We selected DNA solution of three samples (X, Y, and Z) out of 50 samples and created mixed samples at 1:1 to 1:10 of DNA concentration ratios (10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10) using two of the samples (X + Z and Y + Z). The total amount of DNA in the mixed samples was 1 ng. STR nucleotide sequence analysis was performed using NGS. To compare with the result of STR-NGS analysis, the amplification reaction was also performed using the GlobalFiler TM PCR Amplification Kit using the prepared mixed samples, and the amplified product was analysed via capillary electrophoresis (CEP) using the 3500xL Genetic Analyzer (Thermo Fisher Scientific). GeneMapper ID-X Software v1.4 (Thermo Fisher Scientific) was used for electrophoresis data, the analytical threshold was analysed using 175 RFU, which is the default value of 3500xL Genetic Analyzer. Table 1 shows the tested loci and analysis items of the GlobalFiler TM PCR Amplification Kit. Samples A, B, and C had the same STR types but different nucleotide sequences at D8S1179, D21S11, and D2S1338.
3500xl genetic analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, United Kingdom
The 3500xL Genetic Analyzer is a capillary electrophoresis instrument designed for DNA sequencing and genetic analysis. It features 8 capillaries and can process multiple samples simultaneously. The system utilizes laser-induced fluorescence detection for accurate and sensitive DNA fragment analysis.
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Lab products found in correlation
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (55 mentions)
Genemapper software, by Thermo Fisher Scientific (13 mentions)
Qiaamp dna mini kit, by Qiagen (9 mentions)
Genemapper id x software v1, by Thermo Fisher Scientific (9 mentions)
Hi di formamide, by Thermo Fisher Scientific (8 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (7 mentions)
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (6 mentions)
Bigdye xterminator purification kit, by Thermo Fisher Scientific (6 mentions)
Pop 4 polymer, by Thermo Fisher Scientific (4 mentions)
Amplitaq gold 360 master mix, by Thermo Fisher Scientific (4 mentions)
Genemapper id x v1, by Thermo Fisher Scientific (4 mentions)
Qiaamp dna blood mini kit, by Qiagen (4 mentions)
Qiaquick pcr purification kit, by Qiagen (4 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (4 mentions)
Exonuclease 1, by Thermo Fisher Scientific (4 mentions)
Bigdye terminator v3, by Thermo Fisher Scientific (4 mentions)
Bigdye terminator 3.1 kit, by Thermo Fisher Scientific (4 mentions)
Geneamp pcr system 9700 thermal cycler, by Thermo Fisher Scientific (3 mentions)
Genemapper id x software, by Thermo Fisher Scientific (3 mentions)
Sequencing analysis software, by Thermo Fisher Scientific (3 mentions)
287 protocols using 3500xl genetic analyzer
1
Comparing STR Profiles via NGS and CE
2
Genetic Analysis of Hereditary Multiple Exostoses
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Total genomic DNA was available from all patients and parents of patient 4 and patient 9. All exons of EXT1 (ENSG00000182197; ENST00000378204) and EXT2 (ENSG00000151348; ENST00000395673) genes were amplified by PCR from genomic DNA isolated from leukocytes and analyzed by direct sequencing using 3500xL genetic analyzer (Applied Biosystems, USA). PCR primers are available upon request. All samples were tested for large deletions/duplications using the SALSA MLPA P215-B2 EXT probe mix (MRC-Holland, Amsterdam, Netherlands) according to the manufacturer's instructions. Fragment analysis was performed on 3500xL genetic analyzer (Applied Biosystems, USA), and the MLPA data were analysed using Coffalyser.Net software (MRC-Holland, Netherlands).
Hereditary Multiple Exostoses in 9 Families
Hereditary Multiple Exostoses in 9 Families
3
Multiplex PCR of 27 STR Loci
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DNA sample of 1 ng was used to conduct the multiplex PCR of 27 loci according to the following specification. First, we prepared 10 μL PCR cocktail comprising 5 μL STRtyper-27 comp Master Mix, 2.5 μL STRtyper-27 comp Primer Mix, 2.5 μL ddH2O, and 1 ng DNA sample. Second, PCR was conducted on the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, United States ) under reaction conditions of initial denaturation at 95°C for 5 min; 28 cycles of 94°C for 10s, 61°C for 60s, and 70°C for 30 s; and 60°C for 15 min. Third, we mixed 1 μL amplified product/STRtyper-27 comp Allelic Ladder Mix with 8.75 μL deionized HiDi Formamide and 0.25 μL ILS-500 (HEALTH Gene Technologies) and then denatured the mixture at 95°C for 3 min, followed by chilling at 4°C for 3 min. Finally, the mixture was electrophoresed and separated by the 3500xL Genetic Analyzer (Thermo Fisher Scientific). STR typing of each locus was determined by the GeneMapper® ID-X Software v1.5 (Thermo Fisher Scientific) in comparison with the allelic ladder.
