Abi prism 377 dna sequencer

Manufactured by Thermo Fisher Scientific

Sourced in United States, Malaysia, Germany, United Kingdom

The ABI PRISM 377 DNA Sequencer is a laboratory instrument used for DNA sequencing. It employs the Sanger sequencing method to determine the order of nucleotides in DNA samples. The core function of the ABI PRISM 377 is to generate DNA sequence data.

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83 protocols using abi prism 377 dna sequencer

Microsatellite and OPA1 Gene Analysis Protocol

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DNA was extracted from blood by a standard salting out method. For amplification of microsatellite markers (D3S3642, D3S3590, D3S2305, D3S3562, D3S2748) PCR primer sequences were retrieved from the Ensembl Genome Browser (http://www.ensembl.org). Forward primers were 5’labeled with 6-carboxyfluorescein (6-FAM) and reverse primers were unlabeled. PCR products were mixed with a fluorescent size marker (Applied Biosystems, Foster City, CA, USA) and analyzed on ABI PRISM 377 DNA sequencer (Applied Biosystems). Fragment lengths were analyzed with the Gene Mapper software (Applied Biosystems).
For the amplification of all OPA1 exons PCR primers were designed with the Primer 3 Plus public domain software (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi) and the OPA1 gene NG_011605.1 reference sequence. The OPA1 exon amplicons were sequenced using the BigDye Termination cycle sequencing kit v3.1 (Applied Biosystems) and analyzed on ABI PRISM 377 DNA sequencer (Applied Biosystems). The results were visualized by the Variant Reporter Software v1.1 (Applied Biosystems). PCR primers and reaction conditions are available upon request.

Ścieżyńska A., Ruszkowska E., Szulborski K., Rydz K., Wierzbowska J., Kosińska J., Rękas M., Płoski R., Szaflik J.P, & Ołdak M. (2017). Processing of OPA1 with a novel N-terminal mutation in patients with autosomal dominant optic atrophy: Escape from nonsense-mediated decay. PLoS ONE, 12(8), e0183866.

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Plasmid Isolation and Confirmation

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Recombinant clones were selected for plasmid isolation as per the manufacturer’s protocol using the GenElute HP Plasmid Miniprep Kit (Sigma). Isolated plasmids were analyzed on 0.8% agarose gel. PCR using vector-specific and gene specific primers was done to confirm the presence of insert. The recombinant plasmids were sequenced in ABI Prism 377 DNA sequencer (Applied Biosystem) at SciGenom, India.

Athira P.P., Anooja V.V., Anju M.V., Neelima S., Archana K., Muhammed Musthafa S., Antony S.P., Bright Singh I.S, & Philip R. (2022). A hepatic antimicrobial peptide, hepcidin from Indian major carp, Catla catla: molecular identification and functional characterization. Journal of Genetic Engineering & Biotechnology, 20, 49.

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Genetic Profiling of Psychiatric Disorders

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According to the previous studies [11 (link)–23 (link)], the SNPs which were found to be related to the development of psychiatric disorders were selected from the 1000 genome database of Han Chinese in Beijing, China (CHB) (http://www.internationalgenome.org/). We focused on eight SNPs in the OXTR gene, including rs1042778, rs13316193, rs2254298, rs2268494, rs237889, rs237897, rs53576, rs6770632.
Venous blood samples were collected from 765 subjects (342 subjects in BP group and 423 subjects in non-BP group). Genetic DNA was extracted using Sunshinebio TM blood genomic DNA Extraction Kit (Sunshinebio, Nanjing, China) following the manufacturer’s instructions. The extracted DNA was diluted to a concentration of 50 ng/μL. Genotyping of the OXTR SNPs applied multiple PCR technology and high-throughput sequencing technology. The primers showed in Additional file 1: Table S1. Multiple PCR amplification was performed using PERKIN ELMER Gene Amp PCR system 9600 (Applied Biosystems, Shanghai, China). Different samples were distinguished by different Barcode primers, and the high flux sequencing of the amplified products was performed using ABI PRISM 377 DNA Sequencer (Applied Biosystem, Shanghai, China). Several samples failed to be genotyped, but the call rates for all SNPs almost reached 95% (Table 2).

Zhang M., Liu N., Chen H, & Zhang N. (2020). Oxytocin receptor gene, childhood maltreatment and borderline personality disorder features among male inmates in China. BMC Psychiatry, 20, 332.

