Abi prism 3130xl analyzer

Manufactured by Thermo Fisher Scientific

Sourced in United States

The ABI Prism 3130XL Analyzer is a capillary electrophoresis system designed for genetic analysis. It features 16 capillaries and is capable of performing DNA sequencing and fragment analysis.

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ABI PRISM 3130xl Genetic Analyzer (327 citations) ABI 3130XL (189 citations) ABI PRISM 3130xl (99 citations) ABI PRISM 3130xl DNA Analyzer (10 citations) ABI PRISM 3130xl Genetic Analyzer system (8 citations) ABI Prism 3130xl Genetic Analyzer capillary sequencer (7 citations)

9 protocols using abi prism 3130xl analyzer

Molecular Analysis of Meningococcal and Pneumococcal Infections

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The PCR was performed in 25 µl of a reaction mixture containing 2 µl of isolated
DNA, 10 pmol of each primer, 4 nmol of each deoxynucleotide, 1.5 U of Taq DNA
Polymerase (Sigma-Aldrich, USA), 1 × PCR reaction buffer (containing 15 mM
MgCl2; Sigma-Aldrich, USA) and additionally 25 mmol MgCl₂. PCR
products were further purified with thermosensitive Exonuclease I and FastAP
Alkaline Phosphatase (Fermentas, Thermo Fisher Scientific, USA) and sequenced
with BigDye® Terminator v3.1 Cycle Sequencing Kit on an ABI Prism 3130XL
Analyzer (Applied Biosystems, Foster City, CA, USA) according to the
manufacturers’ protocols.
The sequences were compared between cases and healthy family members and the
general population separately for N. meningitidis and
S. pneumoniae cases.
The study was performed with the approval of the Poznan Medical University
Ethical Committee and a written informed consent was obtained from all of the
parents.

Gowin E., Świątek-Kościelna B., Kałużna E., Strauss E., Wysocki J., Nowak J., Michalak M, & Januszkiewicz-Lewandowska D. (2018). How many single-nucleotide polymorphisms (SNPs) must be tested in order to prove susceptibility to bacterial meningitis in children? Analysis of 11 SNPs in seven genes involved in the immune response and their effect on the susceptibility to bacterial meningitis in children. Innate Immunity, 24(3), 163-170.

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Detecting Genomic Copy Number Variations

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If the panel did not detect causal variants, multiplex ligation-dependent probe amplification (MLPA) and/or comparative genomic hybridization array (aCGH) were done. MLPA was used to search for genomic copy number variations in NIPBL gene. The SALSA P141/P142 NIPBL MLPA kit (MRC-Holland, Amsterdam, The Netherlands) was used following the manufacturer’s instructions, the reaction products were separated by capillary electrophoresis on Abi Prism 3130XL Analyzer (Applied Biosystems) and the results obtained were analysed using GeneMapper software (Thermo Fisher Scientific). aCGH analyses were performed with the qChip Post oligonucleotide microarray (Quantitative Genomic Medicine Laboratories, Barcelona, Spain).

Latorre-Pellicer A., Gil-Salvador M., Parenti I., Lucia-Campos C., Trujillano L., Marcos-Alcalde I., Arnedo M., Ascaso Á., Ayerza-Casas A., Antoñanzas-Pérez R., Gervasini C., Piccione M., Mariani M., Weber A., Kanber D., Kuechler A., Munteanu M., Khuller K., Bueno-Lozano G., Puisac B., Gómez-Puertas P., Selicorni A., Kaiser F.J., Ramos F.J, & Pié J. (2021). Clinical relevance of postzygotic mosaicism in Cornelia de Lange syndrome and purifying selection of NIPBL variants in blood. Scientific Reports, 11, 15459.

