We will use a thermophilic (Tth) ligase in a DNA amplification technique known as ligase chain? reaction (LCR), a one-step ligase detection reaction (LDR) to revolutionize DNA diagnostics. The key here is? that the Tth ligase is incredibly sensitive to mismatches at the ligation juncture with error rates < 1 in 103.? This is because the ligase couples the high selectivity of hybridization of two DNA primers to a template with? enzyme recognition and ligation of two perfect hybridizations. Because of this, the ligase reaction (LCR or? LDR) can actually be used as a very selective and sensitive discrimination event in many DNA diagnostic? applications, including identification of point mutations in high background of wild type DNA sequences (up? to 1 in 1000), and nucleotide repeat analysis. Barany and coworkers have recently demonstrated that it is? possible to multiplex PCR/LDR detection of all the known 19 point mutations associated with colorectal? cancer in codons 12, 13 and 61 of K-ras with a sensitivity of 1 mutation in the presence of 500 wild type? sequences.? The goal of this research project is to develop ultrabright near-IR dyes that are water-soluble and can be? conjugated to biomolecules for use in multiplexed genetic-based assays using near-IR, time-resolved? fluorescence.
The specific aims of the project are:? 1. Prepare asymmetrical phthalocyanines (Pc) which contain water-soluble groups and also a? single functional group for labeling nucleic acids and? 2. Perform basic photophysical measurements on the novel dye sets to determine their? compatibility with typical genetic assay conditions and reagents.? 3. Covalently label oligonucleotides for application in LDR-based mutation detection schemes.
