Christopher S Hong, Chunzhang Yang, Zhengping Zhuang

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Presentation transcript:

Application of Peptide Nucleic Acid-based Assays Toward Detection of Somatic Mosaicism Christopher S Hong, Chunzhang Yang, Zhengping Zhuang Molecular Therapy - Nucleic Acids Volume 5, (January 2016) DOI: 10.1038/mtna.2016.22 Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Optimization and application of IDH1 PNA. (a) Gel electrophoresis of PCR products derived from IDH1 (codon 132) sequencing of wild-type DNA with serial dilutions of 100 nmol/l IDH1-specific PNA. The far left column is a 1-kb DNA ladder. Subsequently, from left to right are 1,000-fold (1/1,000), 250-fold (1/250), etc. dilutions of 100 nmol/l PNA. The band intensity is significantly reduced from 1/1,000 to 1/250 and is completely absent at 1/62.5, suggesting complete blockage of amplification by the PNA. A dilution of 1/1,000 was chosen as the appropriate dilution for this IDH1-specific PNA. (b) Sanger sequencing of IDH1 (R132) from leukocyte DNA is shown with and without PNA. Direct sequencing (top panel) demonstrated wild-type IDH1 R132 status. However, addition of the IDH1-specific PNA revealed an IDH1 (R132H) mutation, suggestive of IDH1 somatic mosaicism. PNA, peptide nucleic acids. Molecular Therapy - Nucleic Acids 2016 5, DOI: (10.1038/mtna.2016.22) Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Determination of HIF2A PNA sensitivity. (a) Gel electrophoresis of PCR products derived from HIF2A sequencing of HIF2A wild-type plasmids. The far left column is a 1-kb DNA ladder. Subsequently, from left to right are results from 107 copies/reaction, 106 copies/reaction, 105 copies/reaction, down to 10 copies/reaction on the far right. A total copy number of 106 per reaction was selected as the proper value from which to perform additional PCR with varying proportional mixtures of wild-type and mutant plasmids. (b) Gel electrophoresis of PCR products derived from HIF2A sequencing (with addition of HIF2A-specific PNA) of different proportions of wild-type and mutant plasmids. The far left column is a 1-kb DNA ladder. From left to right are results from plasmid mixtures containing 100% mutant plasmids, 10% mutant plasmids, 5, 2.5, 1.25, 0.6, 0.3, and 0%. Bands were excised and sent for sequencing to determine sensitivity of the HIF2A-specific PNA. (c) Results from experiments similar to b are graphed, demonstrating the enhanced sensitivity of the IDH1-specific PNA-PCR assay compared with standard sequencing. The IDH1-specific PNA was deemed to be sensitive enough to detect 5% of mutant DNA or 10% of mutated cells. HIF2A, hypoxia-inducible factor 2A; PNA, peptide nucleic acids. Molecular Therapy - Nucleic Acids 2016 5, DOI: (10.1038/mtna.2016.22) Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy Terms and Conditions