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UBIAD1 Gene Variant Screening Protocol

Cited 2 times

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Genomic DNA was isolated from blood samples (n = 37) with a standard salting-out procedure. DNA pathogenic variant testing was performed by PCR amplifying and Sanger sequencing of the UBIAD1 gene coding regions (exons 1 and 2). The PCR primer sequences designed using the reference sequence NG_009443.1 and amplification conditions are available upon request. DNA samples were purified with exonuclease I and FastAP thermosensitive alkaline phosphatase (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s protocol, sequenced directly using ABI Prism 377 DNA Sequencer (Thermo Fisher Scientific) and BigDye Terminator v1.1 Cycle Sequencing Kit (Thermo Fisher Scientific) and analyzed with the Variant Reporter DNA analysis software v1.1 (Thermo Fisher Scientific).
Pathogenicity of the novel non-synonymous single nucleotide UBIAD1 variant was predicted using PredictSNP2 [15 (link)], FATHMM [16 (link)], and MutPred2 [17 ], leading and reliable computational approaches [18 (link)] and analyzed for population frequency based on the data from Exome Aggregation Consortium (ExAC, http://exac.broadinstitute.org), 1000 Genomes Project (http://www.1000genomes.org), and NHLBI GO Exome Sequencing Project (ESP, http://evs.gs.washington.edu/EVS; all accessed 06/2018).

Sarosiak A., Udziela M., Ścieżyńska A., Oziębło D., Wawrzynowska A., Szaflik J.P, & Ołdak M. (2018). Clinical diversity in patients with Schnyder corneal dystrophy—a novel and known UBIAD1 pathogenic variants. Graefe's Archive for Clinical and Experimental Ophthalmology, 256(11), 2127-2134.

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Closing Gaps in Abalone Genome Assemblies

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PCR primers (Supplemental Table 2) were manually designed to close gaps between 12 scaffolds, which had been selected by the survey described above. PCR was performed using the KOD FX kit with a pooled DNA sample of diseased abalone obtained from Omaezaki. Amplification was as described above, except that the extension step was elongated to 10 min. PCR products were analyzed using agarose gel electrophoresis, and DNA from positive reactions were purified using the Wizard SV Gel and PCR Clean-Up System (Promega). Direct sequencing was performed using the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the ABI PRISM 377 DNA sequencer (Thermo Fisher Scientific).

Matsuyama T., Takano T., Nishiki I., Fujiwara A., Kiryu I., Inada M., Sakai T., Terashima S., Matsuura Y., Isowa K, & Nakayasu C. (2020). A novel Asfarvirus-like virus identified as a potential cause of mass mortality of abalone. Scientific Reports, 10, 4620.

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CTSK gene amplification and sequencing

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The amplified PCR products for CTSK gene were purified and sequenced with direct sequencing of the DNA Cycle sequencing System (ABI Prism kit) with the dideoxy-chain termination method and applied on an autosequencer (ABI Prism 377 DNA sequencer).

Arman A., Bereket A., Coker A., Kiper P.Ö., Güran T., Özkan B., Atay Z., Akçay T., Haliloglu B., Boduroglu K., Alanay Y, & Turan S. (2014). Cathepsin K analysis in a pycnodysostosis cohort: demographic, genotypic and phenotypic features. Orphanet Journal of Rare Diseases, 9, 60.

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Phylogenetic Analysis of E. coli Isolates

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E. coli isolates from each phylotype along with other typing methods (RAPD, ERIC, and BOX-PCR) were selected for 16S rRNA gene PCR using universal primers (27F, 5’-AGAGTTTGATCMTGGCTCAG-3’ and 1492R, 5’-TACGGYTACCTTGTTACGACTT-3′), followed by sequencing at First Base Laboratories SdnBhd (Malaysia) using Applied Biosystems highest capacity-based genetic analyzer (ABI PRISM^® 377 DNA Sequencer) platforms with the BigDye^® Terminator v3.1 cycle sequencing kit chemistry. Initial quality control of the generated raw sequences was performed using SeqMan software (version 6), and aligned with 13 relevant reference sequences retrieved from NCBI database using Molecular Evolutionary Genetics Analysis (MEGA) version 7.0 for bigger datasets [53 (link)]. Evolutionary distances were computed using the Kimura–Nei method, and the phylogenetic tree was constructed by applying the neighbor-joining method [54 (link)]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.

