Abi 3730xl dna analyzer

Manufactured by Thermo Fisher Scientific

Sourced in United States, China, United Kingdom, Germany, Japan, Canada, Cameroon

The ABI 3730xl DNA Analyzer is a high-throughput capillary electrophoresis instrument designed for DNA sequencing. It features 96 capillaries and can generate up to 192 sequencing reads per run. The instrument utilizes laser-induced fluorescence detection to analyze DNA fragments and produce sequence data.

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Lab products found in correlation

618 protocols using abi 3730xl dna analyzer

Molecular Profiling of Tumor Samples

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MSI status was determined by PCR (FP205-02, Tiangen Biochemical Technology Co., Ltd., Beijing, China) using a panel of five microsatellite markers including three dinucleotide (D2S123, D5S346, D17S250) and two mononucleotide (BAT25, BAT26) repeats, and the PCR products after amplification were detected and analyzed by capillary electrophoresis with ABI 3730XL DNA Analyzer (ABI, USA) [31 ]. Microsatellite instability-high (MSI-H) was defined when there were two or more instability markers, MSI-L was defined when there was only one instability marker, and if there was no instability among the five markers, it was judged to be MSS.
The SNP of XRCC1 (rs25487) was determined by PCR and Sanger sequencing by ABI 3730XL DNA Analyzer (ABI, USA).
The human NRAS mutation detection kit (YZYMT-019-C, Wuhan YZY Medical Science & Technology Co., Ltd., Wuhan, China) and the human BRAF V600E detection kit (SMD-02-026, Beijing SinoMDgene Technology Co., Ltd., Beijing, China) were used to detect the relevant mutation sites.

Li Y., Lou J., Hong S., Hou D., Lv Y., Guo Z., Wang K., Xu Y., Zhai Y, & Liu H. (2023). The role of heavy metals in the development of colorectal cancer. BMC Cancer, 23, 616.

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IGF1 Repeat Polymorphism Genotyping

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The IGF1 19-CA repeat polymorphism was genotyped by PCR amplification and subsequent analysis of the PCR products’ length using the 96-capillary ABI 3730xl DNA Analyzer. The PCR was carried out using 100 ng of DNA, 10.75 μl MilliQ, 2.5 μl 10x PCR buffer, 0.875 μl of 50 mM MgCL2, 2 × 0.125 μl of Primer predilution-mix (10 times diluted), 0.5 μl of 10 mM dNTP mix, and 0.125 μl of Platinum Taq Polymerase (Life Technologies, Bleiswijk, the Netherlands). The primers (forward: 5′-ACCACTCTGGGAGAAGGGTA-3′; reverse: 5′-GCTAGCCAGCTGGTGTTATT-3′) were fluorescently labelled with 6-FAM (blue), NED (yellow), and PET (red), which enabled the simultaneous analysis of three samples in a single run on the ABI 3730xl DNA Analyzer. The protocol was carried out in the dark because of the light-sensitivity of the fluorescent labels. The PCR reactions were performed using the following cycles: 94 °C for 10 min, followed by 35 cycles of 94 °C for 30 sec, 55 °C for 30 sec, and 72 °C for 30 sec, followed by 72 °C for 10 min and 4 °C for 30 min. The analysis included 314 duplicate samples and 436 water controls. The reproducibility of the IGF1 19-CA repeat analysis was 93.6%. Genotyping was successful for 70.7% of samples.

Simons C.C., Schouten L.J., Godschalk R.W., van Engeland M., van den Brandt P.A., van Schooten F.J, & Weijenberg M.P. (2015). Genetic Variants in the Insulin-like Growth Factor Pathway and Colorectal Cancer Risk in the Netherlands Cohort Study. Scientific Reports, 5, 14126.