4
Sanger Sequencing for Mutation Validation
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We performed Sanger sequencing to confirm that the mutations identified by exome sequencing segregated with the disease. PCR products were purified using a MultiScreen-PCR plate (Millipore, Billerica, MA) followed by sequencing with a 3500xL Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA).
5
ITS-Based Molecular Identification of Mushroom
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To analyze the nucleotide sequence of the ITS region of the mushroom samples, PCR was performed using ITS Universal Primers ITS 1 (White et al., 1990 ) and ITS 4B (Gardes & Bruns, 1993 (link)) using KOD FX DNA polymerase (Toyobo, Osaka, Japan). The forward primer (ITS1) was 5′-CTTGGTCATTTAGAGGAAGTAA-3′, and the reverse primer (ITS 4B) was 5′-TCCTCCGCTTATTGATATGC-3′.
The PCR products were sequenced using the BigDye 3.1 kit and loaded on a 3500xL Genetic Analyzer (Thermo Fisher Scientific). The obtained nucleotide sequence was analyzed using GENETYX ver 13 (Genetyx, Tokyo, Japan) and BLAST. The nucleotide sequence characteristics of O. japonicus were selected by comparing the nucleotide sequences of the ITS regions of O. japonicus, L. edodes, P. ostreatus, and P. edulis. The primers for the LAMP method that specifically recognize O. japonicus were designed using PrimerExplorer V5 (Table 2 ).
The PCR products were sequenced using the BigDye 3.1 kit and loaded on a 3500xL Genetic Analyzer (Thermo Fisher Scientific). The obtained nucleotide sequence was analyzed using GENETYX ver 13 (Genetyx, Tokyo, Japan) and BLAST. The nucleotide sequence characteristics of O. japonicus were selected by comparing the nucleotide sequences of the ITS regions of O. japonicus, L. edodes, P. ostreatus, and P. edulis. The primers for the LAMP method that specifically recognize O. japonicus were designed using PrimerExplorer V5 (
Primers for detecting O. japonicus for use in LAMP.
| Primer | Sequence |
|---|---|
| F3 primer | 5′-GAAGCTTGGACTGTGGAG-3′ |
| B3 primer | 5′-GTGAAAACAGACGATTAGAGAG-3′ |
| FIP primer | 5′-ACACCAAGGCTTAGGTCCGAACTAGATGTTCTCAGCTCCT-3′ |
| BIP primer | 5′-ATCTACGCCTTGGTGGTTTGATTTGAAATGAAAGCAGACAGA-3′ |
| LF loop primer (optional) | 5′-TAATCCGGTTTCCGCTAATGC-3′ |
| LB loop primer (optional) | 5′-CTCTTTGGTTGGGATAGCTGCAAC-3′ |
6
Validating Exome Sequencing Variants
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To confirm that mutations identified by exome sequencing segregated with the disease, we performed Sanger sequencing. PCR was performed with the primers shown in table e-1. PCR products were purified using a MultiScreen PCR plate (Millipore, Billerica, MA) followed by sequencing using 3500xL genetic analyzer (Thermo Fisher Scientific, Waltham, MA).
7
Genomic Profiling of Tumor Samples
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The QIAamp DNA Mini Kit (Qiagen) was used to extract genomic DNA from freshly frozen tumor tissues, according to the manufacturer’s instructions. The isocitrate dehydrogenase 1 (IDH1) mutation status was determined via real-time polymerase chain reaction (PCR) for the IDH1 mutation using hybridization probes, as previously described.15 (link)IDH2, H3F3A, HIST1H3B, and C228T or C250T TERT promoter (TERTp) mutations were assessed using Sanger sequencing following PCR amplification. Primers used in this study are presented in Supplementary Table 1 . Sanger sequencing was performed using the 3500xL Genetic Analyzer (Thermo Fisher Scientific), according to the manufacturer’s instructions. Sequence data analyses were performed using Sequencing Analysis software v5.4 (Thermo Fisher Scientific). Using this method, cases with H3K27M mutation were defined as cases harboring the mutation in either H3F3A K27 or HIST1H3B K27. Cases wherein H3F3A K27 or HIST1H3B K27 mutation was not found were defined as cases with H3 wild-type. Moreover, BRAF mutational status was assessed in 3 long-term survivors. BRAF V600E mutation was tested using Sanger sequencing.
