Abi 3130xl gene analyzer

Manufactured by Thermo Fisher Scientific

Sourced in United States

The ABI 3130XL Gene Analyzer is a capillary electrophoresis-based genetic analysis instrument. It is designed to perform high-throughput DNA sequencing and fragment analysis. The system utilizes a laser-induced fluorescence detection method to analyze DNA samples.

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Lab products found in correlation

Genescan software, by Thermo Fisher Scientific (1 mentions) Qiaamp dna tissue kit, by Qiagen (1 mentions) Genescan 3.7 analysis software, by Thermo Fisher Scientific (1 mentions)

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ABI 3130XL

4 protocols using abi 3130xl gene analyzer

CDR3 Length Analysis by Capillary Electrophoresis

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CDR3 length analysis was performed by amplification of the BV19 CDR3 region using cDNA as template and BV19 and Fam-labeled CB specific primers (16 (link)).
1–2 ul of amplified products were combined with 9 ul of Formamide/Liz 500 (900 ul Formamide + 50 ul Liz standard). Samples were heat denatured at 90°C for 3 minutes and then loaded on ABI 3130XL Gene Analyzer (Applied Biosystems). GeneScan software (Applied Biosystems) was used for the collection of the data. The files were analyzed using proprietary software which gives the relative frequency of each CDR3 length. The relative frequency of each CDR3 length was generated for each of the six PBMC samples and an average and the standard deviation calculated.

Yassai M.B., Demos W., Janczak T., Naumova E.N, & Gorski J. (2015). CDR3 clonotype and amino acid motif diversity of BV19 expressing circulating human CD8 T cells. Human immunology, 77(1), 137-145.

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Microsatellite Instability Analysis in Paraffin-Embedded Tissues

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DNA was extracted from paraffin-embedded tissue samples using the QIAamp DNA Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. MSI analysis was performed on six mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24, NR-27, and MONO-27; Table 2) using a Microsatellite Instability Detection Kit (Microread Gene Technology Co., Ltd, Beijing, China). To detect potential specimen contamination, pentanucleotide repeat markers (Penta C and Penta D) and a sex locus marker (amelogenin) were also analyzed for background confirmation. Fluorescently labeled primers were used in the PCR assay, and results were analyzed on an ABI 3130XL gene analyzer with the GeneScan 3.7 analysis software (Applied Biosystems, Foster City, California, USA). Samples were considered MSI-H when two or more of the markers displayed MSI. Samples with MSI at only one marker were considered MSI-L, and those with no MSI were considered MSS.

Ren X.Y., Song Y., Wang J., Chen L.Y., Pang J.Y., Zhou L.R., Shen S.J., Cao X., Wang Y.X., Shao M.M., Liang Z.Y., Sun Q, & Wu H.W. (2021). Mismatch Repair Deficiency and Microsatellite Instability in Triple-Negative Breast Cancer: A Retrospective Study of 440 Patients. Frontiers in Oncology, 11, 570623.

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Genetic Analysis of PAH Gene

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Peripheral blood samples were collected from the patients and parents in each of the 643 core families. For the 12 deceased-proband families, samples were collected only from the parents. Genomic DNA was extracted from each sample, and PCR primers were designed to amplify each of the 13 exons of the PAH gene, as well as their flanking introns, using previously published sequencing data [4 (link)]. PCR products were sequenced bi-directionally using an ABI 3130-xl gene analyzer (Life Technologies, Carlsbad, CA, USA). To identify nucleotide variations, sequences were aligned and inspected using a reference sequence from Ensembl (NM_000277). To determine sequence variability, variable sites in patient genes were aligned with the corresponding sites from the respective parents. We excluded non-biological relationships in de novo-variant pedigrees using the Promega PowerPlex 21 HS system (Promega Corporation, Madison, WI, USA).

Liu N., Huang Q., Li Q., Zhao D., Li X., Cui L., Bai Y., Feng Y, & Kong X. (2017). Spectrum of PAH gene variants among a population of Han Chinese patients with phenylketonuria from northern China. BMC Medical Genetics, 18, 108.

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PCR Amplification and Sequencing of MMACHC Gene

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The coding exons and the flanking introns of the MMACHC gene were amplified by polymerase chain reaction (PCR). The PCR primers were described by Lerner-Ellis et al. [6 (link)] (Table 1). The reaction mixture of a final volumn of 25 μL contained 10 × PCR buffer,2.5 mM dNTP, 0.2 μM of each primer, 1U Taq enzyme, and 50 ng DNA template. The amplification program consisted of an initial denaturation at 94 °C for 3 min, followed by 30 cycles at 94 °C for 30s, 57- 62 °C for 40s, and 72 °C for 50s, with a final extension at 72 °C for 8 min. PCR products were checked on a 2 % agarose gel and then purified. The direct sequencing was performed with an ABI3130xl gene analyzer (Life Technologies, USA) using the ABIBigDye3.1 sequencing kit (Life Technologies, USA) according to manufacturer’s instructions. Both strands of the shifted exons were sequenced. Sequences were aligned and inspected to identify nucleotide variations using a reference sequence from Ensemble (NM_015506).Table 1

PCR primers for mutation analysis of MMACHC

Primers	Gene fragments	Sequence	Length
MMACHC-1 F:	Exon 1	GGGATACCGTGATGATACGC	680 bp
MMACHC-1R:	Exon 1	GAACCCAGGAGGATCAGAGG	680 bp
MMACHC-2 F:	Exon 2	TGCATCACATAGCGTCAGTG	467 bp
MMACHC-2R:	Exon 2	AGCCTGGCTTTAGGGTATCA	467 bp
MMACHC-3 F:	Exon 3	TCATGTTTTCCCTTCTGAGGA	395 bp
MMACHC-3R:	Exon 3	CAAAGCTAATTTGTTCTGGGTTG	395 bp
MMACHC-4 F:	Exon 4	AGGCCTAGCTTGCAATGATG	694 bp
MMACHC-4R:	Exon 4	GAAGGCAGATGGGAATTCTG	694 bp

Zong Y., Liu N., Zhao Z, & Kong X. (2015). Prenatal diagnosis using genetic sequencing and identification of a novel mutation in MMACHC. BMC Medical Genetics, 16, 48.

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