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High-Throughput Sanger Sequencing of PCR Amplicons

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The purified PCR amplicon was sequenced according to manufacturer's instruction directly by using modified Sanger's dideoxy terminator cycle sequencing chemistry on an automated capillary-based DNA sequencer (ABI 3730xl DNA Analyzer) in both forward and reverse direction twice using amplified product specific primers. The PCR products were run in a cycle sequencing reaction with thermal cycling conditions as 30 cycles of denaturation (95°C for 20 s), annealing (60°C for 20 s), and extension (60°C for 4 min) followed by hold at 4°C. The purified sequencing products were resolved on a capillary-based automated DNA sequencer (ABI 3730xl DNA Analyzer). Full length sequence reads were obtained by assembly of multiple reads of each fragment using Phred/Phrap and Consed software (Ewing and Green, 1998 (link)). Each fragment was sequenced at least four times and high quality (Phred 20) consensus sequence was used for data analysis.

Thakur S., Singh P.K., Das A., Rathour R., Variar M., Prashanthi S.K., Singh A.K., Singh U.D., Chand D., Singh N.K, & Sharma T.R. (2015). Extensive sequence variation in rice blast resistance gene Pi54 makes it broad spectrum in nature. Frontiers in Plant Science, 6, 345.

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Determination of MSI and SNP Status

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Determination of MSI status was carried out using a panel of five microsatellite markers including two mononucleotide (BAT25, BAT26) and three dinucleotide (D2S123, D5S346, D17S250) repeats by PCR. The PCR process was as follows: 95 °C for 2 min, 40 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30s and 7 min and a final step at 4 °C. PCR products were analyzed by capillary electrophoresis with ABI 3730XL DNA Analyzer (ABI, USA). MSI-H is defined when at least two of the five markers show instability in tumor DNA. MSI-L was defined when one MSI marker showed instability and others were MSS when there was no instability on tumor DNA.
SNP of ERCC1 (c. C354T) and XRCC1 (c.G1196A) were determined by PCR and Sanger sequencing by ABI 3730XL DNA Analyzer (ABI, USA).

Zhang L., Zhao J., Yu B., Song X., Sun G., Han L., Wang L, & Dong S. (2017). Correlations between microsatellite instability, ERCC1/XRCC1 polymorphism and clinical characteristics, and FOLFOX adjuvant chemotherapy effect of colorectal cancer patients. Cancer genetics, 218-219.

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Eranthis Chloroplast Microsatellite Genotyping

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All of the 935 individuals of the four Eranthis species were genotyped at 12 chloroplast microsatellite loci (Ebp01, Ebp40, Ebp27, Ebp31, Ebp25, Ebp12, Ebp10, Ebp06, Ebp38, Ebp11, Ebp28, Ebp32) which were randomly selected from the 24 cpSSR loci isolated by Oh and Oh (2017). The PCR procedure was the same as that of Oh and Oh (2017), and the length of PCR products was measured using the ABI3730xl DNA Analyzer (Applied Biosystems) and GeneMapper v. 3.7 (Applied Biosystems).
In addition, one individual from each population of the four focal Eranthis species (giving a total of 33 individuals) and five outgroup species, were sequenced at two chloroplast noncoding regions, rpl16 intron and petL‐psbE (Shaw et al., 2005; Shaw, Lickey, Schilling, & Small, 2007). Sequencing was conducted in both directions with ABI3730xl DNA Analyzer (Applied Biosystems), and the consensus sequences were created with Sequencher 4.8 (Gene Codes Corp., Ann Arbor, MI, USA).

Oh A, & Oh B. (2019). The speciation history of northern‐ and southern‐sourced Eranthis (Ranunculaceae) species on the Korean peninsula and surrounding areas. Ecology and Evolution, 9(5), 2907-2919.

