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Abi 3500dx genetic analyzer

Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Singapore

The ABI 3500DX Genetic Analyzer is a capillary electrophoresis instrument designed for DNA sequencing and fragment analysis applications. It utilizes fluorescence detection technology to analyze nucleic acid samples.

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45 protocols using abi 3500dx genetic analyzer

1

Screening Hereditary Hearing Loss Mutations

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We used targeted gene capturing and sequencing (MyGenostics GenCap Enrichment Technologies) to screen the mutations in 121 genes that had been reported to cause hereditary hearing loss. PCR primers for c.919‐2A > G of SLC26A4 (NM_000441.1) were designed by Primer 3.0 (http://bioinfo.ut.ee/primer3‐0.4.0/); the forward primer was 5′‐AAGTTCAGCATTATTTGGTTGACA‐3′ and the reverse primer was 5′‐TGGTTGTTTCTTCCAGATCACA‐3′. PCR conditions for c.919‐2A > G of SLC26A4 were as follows: 95°C for 5 minutes, followed by 35 cycles of 94°C for 30 seconds, 56°C for 45 seconds, and 72°C for 45 seconds. The DNA sequencing was performed on an ABI 3500DX Genetic Analyzer (Applied Biosystems).
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2

CRISPR1 Locus Amplification and Sequencing

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CRISPR1 locus amplification was performed in a T3000 thermocycler (Biometra) using CRISPR1-PCR-F and CRISPR1-PCR-R primers targeting flanking regions of the CRISPR1 locus as previously described (18 (link), 19 (link), 29 (link)). Briefly, 0.5 μM each primer, 0.2 mM deoxynucleoside triphosphate (dNTP), 2 mM MgCl2, 0.02 U/μL GoTaq polymerase (Promega), 1× PCR buffer, and a small GBS colony were mixed in a total volume of 25 μL. The PCR mixtures were heated to 94°C for 5 min, followed by 40 cycles of a denaturation step at 94°C for 30 s, an annealing step at 55°C for 30 s, and an elongation step at 72°C for 1 min, ending with a final extension step at 72°C for 7 min. PCR amplification was verified by electrophoretic migration in a 1% agarose gel, and then the PCR products were purified using centrifugal filter units (Millipore Corporation) in accordance with the manufacturer’s recommendations.
CRISPR1 locus sequencing from purified PCR products was then performed by the Sanger sequencing technique on an ABI 3500 Dx genetic analyzer (Applied Biosystems, Thermo Fisher Scientific), using CRISPR1-SEQ-F and CRISPR1-SEQ-R internal sequencing primers and BigDyeTerminator mix v3.1 (Applied Biosystems) as previously described (19 (link)).
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3

Evaluating Variant Effects on mRNA

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Total RNA was isolated from peripheral blood lymphocytes of the patient and his parents using RNeasy Mini Kit (Qiagen, GmbH, Hilden, Germany). A total of 500 ng RNA was reverse-transcribed into cDNA using random hexamer primers and a Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics, Indianapolis, IN, USA). PCR for evaluation of the variant effect on mRNA was performed using forward primer and reverse primer mapped on exon 1 and 3, respectively; β-actin was used as internal control (Supplementary Materials, Table S1). PCR products were analyzed by standard gel electrophoresis and purified by gel extraction. Finally, Sanger sequencing was carried out using Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Warrington, Cheshire, UK) and run on an ABI 3500 Dx Genetic Analyzer (Applied Biosystems).
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4

Measles Virus N-450 Genotyping

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Measles samples were genotyped by sequencing the N-450 region, using primers that have been previously described [12 (link)]. Sequencing reactions were performed on the Applied GenAmp 2700 Peltier Thermal Cycler (Applied Biosystems, ThermoFisher, Waltham, MA, USA) using the ABI BigDye1 Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), following the manufacturer’s protocols. Sequencing was performed on the purified amplicon template (Qia-quick PCR purification kit, Qiagen) using both forward and reverse primers. The fluorescent-labeled fragments were purified from the unincorporated terminators with the BigDye XTerminator1 Purification Kit (Applied Biosystems). The samples were injected for electrophoresis in an ABI 3500 Dx Genetic Analyzer (Applied Biosystems).
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5

Whole Exome Sequencing and Variant Analysis

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WES was carried out using an Agilent SureSelect Human All Exon V6 Kit (Agilent Technologies Inc., USA) on an Illumina NovaSeq 6000 platform (Illumina Inc., CA, USA). Data and bioinformatic analyses were performed according to a method described by a previous study (Luo et al., 2019 (link)). Candidate variants were confirmed in the parents in each family by Sanger sequencing. PCR products were bi-directionally sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) on an ABI 3500DX Genetic Analyzer (Applied Biosystems) after purification on 2% agarose gels.
Variants were described according to the nomenclature recommended by the Human Genome Variation Society (www.hgvs.org/). Variants were annotated using ANNOVAR (https://annovar.openbioinformatics.org/en/) and filtered according to their predicted effects and allele frequencies in the public database gnomAD (http://gnomad.broadinstitute.org/). Novel variants were checked in the Human Gene Variant Database (www.hgmd.cf.ac.uk) and ClinVar database (www.ncbi.nlm.nih.gov/clinvar/). InterVar (http://wintervar.wglab.org/) software was used to evaluate the pathogenicity of all variants according to the standards and guidelines of the American College of Medical Genetics and Genomics (Li and Wang, 2017 (link)).
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6

