Megabace500

Manufactured by GE Healthcare

Sourced in United Kingdom

The MegaBACE500 is a high-throughput DNA sequencing system designed for large-scale genomic research. It is capable of processing multiple DNA samples simultaneously, generating accurate and reliable sequencing data. The MegaBACE500 utilizes capillary electrophoresis technology to separate and detect DNA fragments, providing efficient and cost-effective DNA sequencing capabilities.

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Lab products found in correlation

Abi3700, by Thermo Fisher Scientific (1 mentions) Abi 3100, by Thermo Fisher Scientific (1 mentions) Taqman fluorogenic 5 nuclease assay, by Thermo Fisher Scientific (1 mentions) Taq polymerase, by Solgent (1 mentions) Ptc 200 thermal cycler, by Bio-Rad (1 mentions) Dyenamic et dye terminator kit, by GE Healthcare (1 mentions) Illustra gfx pcr dna and gel band purification kit, by GE Healthcare (1 mentions) Pyromark assay design software 2, by Biotage (1 mentions) Abi prism 3100, by Thermo Fisher Scientific (1 mentions) Type it hrm pcr kit, by Qiagen (1 mentions)

6 protocols using megabace500

Molecular Detection of ESBL Genes

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The presence of bla_TEM, bla_SHV and bla_CTX-M genes in phenotypically confirmed ESBL-producing isolates was checked by PCR using the primers and conditions described elsewhere [18 (link)] (Supplementary Table S1).
Amplified products were purified and both strands were sequenced in a Mega-BACE 500 sequencer (GE Healthcare Life Sciences, UK). DNA sequences were compared with curated sequences contained in the Bacterial Antimicrobial Resistance Reference Gene Database (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA313047; accessed on 29 September 2021).

Pintor-Cora A., Álvaro-Llorente L., Otero A., Rodríguez-Calleja J.M, & Santos J.A. (2021). Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae in Fresh Produce. Foods, 10(11), 2609.

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BRCA1/2 Mutation Analysis in Cases

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Genomic DNA was extracted from the participants’ peripheral blood samples. For BRCA1/2 mutation testing in cases, 22 coding exons of BRCA1 and 26 coding exons of BRCA2 were scanned through fluorescence-based conformation sensitive gel electrophoresis (F-CSGE) and denaturing high-performance liquid chromatography (DHPLC). For a subset of PCR products with aberrant patterns, direct sequencing was performed on an ABI3100 or ABI3700 (Applied Biosystems, CA) or a MegaBACE500 (GE Healthcare, UK) genetic analyzer. In this study, the definition of a genetic mutation is restricted to the protein-truncating mutation and the missense mutation, which are known to be associated with the disease. This study also includes the participants with a clinically unverified mutation in BRCA1/2 genes. More detailed information about BRCA1/2 mutation analysis has been described before^{31 (link),37 (link)}. Since this study also includes the cases with unverified mutation on BRCA1/2, we examined whether non-pathogenic variants on these genes are more frequent in the cases compared to controls at p-value of 0.05. And we searched them at the ClinVar database.

Lee J.Y., Kim J., Kim S.W., Park S.K., Ahn S.H., Lee M.H., Suh Y.J., Noh D.Y., Son B.H., Cho Y.U., Lee S.B., Lee J.W., Hopper J.L, & Sung J. (2018). BRCA1/2-negative, high-risk breast cancers (BRCAX) for Asian women: genetic susceptibility loci and their potential impacts. Scientific Reports, 8, 15263.

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Genotyping of HO-1 Genetic Variants

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Genomic DNA was isolated from peripheral blood lymphocytes. The genotyping of the HO-1 promoter polymorphism T(-413)A (rs2071746) was performed using the TaqMan fluorogenic 5′ nuclease assay (ABI, Foster City, CA) as previously described.^{19 (link)} Forty-eight duplicate samples and negative controls were included to ensure the accuracy of genotyping, and 100% of the duplicates replicated the original genotype. The rate of successful genotyping was 96.5% for rs2071746 and 98.2% for rs3761439.
The length of the (GT)_n microsatellite polymorphism in the HO-1 gene promoter was determined as previously described with slight modification.^{20 (link)} The (GT)_n repeat segment was amplified in a volume of 20 μL containing 50 ng of genomic DNA, 0.5 pmol of each primer, 1× polymerized chain reaction (PCR) buffer, 200 μM dNTP, 1.5 mM MgCl₂, 1× Band Doctor, and 0.5 U Taq polymerase (Solgent, Daejeon, Korea). The reaction consisted of denaturation at 95°C for 2 min, followed by 32 cycles of 95°C for 20 sec, 53°C for 40 sec, and 72°C for 1 min, with a final extension at 72°C for 5 min using PTC-200 thermal cycler (Bio-Rad, Hercules, CA). The PCR products were analyzed by capillary on a MegaBACE500 (GE Healthcare, Piscataway, NJ) together with an allelic ladder after denaturation at 96°C for 5 min.