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Molecular Characterization of KEAP1 Gene

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Total RNA was extracted and used to synthesize the first chain cDNA according to the procedure described in the real-time PCR analysis description. cDNA was used as a template to synthesize six segments of the KEAP1 gene using the following cycling conditions: initial denaturing step at 94 °C for 5 min followed by 30 cycles at 94 °C for 40 s, 60 °C for 40 s, and 72 °C for 55 s. Products were separated on 1 % agarose gels, and bands were visualized with ethidium bromide. The products were sequenced using the ABI3730 XL DNA Analyzer (Applied Biosystem Japan, Tokyo, Japan) and analyzed with Chromas 2.4.3 software. Primers used to amplify the KEAP1 gene fragments were:

Fragment 1, forward AGAGGTGGTGGTGTTGCTTAT

Reverse TGGAGATGGAGGCCGTGTA

Fragment 2, forward CAGGTCAAGTACCAGGATG

Reverse GATGAGGGTCACCAGTTG

Fragment 3, forward ATCGGCATCGCCAACTTC

Reverse AGGTAGCTGAGCGACTGT

Fragment 4, forward CAGAAGTGCGAGATCCTG

Reverse GCTCTGGCTCATACCTCT

Fragment 5, forward GCCCTGGACTGTTACAAC

Reverse GTCTCTGTTTCCACATCGTA

Fragment 6, forward GCTGTCCTCAATCGTCTC

Reverse AGTTCTGCTGGTCAATCTG

NRF2 exon2, forward TCGTGATGGACTTGGAGCTG

Reverse AGCATCTGATTTGGGAATGTG

Sections were spliced together after manual inspection and compared with the reference sequence with BLAST to identify potential mutations.

Tian Y., Wu K., Liu Q., Han N., Zhang L., Chu Q, & Chen Y. (2016). Modification of platinum sensitivity by KEAP1/NRF2 signals in non-small cell lung cancer. Journal of Hematology & Oncology, 9(1), 83.

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Pharmacokinetics and Genotyping of Busulfan

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Blood samples for PK analysis and genotyping were withdrawn from central venous lines, in heparinized glass tubes, pre-infusion, 0.5, 1, 2, 2.5, 4 and 6 hours after the first infusion. Plasma concentrations of Bu were determined using a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).1 (link) The lower limit of quantitation was 10 ng/mL and the range of quantitation was from 10 to 10,000 ng/mL.
Pretransplant genomic DNA was isolated and extracted from whole blood prior to the first Bu infusion. Improve potassium iodide methods was applied for DNA extraction from whole blood. Ammonium chloride was used to destroy red blood cells, and potassium iodide was used to destroy white blood cells and their nuclear membranes for a short time. Then, proteins, lipids and residual cell debris are precipitated by chloroform/isopropanol. Finally, DNA is precipitated by isopropanol, and was washed by ethanol. The extracted genomic DNA was dissolved with TE. After confirming the DNA concentration and purity, PCR amplification and purification of PCR product were performed. GST genotypes of patients, GSTA1 (rs3957356 and rs3957357, which defines haplotype *A and *B) and GSTM1 (rs3754446), were detected with the ABI 3730XL DNA Analyzer (Applied Biosystem).26 (link)

Supporting Information Table 2

displays the primer sets and Tm used for the genotyping assays.

Yuan J., Sun N., Feng X., He H., Mei D., Zhu G, & Zhao L. (2021). Optimization of Busulfan Dosing Regimen in Pediatric Patients Using a Population Pharmacokinetic Model Incorporating GST Mutations. Pharmacogenomics and Personalized Medicine, 14, 253-268.

