A Novel Long-Range PCR Sequencing Method for Genetic Analysis of the Entire PKD1 Gene Ying-Cai Tan, Alber Michaeel, Jon Blumenfeld, Stephanie Donahue,

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A Novel Long-Range PCR Sequencing Method for Genetic Analysis of the Entire PKD1 Gene Ying-Cai Tan, Alber Michaeel, Jon Blumenfeld, Stephanie Donahue, Tom Parker, Daniel Levine, Hanna Rennert The Journal of Molecular Diagnostics Volume 14, Issue 4, Pages 305-313 (July 2012) DOI: 10.1016/j.jmoldx.2012.02.007 Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Top: Map of the PKD1 gene showing the position of the nine pairs of primers used for LR PCR amplification of the entire gene. The structure of the PKD1 gene is shown, with genes 1 to 46 indicated. Black line indicates the duplicated region of PKD1. Bottom: An example of LR PCR amplification of the DNA fragments (2 to 6 kb long) containing all exons and flanking regions of PKD1 analyzed on a 0.5% agarose gel with ethidium-bromide staining. M, size marker. The Journal of Molecular Diagnostics 2012 14, 305-313DOI: (10.1016/j.jmoldx.2012.02.007) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Titration of the amount of input LR PCR DNA in sequencing reactions. The input DNA varied from 49 to 490 ng. An example of an electropherogram of exon11 (fragment Gene8–12) is shown. There are two variants in the regions PKD1 c.2700 G>A (p. =) and PKD1 c.2730 C>T (p. =). Dashed boxes indicate heterozygous mutation in PKD1. The Journal of Molecular Diagnostics 2012 14, 305-313DOI: (10.1016/j.jmoldx.2012.02.007) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 A typical electropherogram of Sanger sequencing using 400 ng of LR PCR product. There is a clean background with low signal to noise, and the three variants [PKD1 c.9195 G>C (p. =), c.9196 T>C (p.F3066L), and c.9330 T>C (p. =)] in the region could be easily called. Dashed circles indicate heterozygous mutation in PKD1. The Journal of Molecular Diagnostics 2012 14, 305-313DOI: (10.1016/j.jmoldx.2012.02.007) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions