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Detection of Genomic Variations in BRCA1 and BRCA2 Genes by Long-Range PCR and Next-Generation Sequencing Imma Hernan, Emma Borràs, Miguel de Sousa Dias, María José Gamundi, Begoña Mañé, Gemma Llort, José A.G. Agúndez, Miguel Blanca, Miguel Carballo The Journal of Molecular Diagnostics Volume 14, Issue 3, Pages (May 2012) DOI: /j.jmoldx Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions
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Figure 1 Nextera technology to prepare Roche Titanium–compatible libraries. A: A limited-cycle PCR with a four-primer reaction adds Roche Titanium–compatible adaptor sequences. The GS Junior Titanium Primers (black lines), an optional MID (circle), and the transposon sequence (gray line) are shown. B: Nucleotide sequences of the GS Junior Titanium Primers (P1 and P2) are shown, together with 454-compatible adaptor sequences (Ad1 and Ad2), followed by the key (underlined), an optional MID, and the transposon sequence (gray). The Journal of Molecular Diagnostics , DOI: ( /j.jmoldx ) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions
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Figure 2 The PCR for BRCA1 and BRCA2. A: PCR plate layout used to amplify the 11 long-range fragments (A–K) in a single PCR program with a gradient temperature annealing step (shown at the bottom of the diagram). The row and column schemes refer to DNA samples and amplified PCR fragments, respectively. NTC, nontemplate control. B: Agarose gel electrophoresis, 1%, of LR-PCR fragments (A–K). MWM, molecular weight marker. The Journal of Molecular Diagnostics , DOI: ( /j.jmoldx ) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions
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Figure 3 Sequencing depth of BRCA1 and BRCA2 amplicons. Data are the average depth of coding and intronic flanking regions (30 bp) of each LR-PCR fragment for single (dark gray) and multiple (light gray) sample runs. The Journal of Molecular Diagnostics , DOI: ( /j.jmoldx ) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions
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