ULTRASENSITIVE NON-ISOTOPIC DETECTION SYSTEM FOR DNA

Project Details

Description

Phase I of this proposal relates to the development of a non-isotopic
detection system for DNA, specifically for use in Southern Blot, gene
mapping experiments. The system will utilize a chemiluminescent substrate
for alkaline phosphatase, AMPPD, which provides state of the art detection
capabilities. Kits will be designed to enzymatically incorporate biotin
into both short oligoprobes and large double stranded probes. Protocols or
reagents detecting these probes using streptavidin alkaline phosphatase and
AMPPD will be provided. Specialized blocking reagents and conditions
designed to minimize background signal will be developed. The
chemiluminescent signal from AMPPD will be detected on both x-ray film and
instant black and white film. We expect to provide equal or lower
sensitivities that those obtained with 32P or 35S labeled probes. In
addition, we expect to obtain results in a few hours, compared to days with
32P. A major advantage of a non-isotopic probe system is the stability of
the reagents. Unlike 32P labeled probes, biotinylated probes have very
long shelf lifes. Phase II of this program will investigate further increases in sensitivity.
Also, a system for direct digital imaging of chemiluminescent blots will be
developed
StatusFinished
Effective start/end date5/1/908/31/05

Funding

  • National Institutes of Health: $82,597.00
  • National Institutes of Health: $89,250.00
  • National Institutes of Health: $96,058.00
  • National Institutes of Health: $90,404.00
  • National Institutes of Health: $98,233.00

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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