Saha O., Hoque M.N., Islam O.K., Rahaman M.M., Sultana M, & Hossain M.A. (2020). Multidrug-Resistant Avian Pathogenic Escherichia coli Strains and Association of Their Virulence Genes in Bangladesh. Microorganisms, 8(8), 1135.

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Radish virus isolation and sequencing

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Leaf samples of radish from Heilongjiang, Jilin and Shandong provinces from 2005 to 2010 were collected. All the samples were biologically purified by three cycles of single lesion isolation in Chenopodium amaranticolor and propagated in B. rapa. Inoculated plants were maintained in a glasshouse at 25 °C.
Total RNAs were extracted from 100 mg TuMV-infected B. rapa leaves with the Invitrogen Trizol Kit following instructions of the manufacturer. The 3-terminus of TuMV (~1.1 kb) were amplified with RT-PCR using primers CP-F (5′-ATC TTC GAA GAT TAC GAA GA-3′) and CP-R (5′-CCT TGC TTC CTA TCA AAT G-3′) [29 ]. The fragments were cloned into pMD18-T vector (TaKaRa Biotechnology Dalian Co, Ltd) and sequenced by a ABI PRISM™ 377 DNA Sequencer. For each isolate, at least four clones from two separate PCR were sequenced. In case of any inconsistence, at least two more clones will be sequenced to obtain the consensus sequence.

Li X., Zhu T., Yin X., Zhang C., Chen J., Tian Y, & Liu J. (2017). The genetic structure of Turnip mosaic virus population reveals the rapid expansion of a new emergent lineage in China. Virology Journal, 14, 165.

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Molecular Identification of Microbes

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The purified DNA was tested in four individual PCR amplifications using primers targeting a portion of the disulfide bond formation protein (dsb) gene, the 16S rRNA gene, the heat shock protein (groEL) gene and the glycoprotein trp36 (trp36) gene. The reactions (20 µL) contained 2 µL of template DNA, 0.5 mM of each primer, and 10 µL of 2 × EasyTaq PCR SuperMix (TransGen, Beijing, China). Detailed information about the primers and PCR condition is shown in Table 1. The positive PCR product was subjected to DNA sequencing (ABI PRISM 377 DNA sequencer). The full sequence for both strands of each DNA template was determined to ensure maximum accuracy of the data.

Li J., Liu X., Mu J., Yu X., Fei Y., Chang J., Bi Y., Zhou Y., Ding Z, & Yin R. (2019). Emergence of a Novel Ehrlichia minasensis Strain, Harboring the Major Immunogenic Glycoprotein trp36 with Unique Tandem Repeat and C-Terminal Region Sequences, in Haemaphysalis hystricis Ticks Removed from Free-Ranging Sheep in Hainan Province, China. Microorganisms, 7(9), 369.

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Transposon Insertion Site Identification

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AP-PCR was carried out in two rounds, each with a different primer pair (S1 Table). In the first round, primer TN5EXT (sequence unique to the right end of the transposon) and an arbitrary primer ARB1 were used [54 (link)]. PCR was performed as follows: (i) 5 min at 95°C; (ii) six cycles of 30 s at 95°C, 30 s at 30°C, and 1 min 30 s at 72°C; (iii) 30 cycles of 30 s at 95°C, 30 s at 45°C, and 2 min at 72°C; and (iv) a final extension step of 5 min at 72°C. The second round of PCR was performed using 2 μl of the first round reaction as template with primers TN5INT (specific to the right arm of the transposon) and ARB3 (specific to the 5’ end of primer ARB1) [54 (link)]. PCR was performed as follows: (i) 1 min at 95°C; (ii) 30 cycles of 30 s at 95°C, 30 s at 52°C, and 2 min at 72°C; and (iii) a final extension step of 5 min at 72°C. The PCR products were separated by agarose gel electrophoresis and the most-intense bands were purified (Invitrogen PureLink Gel extraction kit) and sequenced using primers TN5INT and ARB3 with an ABI PRISM 377 DNA Sequencer (DNA Sequencing and Oligonucleotide Synthesis Laboratory, IBB PAN, Warsaw). The sequences were compared with sequence databases using BLAST programs from The National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

Raczkowska A., Trzos J., Lewandowska O., Nieckarz M, & Brzostek K. (2015). Expression of the AcrAB Components of the AcrAB-TolC Multidrug Efflux Pump of Yersinia enterocolitica Is Subject to Dual Regulation by OmpR. PLoS ONE, 10(4), e0124248.

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