Multiplex Fluorescent PCR for MSI Profiling

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Total DNA was isolated from FFPE tumor and paired normal tissue samples though the DNA extraction kit (TIANGEN, Beijing, China) following the manufacturer's recommendation and was used for subsequent multiplex fluorescent PCR. MSI status was assessed with the amplification of six mononucleotide repeat markers (BAT25, BAT26, NR21, NR24, MONO27, and NR 27) described either in NCI (National Cancer Institute) - or Promega- panel. In addition, the final panel also contained one gender loci (Amel) and two pentanucleotide repeat markers (Peta C and Peta D) as internal controls. Co-amplification of these targets was performed on ABI 7500 using a 25 μl reaction volume advised by MSI-testing reagent kit (SINOMDgene, Beijing, China). The PCR conditions were carried out according to the operation protocols. Fluorescent PCR products were analyzed by capillary electrophoresis using an ABI 3500DX Genetic Analyzer (Applied Biosystems) and Genemaker software 2.0 (SINOMDgene, Beijing, China).
Tumors with instability at two or more of these 6 markers were defined as MSI-H, while those without instability or showing instability at only one marker were classified as MSS and Low- Microsatellite Instability(MSI-L) tumors, respectively.
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7

Sanger Sequencing of Regulatory Region

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For Sanger sequencing of the 500-bp candidate regulatory region (hg38 coordinates chr1:3,774,672–3,775,171), we used the following primers: forward primer 1: 5′-TCTGTAGGGCCTCCCCAA-3′, forward primer 2: 5′-ACAGGGACCGGTCTAGCTGTA-3′, reverse primer 1: 5′-CTCCTAACAGCAGCCTCAGAG-3′ and reverse primer 2: 5′-TGTTAAGTCAGGCGACAGACC-3′. PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit on an ABI 3500 Dx Genetic Analyzer (Applied Biosystems). Additional Sanger sequencing was done at GATC Biotech, Germany. For peak calling and base determination we used CodonCode Aligner v4.1 software (CodonCode Corporation, USA).
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8

Targeted Sequencing of Deafness Genes

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TCOF1, POLR1D, and POLR1C were screened in the probands by targeted gene capture and sequencing of the 121 deafness‐related genes by MyGenostics Corporation (MyGenostics GenCap Enrichment technologies). The data analysis and bioinformatics analysis procedures were performed as previously described.10The candidate gene variants were observed in the probands in each family by Sanger sequencing. Polymerase chain reaction (PCR) primers were prepared and the amplification conditions for Sanger sequencing were as described previously.9 DNA sequencing was performed on an ABI 3500DX Genetic Analyzer (Applied Biosystems).
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9

T-cell Clonality Analysis by PCR

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T‐cell clonality was analyzed by polymerase chain reaction (PCR) amplification on the basis of BIOMED‐2 protocols.9 (link) Primers (Yuanqi Biotech) were used for TCR‐β, TCR‐γ, and TCR‐δ chain detection. Clonality analysis of the PCR products was performed using an ABI 3500DX genetic analyzer (Applied Biosystems). All experiments included appropriate positive and negative controls. A clear single‐peak fluorescence signal at the target position was determined as a monoclonal rearrangement, whereas a continuous multipeak fluorescence signal was judged as a polyclonal rearrangement.
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10

Microsatellite Instability Profiling in Tumor-Normal Pairs

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DNA was extracted from paired tumor and normal tissues. Multiplex PCR was conducted using the Microsatellite Instability Detection Kit (Microread, China), which allowed for the detection of six markers: BAT25, BAT26, NR-21, NR-24, NR-27, and Mono27 by co-amplification using 5 μl of DNA with a concentration of 3 ng/ml. The PCR program was as follows: denaturation at 95 °C for 5 min and 40 cycles of 95 °C for 30 s, 60 °C for 1 min and 70 °C for 1 min, followed by 15 °C for 40 min. The amplicons were subjected to capillary electrophoresis on an ABI 3500 DX Genetic Analyzer (Applied Biosystems), followed by analysis using the GeneMapper v4.1 software (Applied Biosystems). If the peak for a certain marker (referred to as a left shift or right shift) was present in a tumor sample but absent in the corresponding normal sample, that marker was considered instable. MSI status was classified into three categories: MSI-H, MSI-low (MSI-L), and MSS depending on the number of instable markers (≥2, ≥1, 0).
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