To verify the authenticity of mutations detected from WGS analysis, we first designed primer pairs to obtain amplicons covering the mutations by PCR. In brief, we performed 30 cycles of PCR in a 20 μL mixture containing 100 ng DNA, 1 μM of each primer, 1X buffer, 0.25 mM of dNTP, and 0.5 U of Power Taq polymerase (Genomics, New Taipei City, Taiwan) and with the annealing temperature of 63 °C. An aliquot of the amplicon was purified and subjected to Sanger sequencing using the BigDye Terminator kit v3.1 (Applied Biosystems, Foster City, CA, USA). We used the forward primer for sequencing.
Power taq polymerase
Manufactured by Genomics Plc
Power Taq polymerase is a thermostable DNA polymerase enzyme used for polymerase chain reaction (PCR) amplification of DNA. It possesses 5' to 3' DNA polymerase activity and 3' to 5' exonuclease proofreading activity.
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Lab products found in correlation
5 protocols using power taq polymerase
1
Validating Mutations from WGS Analysis
2
Sanger Sequencing of Genomic Mutations
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We designed primer pairs to obtain amplicons that covered the mutation by polymerase chain reaction (PCR)-based Sanger sequencing to verify the authenticity of mutations identified from whole-genome sequencing analysis. In brief, 30 cycles of PCR were performed in a 20 mL mixture containing 100 ng DNA, 1 μM of each primer, 1X buffer, 0.25 mM of dNTP, and 0.5 U of Power Taq polymerase (Genomics, New Taipei City, Taiwan). An aliquot of the amplicon was purified and subjected to Sanger sequencing using the BigDye Terminator kit v3.1 (Applied Biosystems, Foster, CA, USA). The primer sequences, optimal annealing temperature, and size of each amplicon are summarized in Table 2 .
3
Sanger Sequencing for Mutation Verification
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We also used Sanger sequencing to verify the authenticity of the mutations identified from the whole-exome sequencing analysis. In brief, 30 cycles of PCR were performed in a 20 μL mixture containing 100 ng DNA, 1 μM each of the forward primer and the reverse primer, 1X buffer, 0.25 mM of dNTP, and 0.5 U of Power Taq polymerase (Genomics, New Taipei City, Taiwan). An aliquot of the amplicon was purified and subjected to Sanger sequencing using the BigDye Terminator kit v3.1 (Applied Biosystems, Foster, CA, USA).
4
Sanger Sequencing of Mutation Amplicons
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We designed primer pairs to obtain amplicons covering the mutations by polymerase chain reaction (PCR) to verify the authenticity of mutations identified from NGS analysis. The primer sequences, optimal annealing temperature, and amplicon size are listed in Table 1 . We used the forward primer for sequencing. In brief, we performed 30 cycles of PCR in a 20 μL mixture containing 100 ng of DNA, 1 μM of each primer, 1X buffer, 0.25 mM of dNTP, and 0.5 U of Power Taq polymerase (Genomics, New Taipei City, Taiwan). An aliquot of the amplicon was purified and subjected to Sanger sequencing using a BigDye Terminator Kit v3.1 (Applied Biosystems, Foster, CA, USA).
5
Sanger Sequencing Validation of Genomic Variants
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To verify the authenticity of mutations identified from whole-genome sequencing analysis, we designed primer pairs to obtain amplicons that covered the variants of interest using polymerase chain reaction (PCR)-based Sanger sequencing. In brief, 30 cycles of PCR were performed in a 20 μL mixture containing 100 ng DNA, 1 μM of each primer, 1X buffer, 0.25 mM of dNTP, and 0.5 U of Power Taq polymerase (Genomics, New Taipei City, Taiwan). An aliquot of the amplicon was purified and subjected to Sanger sequencing using the BigDye Terminator kit v3.1 (Applied Biosystems, Foster, CA, USA). We have summarized the primer sequences, optimal annealing temperature, and the size of each amplicon in Table 3 .
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