Cytosine deamination of DNA prior to and during amplification, library preparation, and sequencing is the largestsource of errors (C to A) in next-generation sequencing (NGS) data. Thermocycling conditions during PCR sig-nificantly accelerates cytosine deamination. These errors impede the detection of low-abundance variants suchas driver mutations for small primary and secondary tumors and lower fidelity for complex samples such asmicrobiomes and are particularly acute for damaged samples such as formalin-fixed, paraffin-embedded (FFPE)tissue samples. The NGS-based molecular diagnostics market alone is estimated to reach $2.3B by 2025 andneeds better enzymes.We have discovered a new group of thermostable proofreading DNA polymerase A enzymes with uracil-DNAglycosylase (UDG) activity (UDG-DNAP). Preliminary data shows that one of these enzymes, UP19, possessesvery strong 3' exonuclease activity, UDG activity for removing uracil from DNA, and is capable of robust PCRamplification under normal thermocycling conditions. In this proposal, we will further develop this enzyme with agoal of commercializing a first-in-class, thermostable proofreading DNA polymerase A with intrinsic UDG activityfor correcting uracil mistakes in DNA. Specific Aim 1 seeks to characterize and optimize UP19 in standard PCRreactions against industry standard control enzymes. Specific Aim 2 seeks to measure the fidelity of UP19against industry standard enzymes using an Illumina platform and high-quality DNA from flash-frozen tumorsamples as the template. Specific Aim 3 seeks to measure uracil error correction of UP19 using FFPE treatedsamples and the resulting sequence error rate against industry standard enzymes and a standalone mesophilicUDG enzyme. As a result of this project, we will have a better understanding of this novel enzyme family andbetter understand the applicability of UP19 for NGS applications. The ultimate goal will be to develop a betterenzyme for the NGS market that will reduce error rates and misdiagnoses.1
Public Health Relevance Statement: NARRATIVE
The proposed research seeks to develop and benchmark a new, first-in-class, thermostable proofread-
ing DNA polymerase A with uracil-DNA glycosylase (UDG) activity (UDG-DNAP). The workflow will
allow us to optimize PCR amplification conditions, benchmark enzyme fidelity against current industry
standard polymerases using high-quality DNA templates, and assess error rates in comparison with
industry standard polymerases with damaged DNA templates in the form of DNA from paraffin-embed-
ded, formalin-fixed tumor samples. The goal of the research will be to develop and market this new
enzyme as a robust, higher fidelity, enzyme for NGS applications with greatly increased accuracy on
damaged DNA templates.
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Project Terms: Base Sequence ; Nucleotide Sequence ; nucleic acid sequence ; Biological Sciences ; Biologic Sciences ; Bioscience ; Life Sciences ; Cells ; Cell Body ; Cytosine ; Deamination ; DNA ; Deoxyribonucleic Acid ; DNA Damage ; DNA Injury ; DNA-Directed DNA Polymerase ; DNA Polymerases ; DNA-Dependent DNA Polymerases ; DNA Repair ; DNA Damage Repair ; Unscheduled DNA Synthesis ; Enzymes ; Enzyme Gene ; Exonuclease ; Family ; Feasibility Studies ; Freezing ; Genes ; Goals ; Gold ; Institutes ; Libraries ; Methods ; Mutation ; Genetic Alteration ; Genetic Change ; Genetic defect ; genome mutation ; Neoplasm Metastasis ; Metastasis ; Metastasize ; Metastatic Lesion ; Metastatic Mass ; Metastatic Neoplasm ; Metastatic Tumor ; Secondary Neoplasm ; Secondary Tumor ; cancer metastasis ; tumor cell metastasis ; Nevada ; Noise ; Pathology ; Research ; Research Personnel ; Investigators ; Researchers ; Schools ; Testing ; Uracil ; Work ; dinitroaminophenol ; DNAP ; dinitroaminophenyl group ; spleen exonuclease ; 3' exonuclease ; phosphodiesterase II ; spleen phosphodiesterase ; uracil-DNA glycosylase ; Ung DNA glycosylase ; Ura-DNA glycosidase ; Ura-DNA glycosylase ; uracil N-glycosidase ; uracil N-glycosylase ; uracil-DNA glycosidase ; Measures ; DNA polymerase A ; Morphologic artifacts ; Artifacts ; Paraffin Embedding ; DNA Sequence ; Sequence Analysis ; SEQ-AN ; Sequence Analyses ; base ; replicase ; improved ; Acute ; Clinical ; repaired ; repair ; Phase ; Variant ; Variation ; Chemicals ; Licensing ; Error Sources ; Letters ; tool ; Diagnostic ; Frequencies ; Complex ; Protocol ; Protocols documentation ; Reaction ; Best Practice Analysis ; Benchmarking ; Performance ; thermophile ; thermophilic organism ; thermolability ; thermostability ; DNA Replication ; DNA Synthesis ; DNA biosynthesis ; microbial ; Biopsy Sample ; Biopsy Specimen ; Primary Tumor ; Primary Neoplasm ; novel ; DNA amplification ; Sampling ; theories ; Formalin ; Bio-Informatics ; Bioinformatics ; DNA Proofreading ; DNA Replication Proofreading ; Effectiveness ; Tissue Sample ; Polymerase ; Data ; Detection ; Small Business Innovation Research Grant ; SBIR ; Small Business Innovation Research ; Preparation ; Molecular ; Process ; Development ; developmental ; microbiome ; next generation ; comparative ; prototype ; tumor ; next generation sequencing ; NGS Method ; NGS system ; next gen sequencing ; nextgen sequencing ; personalized medicine ; personalization of treatment ; personalized therapy ; personalized treatment ; Industry Standard ; molecular diagnostics ; clinical diagnostics ; DNA sequencing ; DNA seq ; DNAseq ; driver mutation ; driver lesion ;
