DNA DAMAGE QUANTITATION BY SINGLE MOLECULE LASER SIZING

Project: Research project

Project Details

Description

DESCRIPTION: (Applicant's Description)
Using single-molecule sizing by laser-induced fluorescence from DNA moving
through a microfabricated channel, this project will develop novel
technologies for quantifying DNA damage produced by ionizing radiation and
other carcinogens. Damages include DNA strand breaks and oxidative damage to
DNA bases, and clusters containing such lesions. This technology will reduce
the cost and improve the sensitivity and throughput for quantifying DNA
damage. It will quantify low levels of damage and can be applied to DNA
damaged in situ, thus facilitating basic research on DNA damage and repair and
the relationship of these processes to mutation induction and carcinogenesis.
Potential clinical applications include determining the ability of normal and
tumor cells to repair damage, thus permitting identification of individuals
who may be at elevated risk or the optimum agents to use against a particular
cancer.

In the R21 phase, we shall assemble a laser system, optics and microfluidic
DNA transport system and demonstrate its ability to count single DNA molecules
and accurately determine their size. In addition, we shall demonstrate the
ability of this system to determine the number average molecular lengths and
frequencies of ionizing radiation induced strand breaks in populations of DNA
molecules of known lengths.

In the R33 phase, the emphasis will shift to focus on the human DNAs from
cells and tissues found both in research and clinical settings. We will work
with the much larger DNA molecules available from human cells, because the
sensitivity of lesion detection increases with the size of the molecules that
can be analyzed. We shall also extend the range of lesions that can be
quantified, with particular emphasis on double-strand breaks and multiply
damaged sites containing heterogeneous mixtures of strand breaks, oxidized
bases and adducts, which are difficult for cells to repair accurately. We
shall also work towards reducing the quantity of DNA required for a
determination of lesion frequency to the level found in individual human
cells. The basic technology for single-molecule sizing will be advanced in
the R33 phase, first by improving the sensitivity of detection of fluorescence
from single DNA molecules by photon counting and phase-sensitive detection.
Improvements in fluorescence detection achievable with near-infrared
fluorescence will be studied, and near-infrared fluorescent DNA labels
developed, leading to a compact system optimized for high sensitivity and
throughput and low cost.
StatusNot started