DEVELOPMENT OF ULTRABRIGHT WATER-SOLUBLE DYES CONJUGATED TO BIOMOLECULES

Southern University At Shreveport, La, Shreveport LA

Investigators

Abstract

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