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Genetic Characterization of Scleractinian-Associated Hydroids

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The total genomic DNA of 63 ethanol-fixed Zanclea samples from 13 scleractinian genera was extracted following a protocol modified from Zietara et al. [58 ]. Three different molecular markers were amplified: (1) a ~300 bp portion of the nuclear 28S ribosomal DNA gene (28S), (2) a ~400 bp portion of the mitochondrial 16S ribosomal RNA gene (16S), and (3) a ~700 bp portion of the mitochondrial cytochrome oxidase subunit I gene (COI). The first two regions of DNA have been extensively used to infer phylogenetic relationships among hydroids in numerous previous molecular studies [26 (link), 28 , 44 , 45 , 59 –61 ]. We also selected the barcoding region of COI gene because it turned out to be useful for species delimitation in Hydrozoa [40 (link), 62 (link)]. 16S and 28S genes were amplified using hydroid-specific primers and the protocols proposed by Fontana et al. [26 (link)]. The barcoding region of COI gene was amplified using universal primers LCO1490 and HCO2198 and the protocol proposed by Folmer et al. [63 (link)]. All PCR products were purified and directly sequenced in forward and reverse directions using an ABI 3730xl DNA Analyzer (Applied Biosystem, Foster City, CA, USA). The sequences obtained in this study were deposited with the EMBL, and the accession numbers are listed in Table 1.

Montano S., Maggioni D., Arrigoni R., Seveso D., Puce S, & Galli P. (2015). The Hidden Diversity of Zanclea Associated with Scleractinians Revealed by Molecular Data. PLoS ONE, 10(7), e0133084.

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Molecular Characterization of Streptomyces by 16S rRNA Analysis

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Molecular characterization of Streptomyces was carried out by 16S rRNA analysis. The cells from the biomass were harvested by centrifugation, washed, and re-suspended in TE buffer (10 mM Tris/HCl, 1 mM EDTA; pH 8.0). Genomic DNA was extracted by a standard protocol and the PCR amplification was conducted in Eppendorf Master Cycle gradient AG22331 (Model No. 5331), using appropriate forward primer 27F (5' AGA GTT TGA TCC TGG CTC AG 3') and reverse primer 1525R (5' AGA AAG GAG GTG ATC CAG CC 3') in the E. coli numbering system [8 (link), 9 ]. The amplified products were sequenced in Applied Biosystem Sequencer (ABI 3730XL DNA Analyzer) with appropriate primers.
This 16S rRNA gene sequence was used for phylogenetic analysis. The 16S rRNA gene sequence related taxa were acquired from the GenBank database and the primer was designed using the Primer 3 software. Multiple sequence alignment was conducted using the Clustal W program and the phylogenetic tree was constructed using the neighbor-joining method [10 (link), 11 (link)] using the MEGA7 software (https://www.megasoftware.net/) and phylogeny program (http://www.phylogeny.fr/). The evolutionary distances for the neighbor-joining and maximum likelihood tree were calculated by Kimura’s two-parameters method. The topologies of each tree have been evaluated using bootstrap resampling methods based on 1,000 replications.

Ghosh M., Gera M., Singh J., Prasad R, & Pulicherla K.K. (2020). A Comprehensive Investigation of Potential Novel Marine Psychrotolerant Actinomycetes sp. Isolated from the Bay-of-Bengal. Current Genomics, 21(4), 271-282.

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Enterovirus Serotype Identification via RT-PCR

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The nested RT-PCR yielded about a 389bp amplicon. The final PCR products were subjected to sequencing in both directions using the ABI 3730 XL DNA Analyzer (Applied Biosystem Inc., Foster City, CA). Nucleotide sequences of 5’-UTR were checked by the BLAST search in the NCBI database to identify the enterovirus serotype with the highest identity. Obtained sequences were assembled using SeqMan software (version7.1.0). Phylogenetic tree was constructed by neighbor-joining method with 1000 bootstrap replications using MEGA6.0. Reference sequences representing EV71, CVA (2, 4, 6, 10, 12, 16), CVB4, Echo (3, 9, 25) and HEV-C were downloaded from NCBI database and selected for phylogenetic analysis.

Guan H., Wang J., Wang C., Yang M., Liu L., Yang G, & Ma X. (2015). Etiology of Multiple Non-EV71 and Non-CVA16 Enteroviruses Associated with Hand, Foot and Mouth Disease in Jinan, China, 2009—June 2013. PLoS ONE, 10(11), e0142733.

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