All potential functional variants were validated with Sanger sequencing in an Applied Biosystems 3130 capillary sequencer using individual primers in both directions to obsolete false positive errors. The technically uncovered 1304 bp regions of the 13 targeted genes were carefully sequenced directly by Sanger sequencing. No more functional variant was detected in all 120 patients. To evaluate the sensitivity and false-positive-rate of this panel, we randomly selected seven genes (MYH7, MYBPC3, ACTC1, PRKAG2, MYOZ2, ACTN2, JPH2) and sequenced all exons and flanking regulation regions directly by Sanger sequencing in eight subjects from the 120 HCM patients.
3130 capillary sequencer
Manufactured by Thermo Fisher Scientific
The 3130 Capillary Sequencer is a genetic analysis instrument designed for DNA sequencing applications. It features a capillary-based electrophoresis system that separates and detects fluorescently labeled DNA fragments. The 3130 Capillary Sequencer provides automated sample loading, data collection, and analysis capabilities for DNA sequencing workflows.
Automatically generated - may contain errors
Lab products found in correlation
Exonuclease 1 en 0582, by Thermo Fisher Scientific (2 mentions)
Abi prism big dye terminator kit version 3, by Thermo Fisher Scientific (2 mentions)
Shrimp alkaline phosphatase, by Thermo Fisher Scientific (1 mentions)
Pcr buffer, by Thermo Fisher Scientific (1 mentions)
Exonuclease 1, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
Multina, by Shimadzu (1 mentions)
6 protocols using 3130 capillary sequencer
1
Sanger Sequencing Validation of HCM Gene Variants
2
Targeted HRM-PCR Mutation Detection
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DNA was isolated from whole blood. HRM-PCR was performed using Qiagen Type-it PCR Polymerase Mix (Qiagen, Hilden, Germany) in a Rotor Gene Q thermocycler (Qiagen). Reaction results were analyzed using the Rotor-Gene Q Series Software Version 2.2.3 relative to control samples. All mutations detected using the HRM-PCR technique were confirmed using capillary sequencing using the 3130 Capillary Sequencer (Applied Biosystems/ThermoFisher Scientific). Details are described in S1 Supplementary Methods .
3
PCR Amplification and Sequencing Protocol
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Selected fragments were amplified by PCR using Taq polymerase (Invitrogen) in a Biometra T Professional Gradient 96 cycler. Amplification mixtures contained 10 pmol of each primer, PCR buffer (Invitrogen), 1.5 mM MgCl2, 50 ng of genomic DNA, 200 µM dNTPs, 2.5 U Taq polymerase (Invitrogen), and water to a final volume of 25 µl. After denaturing at 94°C for 2 minutes, thermal cycling was performed for 35 cycles at 94°C for 30 seconds, followed by 30 seconds at a temperature set to 5°C less than the melting temperature of the selected primers, followed by 72°C for 30 seconds. Reactions were finished by a 5 minute incubation at 72°C. Amplification products were checked in 1.2% agarose gels stained with ethidium bromide to verify the presence of a single amplification product. Next, an aliquot (10 µl) of the amplification reaction was treated with 1 U of Exonuclease I (Fermentas) and 10 U of Shrimp Alkaline Phosphatase (Fermentas) for 45 minutes at 37°C and then for 30 minutes at 80°C to inactivate these enzymes. Subsequently two sequencing reactions were prepared, each with one of the primers used for the amplification of the product. Sequencing was carried out in an Applied Biosystems 3130 capillary sequencer using a Big-Dye terminator cycle sequencing kit, according to the instructions of the manufacturer.
4
Capillary Sequencing and DNA Quantification
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The curves diverging in shape from the control curve (WT) were verified by applying capillary sequencing. The concentration of DNA in these samples was measured using the electrophoresis device Multina (Shimadzu, Japan). For the Sanger sequencing reaction, PCR amplification products were purified using 10 U (Cat. No. EN 0582) of exonuclease I and 1 U of phosphatase Fast-AP (Cat. No. EF 0651) (Thermo Fisher). The reaction was incubated for 15 min at 37 °C, followed by 20 min at 80 °C.
Sequencing reactions were performed using forward and reverse sequence-specific primers (described above) and the ABI-PRISM Big Dye Terminator version 3.1 kit (Applied Biosystems, Foster, CA; Cat. No. 4337450), according to the manufacturer’s instruction manual. Sequencing reactions were analyzed using the 3130 Capillary Sequencer (Applied Biosystems, Foster, CA). The generated sequences were compared relative to the reference sequence RefSeq (NM_007272.2), using the NCBI Blast Nucleotide program.
Sequencing reactions were performed using forward and reverse sequence-specific primers (described above) and the ABI-PRISM Big Dye Terminator version 3.1 kit (Applied Biosystems, Foster, CA; Cat. No. 4337450), according to the manufacturer’s instruction manual. Sequencing reactions were analyzed using the 3130 Capillary Sequencer (Applied Biosystems, Foster, CA). The generated sequences were compared relative to the reference sequence RefSeq (NM_007272.2), using the NCBI Blast Nucleotide program.
5
Confirming CHEK2 Mutations by Sanger Sequencing
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All mutations (I157T, IVS2+1G > A, and delC1100) detected by TaqMan PCR and allele-specific PCR were confirmed by capillary sequencing. PCR amplification products, purified by incubation with 10 U of exonuclease I (EN 0582) and 1 U of phosphatase Fast-AP (EF 0651) (both from ThermoFisher Scientific, Waltham, MA) for 15 min at 37 °C, followed by 20 min at 80 °C, were used as template for Sanger sequencing reactions. Sequencing reactions were performed using forward and reverse sequence-specific primers (CHEK2_EK3_F & CHEK2_EK3_R; F2 CONTROL & R2 CONTROL; CHEK2_1100delC_3_F & CHEK2_1100delC_3_R) and an ABI PRISM Big Dye Terminator Kit, version 3.1 (catalogue number: 4337450, Applied Biosystems/ThermoFisher Scientific), according to the manufacturer’s instructions. Sequencing results were analyzed using a 3130 Capillary Sequencer (Applied Biosystems/ThermoFisher Scientific). The sequences generated were compared with the reference sequence (NM_007194.4) using the NCBI BLAST Nucleotide tool. We decided to use Sanger sequencing instead of next-generation sequencing tests (NGS) in the analysis because the sensitivity of Sanger sequencing is at the level of 20% of allelic frequency so it is sufficient to detect germline mutation which are at the level of about 50% allelic frequency [37 (link)].
6
Sanger Sequencing Protocol for Tumor Mutation Verification
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Thanks to the close cooperation with our Genetic Clinic, mutations detected in tumor tissue were verified (germline vs. somatic) using DNA isolated from blood sample when it was available from the same patient. The Sanger Sequencing reaction used PCR amplification products that were purified using 10 U of exonuclease I (EN 0582) and 1 U of phosphatase Fast-AP (EF 0651) (both from ThermoFisher Scientific, Waltham, MA). The reaction was incubated for 15 min at 37°C, followed by 20 min at 80°C. Sequencing reactions were performed using forward and reverse sequence-specific primers and the ABI PRISM Big Dye Terminator Kit, version 3.1 (catalogue number 4337450, Applied Biosystems/ThermoFisher Scientific), according to the manufacturer's instructions. The sequencing results were analyzed using the 3130 Capillary Sequencer (Applied Biosystems/ThermoFisher Scientific). The generated sequences were compared to the reference sequence using the NCBI Blast Nucleotide program.
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