8
CGG Repeat Analysis and FMR1 Methylation
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CGG sizing was assessed by AmplideX® PCR assay (Asuragen, Austin, TX, USA), a sensitive and efficient amplification method [17 (link)]. This CGG Repeat Primed (RP) PCR protocol uses three primers and fragment sizing on a Genetic Analyzer (Thermo Fisher), allowing detection of AGG interruptions within the FMR1 allele. PCR reagents include gene-specific and CGG primers, a polymerase mix buffer for amplification of the CGG repeat region in the FMR1 gene and a ROX 1000 Size Ladder for sizing by capillary electrophoresis. Amplified products were visualized by capillary electrophoresis (3500xL Genetic Analyzer, Thermo Fisher, Waltham, MA, USA) and separated using POP-7TM polymer (Thermo Fisher), following manufacturer’s instructions. The size of the PCR products has been converted to the estimated number of CGG repeats using size and mobility conversion factors. The reference sequence of the FMR1 gene used for sizing the CGG repeat is LRG_762 (identical to NG_007529.2).
Evaluation of genomic DNA methylation was performed through MS-MLPA on prenatal diagnosis samples and peripheral blood leukocytes of III-2 (Family 2), using the specific assay for FMR1-AFF2 locus in Xq27.3 (code ME029B2), following the manufacturer’s protocol (MRC Holland).
Evaluation of genomic DNA methylation was performed through MS-MLPA on prenatal diagnosis samples and peripheral blood leukocytes of III-2 (Family 2), using the specific assay for FMR1-AFF2 locus in Xq27.3 (code ME029B2), following the manufacturer’s protocol (MRC Holland).
9
Repeat expansion detection in NOTCH2NLC
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Blood samples were processed to extract genomic DNA following a standard phenol-chloroform method. We performed a repeat-primed PCR protocol to test repeat expansions in the 5′UTR of NOTCH2NLC as reported in the literature.30 (link) A saw-tooth tail pattern in the electropherogram was considered to indicate a repeat expansion. Fluorescence amplicon-length PCR was used to detect the length of GGC repeat expansions. Electrophoresis was performed on a 3500xL Genetic Analyzer (Thermo Fisher Scientific). The data were analyzed using GeneMapper software (Thermo Fisher Scientific). The length of the highest signal peak of the expanded allele was used to calculate the repeat number.31 (link) Long-read whole-genome sequencing was performed using a PromethION sequencer on 2 individuals who had a saw-tooth tail repeat-primed PCR, but no peak was seen on amplicon-length PCR. Library preparation was performed using ligation sequencing 1D kits (SQK-LSK109, Oxford Nanopore Technologies, UK) according to the manufacturer's protocol. About 800 ng DNA libraries were constructed and sequenced on the PromethION platform (Oxford Nanopore Technologies, UK).
10
Detecting NOTCH2NLC Repeat Expansion
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Genomic DNA was extracted from peripheral blood using an automated DNA extraction system (QuickGene-Auto240L, Kurabo, Japan). Repeat expansion in NOTCH2NLC was examined by repeat-primed PCR, followed by electrophoresis on a 3500xl Genetic analyzer (ThermoFisher Scientific, Waltham, MA, USA), as described previously [6 (link)]. Briefly, PCR was performed in a total volume of 10 μL of reaction solution containing 0.25U PrimeSTAR GXL DNA Polymerase; 1 × PrimeSTAR GXL Buffer; 200 µM of dATP, dTTP, dCTP (Takara Bio, Shiga, Japan), and 7-Deaza-2′-deoxy-guanosine-5′- triphosphate (Sigma-Aldrich, St. Louis, MO, USA); 5% dimethyl sulfoxide (Sigma-Aldrich); 1 M betaine (Sigma-Aldrich); 0.3 µM of each primer mix; and 100 ng of genomic DNA. The presence of a sawtooth pattern in the electropherogram was regarded as an abnormal repeat expansion. NOTCH2NLC repeat size was further determined by fluorescence amplicon-length PCR as described previously [6 (link)]. GeneMapper software (ThermoFisher Scientific) was used to determine GGC repeat length.
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