Lee E.Y., Lee Y.H., Kim S.H., Chung K.S., Kwon O., Kim B.S., Nam C.M., Park C.S., Lee B.W., Kang E.S., Cha B.S, & Lee H.C. (2015). Association Between Heme Oxygenase-1 Promoter Polymorphisms and the Development of Albuminuria in Type 2 Diabetes: A Case–Control Study. Medicine, 94(43), e1825.

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Screening for Mutations in Cancer Genes

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In order to screen for potential mutations in PIK3CA (exons 9 and 20), PTEN (exons 5–8), KRAS (exons 2 and 3), and BRAF (exons 11 and 15), radiolabelling for SSCA was performed. For the radiolabelling, a second PCR was performed on MJ Research PTC-200 (GMI, Ramsey, MN) in which 1 μL of the primary PCR product was amplified for 12–15 cycles in the presence of 1.0 μCi of [α-³²P]-dATP (3000 Ci/mmol) (PerkinElmer Sverige AB, Upplands Väsby, Sweden). Samples were thereafter diluted 1:10 with denaturing loading solution (98% formamide, 9.8 mmol/L EDTA (pH 8.0), 0.098% bromphenol blue and xylene cyanol). Following dilution, samples were denatured at 95°C for 3 min and loaded onto a 6% polyacrylamide gel and/or MDE™ gel (Bio-Rad, Hercules, CA and Cambrex Bioscience Rockland Inc., Rockland, ME, respectively). Fragments were separated at 3–6 W constant power at room temperature for 15–17 h. Gels were transferred to 3 mm paper, dried and autoradiographed for 5–48 h. Shifted fragments were excised, reamplified, and purified for sequencing on a MegaBACE™ 500 (GE Healthcare Bio-Sciences AB). In order to label samples, a DYEnamic™ ET Dye Terminator kit (GE Healthcare Bio-Sciences AB) was used according to the manufacturer's protocols.

Stjernström A., Karlsson C., Fernandez O.J., Söderkvist P., Karlsson M.G, & Thunell L.K. (2014). Alterations of INPP4B, PIK3CA and pAkt of the PI3K pathway are associated with squamous cell carcinoma of the lung. Cancer Medicine, 3(2), 337-348.

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HPV Genotype Identification Sequencing

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The HPV DNA-positive samples were sequenced for identification of the present genotypes. The products obtained from nPCR were purified using the Illustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Health Care) and sequenced on a MegaBACE 500 (GE Healthcare).

Entiauspe L.G., Seixas F.K., Nunes E.M., Rodrigues F.M., Dellagostin O.A., Collares T, & da Silveira M.F. (2014). Uncommon non-oncogenic HPV genotypes, TP53 and MDM2 genes polymorphisms in HIV-infected women in Southern Brazil. The Brazilian Journal of Infectious Diseases, 18(6), 643-650.

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Genetic Profiling of RET Gene

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Genomic DNA was extracted from peripheral blood using guanidine isothiocyanate and following
phenol-chloroform extraction using a standard protocol (Słomski 2008) . Isolates were dissolved in 1× TE buffer and stored at -20°C until analysis. Then, SNPs in the RET gene were analyzed by pyrosequencing or high-resolution melting analysis (HRMA) and Sanger sequencing. Primers were designed using PyroMark Assay Design Software 2.0 (Biotage) and Primer 3 online primer design tool (all primer sequences and melting temperatures are listed in Supplementary Table 2). Templates for pyrosequencing were amplified by PCR as follows: a 5-min initial denaturation at 94°C, then 50 cycles of 94°C for 30 s, annealing for 30 s and 72°C for 60 s, followed by a 5-min final elongation at 72°C. Pyrosequencing reactions were performed on the PSQ96 apparatus using PyroMark Gold Q96 reagents kits (Qiagen) according to the manufacturer's recommendations. HRM analyses were run on the Rotor-Gene Q cycler using the Type-it HRM PCR Kit (Qiagen). The DNA fragments containing the RET gene exons were amplified by PCR (5 min at 95°C, then 40 cycles as follows: 95°C for 10 s, 60°C for 30 s and 72°C for 10 s) and the products were subsequently melted from 75 to 95°C by raising the temperature by 0.1°C at each step. Sanger sequencing was performed on MegaBACE 500 (GE Healthcare) or ABI PRISM 3100 (Applied Biosystems).

Kaczmarek-Ryś M., Ziemnicka K., Pławski A., Budny B., Michalak M., Hryhorowicz S., Hoppe-Gołębiewska J., Boruń P., Gołąb M., Czetwertyńska M., Sromek M., Szalata M., Ruchała M, & Słomski R. (2018). Modifying impact of RET gene haplotypes on medullary thyroid carcinoma clinical course. Endocrine-related cancer, 25(4).

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