5 µL of PCR grade water, 1 µL of BigDye™ (Applied BioSystems, Foster City, USA), 1 µL of sequencing buffer, 0.5 µL of primer diluted to 10 µM and 2 µL of purified PCR product. The cycling conditions included one cycle at 96 °C for 2 min, followed by 30 cycles of 30 s at 96 °C, 15 s at 50 °C and 4 min at 60 °C. Cycle sequencing products were purified using Sephadex G-50 gel filtration. Sequencing was performed on the ABI Prism™ 3500xl automated DNA sequencer (Applied Biosystems USA, Foster City, California, USA) at the University of Pretoria sequencing facility. The reverse and forward sequences obtained were aligned on CLC Main Workbench 8 (Qiagen, Hilden, Germany) and the consensus sequence was used for a BLASTn analysis [68 (link)] against the NCBI nucleotide database [69 (link)].

Queffelec J., Postma A., Allison J.D, & Slippers B. (2022). Remnants of horizontal transfers of Wolbachia genes in a Wolbachia-free woodwasp. BMC Ecology and Evolution, 22, 36.

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Sequencing of Androgen Receptor Coding Region

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We have sequenced the coding region of AR for each patient studied. The reaction mixture for sequencing contained: 15–30 ng of DNA, 1 µL of AR primer (20 µM), 2 µL of BigDye (5x) buffer, and BigDye Terminator v3.1 (Applied Biosystems Life Technologies, Carlsbad, CA, USA) in a final volume of 20 µL. The primer pairs used for the sequencing reaction are listed in Table S2. The products were separated on an ABI Prism 310 (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). Changes in the patient’s sequenced AR fragments were identified with respect to the reference DNA (made available in the NCBI database) using the CLC program Workbench 6.0. The AR sequence of each given individual were compared to the NCBI Reference Sequence NM_000044.3.

Malcher A., Jedrzejczak P., Stokowy T., Monem S., Nowicka-Bauer K., Zimna A., Czyzyk A., Maciejewska-Jeske M., Meczekalski B., Bednarek-Rajewska K., Wozniak A., Rozwadowska N, & Kurpisz M. (2019). Novel Mutations Segregating with Complete Androgen Insensitivity Syndrome and Their Molecular Characteristics. International Journal of Molecular Sciences, 20(21), 5418.

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DNA Extraction, Purification, and Sequencing

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Genomic DNA was purified using Masterpure™ complete DNA/RNA purification kit (Epicentre, Cambio Ltd., Cambridge) and plasmids were purified using E.Z.N.A^® plasmid mini kit (Omega Bio-tek, Inc., Norcross, GA). The DNA concentration was measured using NanoDrop™ 2000c spectrophotometer (Thermo Scientific, DE, USA). The PCR amplification was performed using Phusion^® polymerase (Thermo Fisher Scientific, Waltham, MA, USA) or Taq polymerase (Sigma, St. Louis, MO, USA) in a Arktik™ thermal cycler (Thermo Fisher Scientific, USA). Restriction digestion using XhoI and SpeI restriction enzymes and DNA ligation using T4 DNA ligase were performed as recommended by the manufactures and were obtained from New England Biolabs (Ipswich, MA, USA). The PCR products and the digested DNA fragments were separated using agarose gel electrophoresis and extracted using Montage^® gel extraction kit (Millipore, MA, USA). DNA sequencing was performed using Big Dye (Applied biosystems, CA, USA). The primers used in the PCR and sequencing reactions were synthesized by Sigma-Aldrich and are listed in Table 1.

Maharajan A.D., Hansen H., Khider M, & Willassen N.P. (2021). Quorum sensing in Aliivibrio wodanis 06/09/139 and its role in controlling various phenotypic traits. PeerJ, 9, e11980.

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