# Complex Mechanisms of Mutation and Mutation Avoidance in Living Cells

> **NIH NIH R35** · UNIVERSITY OF NORTH CAROLINA GREENSBORO · 2022 · $82,743

## Abstract

We are requesting an administrative supplement to purchase equipment for R35 award 5R35GM133483
“Complex Mechanisms of Mutation and Mutation Avoidance in Living Cells” (project period: 09/17/2019 -
07/31/2024). We are requesting to purchase a Bio-Rad Automated Droplet Generator AutoDG Instrument (Bio-
Rad item #1864101) for use with a QX200 Droplet Digital PCR (ddPCR) System and an associated Bio-Rad
PX1 PCR Plate Sealer (Bio-Rad item #1814000) in order to significantly enhance the throughput and
reproducibility of sample processing for the QX200 ddPCR system currently housed at the Joint School of
Nanoscience and Nanoengineering (JSNN) of North Carolina A&T State University and UNC Greensboro
(UNCG), where the PI is faculty (at UNCG and JSNN). ddPCR is an advanced PCR technique where individual
nucleic acids from a sample are encapsulated in small oil-water emulsions prior to amplification by PCR, so that
after the PCR cycles the emulsions that originally contained individual nucleic acids can be counted using
fluorescent detection. ddPCR is at this point an established nucleic acid quantification technology that leads in
precision and accuracy. Compared to traditional quantitative PCR (qPCR) methods such as the standard Applied
Biosystems 7500 Real-Time PCR instrument (also at JSNN), ddPCR allows for absolute quantification of specific
nucleic acids in a sample without the need for or variability of standard reference curves; provides increased
sensitivity for the detection of rare mutants by over an order of magnitude (from >5% prevalence using qPCR to
<0.1% prevalence); and exhibits less sensitivity to PCR inhibitors. These attributes will be crucial for emerging
applications in projects related to the R35 grant. ddPPR and these requested equipment are expected to
significantly help to advance and enhance several parallel lines of research associated with the R35 award in
my laboratory. The reason for this is that the limitation of our ddPCR system is in its sample preparation—while
the QX200 can perform nucleic acid quantification of 96 samples at a time, the standard equipment performs
droplet preparation for only 8 samples at a time and this must be performed manually. The AutoDG system
automates the simultaneous preparation of 96 samples, allowing us to maximize usage of this instrument and
associated consumables. This automated processing will be necessary for the R35 research to significantly
improve sample preparation throughput for the QX200 ddPCR system by orders of magnitude while reducing
intra- and inter-user variability in preparation, all of which will be necessary to the completion of the research in
a timely manner, to the highest quality standards, and at the sensitivity necessary to resolve key differences
between experimental conditions. The PX1 PCR plate sealer will be necessary to maintain biosafety level-
appropriate containment of Biosafety level 2 (BSL2) samples prior to and after droplet preparation.

## Key facts

- **NIH application ID:** 10581066
- **Project number:** 3R35GM133483-04S1
- **Recipient organization:** UNIVERSITY OF NORTH CAROLINA GREENSBORO
- **Principal Investigator:** Eric Alan Josephs
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $82,743
- **Award type:** 3
- **Project period:** 2019-09-17 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10581066

## Citation

> US National Institutes of Health, RePORTER application 10581066, Complex Mechanisms of Mutation and Mutation Avoidance in Living Cells (3R35GM133483-04S1). Retrieved via AI Analytics 2026-06-01 from https://api.ai-analytics.org/grant/nih/10581066. Licensed CC0.

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