Fiscal Year:
2002
Title:
DEVELOPMENT OF DNA CHIP WITH HAIRPIN OLIGONUCLEOTIDES
Agency:
HHS
Contract:
1R43RR017102-01
Award Amount:
$85,136.00
Abstract:
DESCRIPTION (provided by applicant): Many of the applications of
oligonucleotide arrays, especially the complex and quantitative applications
such as Sequence By Hybridization (SBH) are hampered by errors resulting from
the variable hybridization properties of the small probes and the stability of
their hybrids. Non-specific hybridization also cause a low signal to noise
ratio. We propose a method that is aimed at addressing these drawbacks by use
of 'snap-back' oligonucleotide probes and novel detection methods. Hairpin
oligonucleotide probes that are composed of mixed backbone of ribose, and
deoxyribose or modified or non-natural phosphodiester linkages such as methyl
phosphonates will be used to setup pseudo-competitive hybridization reactions
to increase specificity. Such oligonudeotide probes will be designed in a way
that would render a region of the probe susceptible to RNase H digestion when
hybridized to the target DNA sequence. The 'snap-back' hairpin oligonucleotide
probe containing methyl phosphonate residues in one strand and RNA in the
opposite strand of the stem will not be digestible by RNase-H. After
hybridization of the RNA strand to the target DNA stand, the RNA strand can be
digested with RNase-H. The hairpins with single stranded region that are left
attached to the substrate will be detected using one of the novel detection
methods such as fluorescent-labeled oligonucleotide mixture. The intact and
un-hybridized probe would snap back rendering themselves un-reactive in the
detection stage, resulting in a low background.
We propose to validate this approach and compare it with the current SBH
methods by sequencing short segments of DNA. Subsequently, these studies will
be extended to typing of rifampicin resistant Mycobacterium tuberculosis. Our
long-term goal for Phase II of the project will be to use the snap-back probes
in genotyping mutations such as those in the presenilin I gene that are related
to Alzheimer. Ultimately, we would extend its application in genotyping
mutations for other genetic diseases in clinical settings and physicians'
offices.
Small Business Information at Submission:
APPLIED GENE TECHNOLOGIES, INC.
APPLIED GENE TECHNOLOGIES, INC. 6190 CORNERSTONE CT E, #101 SAN DIEGO, CA 92121
EIN/Tax ID:
330816239
DUNS:
N/A
Number of Employees:
N/A
Woman-Owned:
No
Minority-Owned:
No
HUBZone-Owned:
No