Engineering a high-fidelity, damage-tolerant polymerase for forensic analysis of damaged DNA samples

As submitted by the proposer: DNA analysis is an integral part of our justice system and is widely used for forensic analysis. It can provide much needed evidence in cases to either implicate or exonerate certain individuals. In some cases however, this analysis cannot be used. The first step of the analysis utilizes polymerase chain reaction (PCR) to amplify the DNA found so that it can then be sequenced for small tandem repeat (STR) analysis. Unfortunately, samples can be degraded, whether it is through storage conditions, the environment, or other factors that can lead to DNA damage. In some instances, this damage is severe enough to block PCR, the first step used in forensic analysis. PCR utilizes a polymerase to replicate DNA efficiently and very accurately. Unfortunately, this DNA polymerase cannot replicate past certain DNA damages, leading to an unfinished reaction unsuitable for STR analysis. There have been previous attempts to overcome this issue, one being the inclusion of translesion synthesis (TLS) polymerases. These are specialized polymerases that can bypass DNA damage, including those that complicate forensic analysis. While they can replicate past the damage, this comes at a cost of much reduced accuracy, which compromises the integrity of the analysis. To overcome this problem, we propose to engineer a polymerase than can bypass DNA damage while maintaining high fidelity. We will begin the engineering process with a thermostable replicative polymerase and then will introduce lesion bypass ability. The process has been split into three distinct phases, with computational and experimental methods to gauge the success at each step. The first phase is introducing to the polymerase the ability to bind damaged DNA templates. The next phase is to give the polymerase the ability to preferentially form the correct Watson-Crick base pair, ensuring high fidelity. The final phase is to confirm that the polymerase can still catalyze the polymerization reaction. Computational techniques will be used at each step to help guide the engineering process and gauge the effectiveness of mutations. Experimental methods will be used to determine if the selected variants are capable of binding the substrates and catalyzing the polymerization. While the first two phases advance the goal of binding the substrates, the final phase ensures an active enzyme. In the end, we hope to have developed a high-fidelity, damage-tolerant polymerase that can bypass DNA damage that currently hinders forensic analysis. This project contains a research and/or development component, as defined in applicable law. nca/ncf

As submitted by the proposer: DNA analysis is an integral part of our justice system and is widely used for forensic analysis. It can provide much needed evidence in cases to either implicate or exonerate certain individuals. In some cases however, this analysis cannot be used. The first step of the analysis utilizes polymerase chain reaction (PCR) to amplify the DNA found so that it can then be sequenced for small tandem repeat (STR) analysis. Unfortunately, samples can be degraded, whether it is through storage conditions, the environment, or other factors that can lead to DNA damage. In some instances, this damage is severe enough to block PCR, the first step used in forensic analysis. PCR utilizes a polymerase to replicate DNA efficiently and very accurately. Unfortunately, this DNA polymerase cannot replicate past certain DNA damages, leading to an unfinished reaction unsuitable for STR analysis. There have been previous attempts to overcome this issue, one being the inclusion of translesion synthesis (TLS) polymerases. These are specialized polymerases that can bypass DNA damage, including those that complicate forensic analysis. While they can replicate past the damage, this comes at a cost of much reduced accuracy, which compromises the integrity of the analysis. To overcome this problem, we propose to engineer a polymerase than can bypass DNA damage while maintaining high fidelity. We will begin the engineering process with a thermostable replicative polymerase and then will introduce lesion bypass ability. The process has been split into three distinct phases, with computational and experimental methods to gauge the success at each step. The first phase is introducing to the polymerase the ability to bind damaged DNA templates. The next phase is to give the polymerase the ability to preferentially form the correct Watson-Crick base pair, ensuring high fidelity. The final phase is to confirm that the polymerase can still catalyze the polymerization reaction. Computational techniques will be used at each step to help guide the engineering process and gauge the effectiveness of mutations. Experimental methods will be used to determine if the selected variants are capable of binding the substrates and catalyzing the polymerization. While the first two phases advance the goal of binding the substrates, the final phase ensures an active enzyme. In the end, we hope to have developed a high-fidelity, damage-tolerant polymerase that can bypass DNA damage that currently hinders forensic analysis. This project contains a research and/or development component, as defined in applicable law. Note: This project contains a research and/or development component, as defined in the applicable law, “and complies with Part 200 Uniform Requirements – 2 CFR 200.210(a)(14). nca/ncf