Specific variants of interest mentioned throughout the text (DICER1, HLTF, LPP, PLK3, RAD51D, RELB, SH3GL2, and SPTAN1) and highlighted as bold in the tables were validated using specific primers for polymerase chain reaction amplification designed with Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/ ) and Sanger sequencing on a 3,500 Dx Genetic Analyzer (Life Technologies, CA, USA), using ABI PRISM 3.1 Big Dye terminator chemistry, according to the manufacturer's instructions. The electrophoretic profiles were analyzed manually. Segregation analysis of the prioritized variants was performed in additional family members when DNA was available. Primer details are available on request.
Abi prism 3.1 big dye terminator chemistry
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI PRISM 3.1 Big Dye terminator chemistry is a reagent system used in DNA sequencing applications. It provides fluorescently labeled dideoxynucleotides for the termination of DNA synthesis during the sequencing process.
Automatically generated - may contain errors
6 protocols using abi prism 3.1 big dye terminator chemistry
1
Primer Design and Sanger Sequencing
2
Sanger Sequencing of Variant Identification
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Specific variants of interest mentioned throughout the text were all confirmed by direct Sanger sequencing on a 3500 Dx Genetic Analyzer (Life Technologies, CA, USA), using ABI PRISM 3.1 Big Dye terminator chemistry (Life Technologies, CA, USA) according to the manufacturer’s instructions.
3
Genetic Variants Analysis Protocol
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For all the patients, the entire coding region of GJB2 was analyzed by Sanger sequencing (primers available upon request). According to the manufacturer’s instructions, DNA was sequenced on a 3500 Dx Genetic Analyzer (Life-Technologies, Carlsbad, CA, USA), using ABI PRISM 3.1 Big Dye terminator chemistry (Life Technologies, Carlsbad, CA, USA). Moreover, GJB6 deletions (D13S1830-D13S1854) were screened by multiplex PCR using the method described by del Castillo et al. 2002 [19 (link)], while The A1555G mtDNA mutation was tested by Restriction Fragment Length Polymorphism (PCR-RFLP) analysis using BsmAI as restriction enzyme, followed by visualization on an agarose gel stained with ethidium bromide.
4
Validation of Variants via Sanger Sequencing
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After filtering the variants based on the FCVPPv2, we visually inspected the WGS data for validity using the Integrative Genomics Viewer (IGV) (26 (link)). The final selection of potentially causative variants was based on a thorough review of available literature. We screened WGS data from the other four NMTC families that were part of our previous study for the selected variants. Selected variants were validated by Sanger sequencing of DNA samples of all available family members using specific primers for polymerase chain reaction amplification designed with Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/ ). Primer details are available on request. Sequencing was performed on a 3500 Dx Genetic Analyzer (Life Technologies, CA, USA) using ABI PRISM 3.1 Big Dye terminator chemistry according to the manufacturer’s instructions. The electrophoretic profiles were analyzed manually. Segregation of the variant with the disease was confirmed.
5
Validating Variant of Interest by Sanger Sequencing
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After filtering the variants based on the FCVPPv2, we visually inspected the WGS data for correctness using the Integrative Genomics Viewer (IGV) [42 (link)]. The final selection was based on a thorough literature review. The selected variant of interest (POT1 p.V29L) was validated by Sanger sequencing of DNA samples of all available family members using specific primers for polymerase chain reaction amplification designed with Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/ ). Primer details are available on request. Sequencing was performed on a 3500 Dx Genetic Analyzer (Life Technologies, Carlsbad, CA, USA) using ABI PRISM 3.1 Big Dye terminator chemistry according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). The electrophoretic profiles were analyzed manually. Segregation of the variant with the disease was confirmed.
6
Sanger Sequencing for Variant Confirmation
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Sanger sequencing was used to confirm candidate variants, and to define genetic inheritance mode of candidate variant as familial segregation testing. All candidate variants were sequenced bidirectionally with ABI PRISM 3.1 Big Dye terminator chemistry (Applied Biosystems, Foster City, CA). Sequencing products were resolved on an ABI PRISM 3130XL sequencer (Applied Biosystems), and chromatograms were analyzed with Sequencher 4.9 (Gene Codes, Ann Arbor, MI).
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