A radically new approach for the fluorescence in situ detection of DNA is proposed, which makes it possible to detect short (about 20-bp-long) single-copy DNA sequences in metaphase chromosome spreads and in interphase nuclei under non-denaturing conditions. The method of fluorescence in situ detection of short sequences (FISDOSS) to be developed will be exceedingly specific because a circular probe will be assembled via ligation of synthetic oligonucleotides on short DNA sequences opened up by specially designed peptide nucleic acids (PNAs). A high sensitivity will be provided by an efficient contamination-immune isothermal method of signal amplification: rolling-circle amplification (RCA) of the assembled circular probes with incorporation of numerous fluorescently labeled nucleotides. All procedures will be performed directly on slides and the final detection of interphase nuclei and metaphase chromosomes will be done by standard techniques using a fluorescent microscope. In Phase I, proof-of-principle experiments will be performed on arbitrarily chosen sites unique for the human genome. The goal of Phase I is to demonstrate, after initial optimization, that the short specific sequences can be effectively and specifically detected within non-denatured metaphase human chromosomes. The method will be extended to parallel multiple detection of various unique sites in the human genome. To demonstrate that FISDOSS is applicable to detect genetic markers of cancer, 12 appropriate sites associated with chronic lymphocytic leukemia (CLL) will be tested. The implementation of the project will yield a convenient fluorescent in situ technique with a great potential for reliable and highly sensitive diagnosis of cancer on the DNA level. ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA112418-02
Application #
7060370
Study Section
Special Emphasis Panel (ZCA1-SRRB-C (O1))
Program Officer
Tricoli, James
Project Start
2005-05-03
Project End
2008-04-30
Budget Start
2006-05-10
Budget End
2008-04-30
Support Year
2
Fiscal Year
2006
Total Cost
$162,236
Indirect Cost
Name
Boston University
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
049435266
City
Boston
State
MA
Country
United States
Zip Code
02215