Method for detecting point mutations in DNA utilizing fluorescence energy transfer
- Lincoln, NE
A method for detecting point mutations in DNA using a fluorescently labeled oligomeric probe and Forster resonance energy transfer (FRET) is disclosed. The selected probe is initially labeled at each end with a fluorescence dye, which act together as a donor/acceptor pair for FRET. The fluorescence emission from the dyes changes dramatically from the duplex stage, wherein the probe is hybridized to the complementary strand of DNA, to the single strand stage, when the probe is melted to become detached from the DNA. The change in fluorescence is caused by the dyes coming into closer proximity after melting occurs and the probe becomes detached from the DNA strand. The change in fluorescence emission as a function of temperature is used to calculate the melting temperature of the complex or T.sub.m. In the case where there is a base mismatch between the probe and the DNA strand, indicating a point mutation, the T.sub.m has been found to be significantly lower than the T.sub.m for a perfectly match probelstand duplex. The present invention allows for the detection of the existence and magnitude of T.sub.m, which allows for the quick and accurate detection of a point mutation in the DNA strand and, in some applications, the determination of the approximate location of the mutation within the sequence.
- Research Organization:
- Center for Biotechnology, University of Nebraska-Lincoln
- Assignee:
- Board of Regents of University of Nebraska (Lincoln, NE)
- Patent Number(s):
- US 6248518
- OSTI ID:
- 873795
- Country of Publication:
- United States
- Language:
- English
Similar Records
PCR amplfication on a microarray of gel-immobilized oligonucleotides : detection of bacterial toxin- and drug-resistent genes and their mutations.
Statistical analysis of fragment mobility shifts obtained from multi-color fluorescent SSCP
Related Subjects
detecting
mutations
dna
utilizing
fluorescence
energy
transfer
fluorescently
labeled
oligomeric
probe
forster
resonance
fret
disclosed
selected
initially
dye
donor
acceptor
pair
emission
dyes
changes
dramatically
duplex
stage
hybridized
complementary
strand
single
melted
detached
change
caused
coming
closer
proximity
melting
occurs
function
temperature
calculate
complex
base
mismatch
indicating
mutation
found
significantly
perfectly
match
probelstand
allows
detection
existence
magnitude
quick
accurate
applications
determination
approximate
location
sequence
resonance energy
dna strand
melting temperature
energy transfer
fluorescence emission
accurate detection
selected probe
complementary strand
fluorescently labeled
acceptor pair
single strand
approximate location
dna utilizing
/435/999/