The investigator proposes to continue developing methods for assaying nucleic acids of pathogens using """"""""molecular beacons."""""""" Molecular beacons are hairpin shaped nucleic acids containing a fluorescent group on one end and a quencher on the other. Thus these sequences do not normally fluoresce. However, upon hybridization of the oligonucleotide to a continuous complementary sequence, the fluorescent group and the quencher are separated from one another and the duplex molecule becomes fluorescent. Thus a solution of hairpin """"""""molecular beacons"""""""" becomes fluorescent in the presence of the complementary sequence, allowing quantitation of the number of complementary molecules, even in a complex mixture, by measuring fluorescence intensity and comparing it to a standard curve. This method was invented by Dr. Kramer in 1996 and forms the basis for the present proposal. In studies conducted during the previous funding period, molecular beacons were reported to be able detect point mutations in human DNA, detect drug resistant mutants in Mycobacteria, and detect gene expression in Mycoplasma. An application called """"""""spectral genotyping"""""""" was developed, in which probes of different colors are used simultaneously to monitor different alleles. The fluorescence spectrum indicates the ratio of different sequences present in a mixture. Dr. Kramer and coworkers carried out a thermodynamic study of hybridization by hairpin molecules, which revealed that inclusion of hairpins can increase specificity compared to linear hybridization probes. Molecular beacons were also used in a multiplex format using fluorescent labels of different colors to detect four pathogenic retroviruses simultaneously in single blood samples. The investigator proposes six Aims in Research Design and Methods. 1) In order to obtain more colors for multiplex assays, """"""""wavelength shifting"""""""" probes will be developed, in which two fluors are attached to one end of the DNA, so that transfer of energy from one to the other yields a new color. The six probes will then be used to simultaneously detect 6 pathogenic viruses, four human retroviruses plus hepatitis B and C, in clinical samples. 2) A computer program will be developed using the thermodynamic data to improve the design of molecular beacons. 3) A two-step method will be developed to identify drug resistant mutations in HIV in clinical samples. A first set of probes will scan the genome for departures from a standard sequence. Once departures are identified, a second set of probes will identify the specific change. In this way large numbers of clinical samples can be characterized for drug resistant changes. 4) Molecular beacons will be employed to improve the design of DNA chips in collaboration with Affymetrix. Probe sequences will be synthesized attached to a solid support as in current chip designs, but molecular beacon hairpins will be designed into each. This will permit testing of unlabeled probe preparations of nucleic acids, since binding of cold nucleic acid will result in fluorescent signals at defined locations on the chip. Present methods, in contrast, require rather intricate labeling of probe sequences. 5) In order to detect particularly rare sequences, such as HIV drug resistant mutants before initiation of treatment, """"""""allele discriminating primers"""""""" will be designed and tested. These primers will exploit the improved discrimination possible with hairpins relative to linear DNAs, but using cold hairpin primers in conventional PCR. Molecular beacons will also be used to visualize the amplification product. Patient samples provided by Dr. David Ho will be used for these tests. 6) Efforts will be made to use molecular beacons to bind sequences in living cells. This is particularly attractive since cells labeled by this method could be analyzed by FACS.
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