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Transcriptome analysis of Tenebrio molitor

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Whole brains, CC/CAs and VNCs from three stages of T. molitor were dissected in physiological saline (PS) (274 mM NaCl, 19 mM KCl, 9 mM CaCl₂, 5 mM glucose, and 5 mM HEPES, pH 7.0, Sigma-Aldrich, Poznań, Poland). Total RNA was extracted from ten samples of each tissue (10× brains, 10× CC/CAs and 10× VNCs) using the Insect RNA MicroPrep™Kit (Zymo Research Corp., Irvine, CA, USA) according to the manufacturer’s protocols. The protocol included the in-column DNase I treatment to remove traces of gDNA and prevent the contamination of the RNA samples. ReverAid™ Reverse Transcriptase (Fermentas, Waltham, MA, USA) was then used to generate cDNA for PCR according to the manufacturer’s protocols.
A Bio-Rad T100 thermocycler (Bio-Rad, Hercules, CA, USA) was employed for PCR using the following cycling conditions: 95 °C for 3 min, followed by 34 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for 60 s. The PCR products were separated using 2% agarose gels in TEA buffer and visualized using ethidium bromide. Gel products were purified using the Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corp., Irvine, CA, USA) and sequenced with BigDye Terminator v3.1 in an ABI Prism 3130XL Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocols.

Lubawy J., Marciniak P, & Rosiński G. (2020). Identification, Localization in the Central Nervous System and Novel Myostimulatory Effect of Allatostatins in Tenebrio molitor Beetle. International Journal of Molecular Sciences, 21(10), 3510.

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Genotyping β-Globin Gene Variants

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Four intragenic β-globin gene polymorphisms, including codon 2 (C/T), IVS II-16 (C/G), IVS II-74 (G/T) and IVS II-81 (C/T), were genotyped using direct DNA sequencing on an ABI PRISM™ 3130 XLanalyzer (Applied Biosystems, Foster City, CA, USA), as described elsewhere [5 (link)]. Only samples with the codon 41/42 (-TTCT) mutation (four polymorphisms) were genotyped, with multiplexing targeted sequencing, using a barcode and a next-generation sequencing platform. Hinf I polymorphism genotyping was conducted using allele-specific PCR, as described elsewhere [17 (link)]. The basic statistics for five polymorphisms were calculated, including the allele frequency, genotype frequency and minor allele frequency (p-value < 0.05), using PEAS V1.0: a package for elementary analysis of SNP data. The Fisher’s exact test revealed the derived allele frequency (DAF), and MINITAB release 14.12.0 statistical software was used for the calculations, indicating a significant difference between the wild type and β-thalassemia genotypes in biological polymorphic markers. A p-value < 0.05 was considered to be statistically significant.

Karnpean R., Tepakhan W., Suankul P., Thingphom S., Poonsawat A., Thanunchaikunlanun N., Ruangsanngamsiri R, & Jomoui W. (2022). Genetic Background Studies of Eight Common Beta Thalassemia Mutations in Thailand Using β-Globin Gene Haplotype and Phylogenetic Analysis. Genes, 13(8), 1384.

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Quantitative Analysis of α-Globin Gene Cluster

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Dosage quantitative analysis of the α-globin gene cluster on chromosome 16p13.3 was carried out in an unidentified Hb H disease and Hb Bart’s hydrops fetalis subjects using an MLPA commercial kit (SALSA MLPA probemix P140-C1 HBA, MRC-Holland, Amsterdam, The Netherlands). MLPA analysis was performed on a SimpliAmp™ thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Fragment analysis was performed using an ABI PRISM™ 3500 analyzer (Applied Biosystems, Foster City, CA, USA). Data analysis was performed using the Coffalyser.Net™ software (MRC Holland). Gap-PCR to detect rare α-thalassemia genotypes (--^SA deletion (NG_000006.1:g.19464_43064del23601) and --^CR deletion (NC_000016.10:g.144,215_188,841)) was conducted after receiving the MLPA results, as previously identified^{11 (link),12 (link)}. Breakpoint confirmation of targeted PCR fragments was performed by direct DNA sequencing using an ABI PRISM™ 3130 XL analyzer (Applied Biosystems).

Jomoui W., Panyasai S., Sripornsawan P, & Tepakhan W. (2023). Revisiting and updating molecular epidemiology of α-thalassemia mutations in Thailand using MLPA and new multiplex gap-PCR for nine α-thalassemia deletion. Scientific Reports, 13, 9850.

