Genomic DNA from BM aspirates were isolated using a Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA). Pyrosequencing was carried out using a PCR primer mix for KRAS exon 2 (codon 12, 13) and NRAS exon 3 (codon 61) (Supplementary Table 1 ). Each PCR mix contained forward and reverse primers (1 μL), 10× PCR buffer (2 μL), dNTP (0.2 μL), water (19.65 μL), Hotstar Taq Polymerase (0.15 μL), and 2 μL of genomic DNA for a total volume of 25 μL. PCR was done on a GeneAmp PCR system 9700 Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA) with an initial activation step at 95°C for 15 min, 45 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec, followed by a final extension cycle at 72°C for 15 min. Pyrosequencing was performed with an 80 μL final volume, containing 40 μL of biotinylated PCR product with high purity water, 37 μL of PyroMark Binding Buffer, and 3 μL of streptavidin beads (GE Healthcare, Uppsala, Sweden). Pyrosequencing was performed using a PyroMark Q96 ID instrument according to the manufacturer’s instructions (Biotage, Uppsala, Sweden).
Pyromark q96 id instrument
Manufactured by Biotage
Sourced in Sweden
The PyroMarkTM Q96 ID instrument is a high-throughput pyrosequencing system designed for DNA analysis. It utilizes a sequencing-by-synthesis approach to generate DNA sequence information.
Automatically generated - may contain errors
Lab products found in correlation
Pyromark gold q96 reagent, by Qiagen (4 mentions)
Rotor gene 6000, by Qiagen (2 mentions)
Psq hs 96a pyrosequencer, by Biotage (2 mentions)
Streptavidin beads, by GE Healthcare (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Geneamp pcr system 9700 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Pcr buffer, by Thermo Fisher Scientific (1 mentions)
Platinum taq dna polymerase high fidelity, by Thermo Fisher Scientific (1 mentions)
Epitect bisulfite kit, by Qiagen (1 mentions)
6 protocols using pyromark q96 id instrument
1
Pyrosequencing of KRAS and NRAS Mutations
2
Molecular Identification of Taenia Species
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The cox1 gene fragments (208 bp) were amplified from Taenia DNA samples. Each PCR reaction had a final volume of 25 µl containing 1×PCR buffer (Invitrogen, Carlsbad, CA) with 0.2 mM of each dNTP, 2 mM MgSO4, 0.2 µM of Cest_F primer, 0.2 µM of biotinylated Cest_R primer, 0.625 U of Platinum Taq DNA polymerase high fidelity (Invitrogen) and 2 µl of the DNA template. The amplification procedure was as follows: 5 min at 94°C for the initial denaturation, 35 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 52°C, and extension for 30 s at 72°C, followed by a final extension for 10 min at 72°C. The molecular sizes of the amplicons were verified by electrophoresis on 1.5% agarose gels. After the PCR amplification, the biotinylated PCR products were placed in 96-well plates containing 0.4 µM Cest_S sequencing primer and processed by using the PyroMark Q96 ID instrument (Biotage) as previously described [22] (link), [23] (link). Following the completion of the pyrosequencing reaction, a pyrogram was produced and analyzed. The readouts were interpreted manually in case the target sequences contained homopolymers longer than four bases.
3
K-Ras Mutation Analysis using Pyrosequencing
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The K-Ras codons 12/13 mutational analysis was evaluated amplifying 5 μL of genomic DNA concentrated 20 ng/μL by Anti-EGFR MoAb response® (KRAS status) CE-IVD marked kits (DIATECH Pharmacogenetics, Italy) on Rotor-GeneTM 6000 (Corbett Research, Australia) following the manufacturer’s instructions. The resulting PCR product was immobilized onto magnetic streptavidin-coated beads via the biotin/streptavidin interaction. The bead/DNA complex was then washed and added to 1.65 pmol of pyrosequencing primer included in the same kit. The primed single-stranded DNA templates were then transferred to the microtitre plate-based PSQ HS 96 A Pyrosequencer (Biotage, Sweden), where real-time sequencing of the sequence surrounding codon 12/13 exon 2 of K-Ras was performed by using PyroMark Gold Q96 reagents (QIAGEN) on PyroMarkTM Q96 ID instrument (Biotage). The results were analyzed using PyroMark Q24 1.0.9 software, and were independently evaluated by two investigators.
4
KRAS and BRAF Mutation Detection
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A total of 5 μl genomic DNA (20 ng/μl) was amplified using anti-EGFR Moab response® (KRAS and BRAF status) conformité Européene-in vitro diagnostic (CE-IVD)-marked kits (Diatech Pharmacogenetics Srl, Jesi, Italy) on a Rotor-GeneTM 6000 (Corbett Research, Sydney, Australia), according to the manufacturer’s instructions. The resulting PCR product was immobilized onto magnetic streptavidin-coated beads (Diatech Pharmacogenetics Srl) via biotin/streptavidin interaction. The bead/DNA complexes were then washed and added to 1.65 pmol pyrosequencing primer, which was included in the same kit. The primed, single-stranded DNA templates were transferred to a microtiter plate-based PSQ HS 96A Pyrosequencer (Biotage AB, Uppsala, Sweden) in which the real-time sequencing of the sequence surrounding codons 12 and 13 of KRAS and codon 600 of BRAF was performed using PyroMark Gold Q96 reagents (Qiagen) on a PyroMarkTM Q96 ID instrument (Biotage AB). The results were analyzed using PyroMark Q24 1.0.9 software.
5
CpG Methylation Analysis of CDKN2B
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CpG methylation in the promoter region of CDKN2B was quantified using pyrosequencing. The EpiTect Bisulfite Kit (Qiagen, Hilden, Germany) was used for bisulfite conversion of genomic DNA; the PyroMark Gold Q96 Reagents (Qiagen) and the PyroMarkTM Q96 ID instrument (Biotage AB, Uppsala, Sweden) were used for pyrosequencing, according to the manufacturer's instructions. A total of seven CpG sites were analyzed per sample. The primers for the analysis of CDKN2B-specific CpG regions were the same as those used by our group in a previous study [20 (link)].
6
Genetic Analysis of EZH2, KRAS, and BRAF
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Researchers who were not aware of EZH2 and H3K27 immunohistochemistry results and were blinded to patients’ clinical data performed genetic analyses. EZH2 SNP [c.626–394C > T (rs3757441)] was analyzed through real-time PCR, as previously described [8 (link)]. KRAS exon 2 (codon 12–13) and BRAF exon 15 (codon 600) mutations were detected via real-time sequencing using PyroMark Gold Q96 reagents (QIAGEN) on PyroMarkTM Q96 ID instrument (Biotage, Sweden), as reported elsewhere [10 (link)]. Results were analyzed with PyroMark Q24 1.0.9 software.
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