PCR ANALYSIS OF RADIATION-INDUCED DNA DELETION SPECTRA

Project: Research project

Project Details

Description

Exposure to ionising radiation is known to cause deleterious effects in
humans, many of which are thought to be mediated by the induction of
somatic and germline mutation. A new type of assay with which to determine
levels of mutation directly from human DNA samples has recently been
developed by the author. The assay uses the Polymerase Chain reaction
(PCR), an enzymatic technique for amplifying genetic sequences to detect
deletion mutations of the mitochondrial genome which occur in human DNA.
Basal (spontaneous) levels of a particular deletion mutation (delta1) were
detected somatic tissues of normal adult humans with this assay, and appear
to increase with age (Cortopassi and Amheim, 1990). The identical,delta1
deletion occurs at high level in some rare, sporadically inherited human
genetic diseases. A PCR-based assay may have several intrinsic advantages
over other existing assays of radiation-induced somatic mutation. The
number of mutant molecules can be directly determined from purified DNA;
therefore, the level of somatic mutation can be determined from any source
from which DNA is available, including tissue culture or fresh, frozen, or
formalin-fixed human mitotic or postmitotic tissues. Because the assay is
DNA-based, no "expression time" subsequent to occurrence of the mutation is
required for detection. The high copy number of mitochondrial DNA (mtDNA)
and deficiency in recombination and repair of mtDNA deletions might make
this system an optimal genetic marker of radiation exposure, since the
effective target size is relatively large, and multiple hits in
mitochondrial targets are known not to be lethal to the cell. Aims of this
proposal include the development of a PCR-based assay of a nuclear gene
(ADA) which is thought to contain a "deletion hotspot", determination of
the effect of ionising radiation on absolute levels of mtDNA and ADA
deletions in human tissue culture, and its effect on relative frequencies
of particular mtDNA deletions (the mutation spectrum) in tissue culture.
Preliminary data which bear on several of these objectives have been
collected. The experimental design of the radiation experiments involves
exposure of cells to the desired radiation dose, preparation of DNA from
the exposed cells, and quantitative determination of the number of deleted
forms relative to normal mtDNA or ADA genes using techniques that have been
developed (Cortopassi and Amheim, 1990).
StatusFinished
Effective start/end date5/1/925/31/96

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $77,658.00
  • National Institutes of Health: $72,703.00

ASJC

  • Medicine(all)

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