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Mitochondrial DNA Barcoding of Daphnia

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To assign individuals to species, we amplified a partial sequence of the mitochondrial cytochrome oxidase subunit I (COI) gene, which is species-specific in Daphnia²⁸, from a subset of current-day populations (16 and 10 individuals from the heated and control lakes, respectively) and the resting eggs from sediment cores (6 eggs). The following primers were used: bcdF01: 5′-CATTTTCHACTAAYCATAARGATATTGG-3′ and bcdR04: 5′-TATAAACYTCDGGATGNCCAAAAAA-3′^{67 (link)}. COI gene sequences were amplified from 1 μL DNA template in 5 μL of PCR reaction mix, containing 2.5 μL Type-it Microsatellite Kit (Qiagen GmbH, Hilden, Germany), 0.25 μmol/L of each primer, and 2 μL of water. Thermocycling parameters were: one cycle of 5 min at 95 °C; 35 steps of 30 s at 95 °C, 1 min at 50 °C, and 1 min at 72 °C; and a final step of 5 min at 72 °C. The PCR product was visualised by electrophoresis on 1% agarose gel, purified using Exonuclease I and Alkaline Phosphatase (Thermo Scientific, San Jose, USA) for 15 min at 37 °C and 15 min at 80 °C, and Sanger sequenced using the BigDye Terminator v3.1 kit and an ABI Prism 3130XL Analyzer (Applied Biosystems, Foster City, CA, USA).

Dziuba M.K., Herdegen-Radwan M., Pluta E., Wejnerowski Ł., Szczuciński W, & Cerbin S. (2020). Temperature increase altered Daphnia community structure in artificially heated lakes: a potential scenario for a warmer future. Scientific Reports, 10, 13956.

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DNA Sequencing with BigDye v3.1

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DNA sequencing was performed with a BigDye v3.1 sequencing kit (Applied Biosystems, Foster City, CA, United States) on a ABI Prism 3130XL Analyzer (Applied Biosystems) in the Molecular Biology Techniques Laboratory, Faculty of Biology, Adam Mickiewicz University in Poznan, Poland.

Pieczynski M., Kruszka K., Bielewicz D., Dolata J., Szczesniak M., Karlowski W., Jarmolowski A, & Szweykowska-Kulinska Z. (2018). A Role of U12 Intron in Proper Pre-mRNA Splicing of Plant Cap Binding Protein 20 Genes. Frontiers in Plant Science, 9, 475.

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Targeted MLPA Analysis for IRD Genes

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MLPA was used to search for genomic copy number variations in 32 patients without causative mutations found after sequencing of 316 IRD genes. We selected 9 genes with high prevalence of reported rearrangements^{38 (link)–40 (link)}.
Patients with a dominant inheritance pattern were analysed using MLPA Retinitis Probemix (P235). This probemix contains PRPF31, RHO, RP1 and IMPDH1 genes.
Patients with heterozygotic mutations in USH2A genes or EYS were also analysed for CNVs, in search of the second mutated allele within these genes (Salsa Mixes P361/2 and P328, respectively).
In addition, patients with an X-linked inheritance pattern, clinically diagnosed with choroideremia or families with only males affected, were analysed for RP2, RPGR and CHM genes (Salsa probemix P366).
MLPA reactions were run according to the manufacturer’s general recommendations (MRC-Holland, Amsterdam, Holland) as previously described^{63 (link)}. The MLPA reaction products were separated by capillary electrophoresis on Abi Prism 3130XL Analyzer (Applied Biosystems) and the results obtained were analysed by GeneMapper software (Thermo Fisher Scientific).

Ezquerra-Inchausti M., Anasagasti A., Barandika O., Garay-Aramburu G., Galdós M., López de Munain A., Irigoyen C, & Ruiz-Ederra J. (2018). A new approach based on targeted pooled DNA sequencing identifies novel mutations in patients with Inherited Retinal Dystrophies. Scientific Reports, 8, 15457.

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DNA Sequencing Protocol Confirmation

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DNA sequencing was performed to confirm the size of the alleles detected during our study using the ABI Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). PCR-amplified fragments were purified using BigDye XTerminator  Purification kit (Applied Biosystems) and visualized in an ABI PRISM 3130XL analyzer using the Sequencing Analysis v.5.3.1 software (Applied Biosystems).

Camacho-Mejorado R., Noris G., Santana C., Magaña J.J., Majluf-Cruz A., Arellano-Galindo J., De la Peña A., Hernández-Juárez J., Calderón-Aranda E.S., Meraz-Ríos M.A, & Gómez R. (2015). Interethnic variation of the MMP-9 microsatellite in Amerindian and Mexican Mestizo populations: considerations for genetic association studies. Genetics and molecular research : GMR, 14(1).

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