) An important goal of the program project is the development of a high sensitivity polymerase chain reaction/restriction endonuclease/ligase detection reaction (PCR/RE/LDR) method to identify specific mutations in the presence of excess normal DNA (Sensitivity of 1 in 100,000 - 1,000,000 cells). To this end, a new class of nucleotide analogues herein termed """"""""convertides"""""""", have been designed to provide a means to convert a wild-type sequence into one which contains a restriction endonuclease recognition site. Convertides are defined as nucleoside analogues that have base modifications or replacements which allow them to pair to one or more of the natural bases in hybridization step (""""""""read""""""""), and also to function as a template for another base in a DNA replication reaction (""""""""write""""""""). A successful convertide will pair to one or more natural bases when annealing to a target, allowing for efficient extension by Tag polymerase (read), and also functions as a template for incorporation of another base the tag polymerase copies the primer-containing strand (write). For performing the 12 possible base conversions, 7 known deoxyribonucleoside analogues (Q1, Q2, Q4-Q8) and 13 newly-designed, modified deoxyribonucleosides (Q3, Q9-20) will be investigated. Specific objectives include: Synthetic routes to deoxyribonucleosides Q3, Q9-20 and their characterization. ([1] H and [13] C NMR, mass spectrometry, absorption spectroscopy, and elemental analysis; Synthesis of 5'- Dimethoxytrityl (DMT)-protected derivatives of the convertides will be prepared and transformed to 3'-phophoramidites and 3'-linked CPG solid supports for incorporation into oligonucleotides for Projects 1 and 3. The effect of the modified nucleotides on duplex structure will be determined by measuring and analyzing the helix-coil transition of sets of duplexes containing each of the Q nucleosides opposite each of the natural nucleosides. Synthesis of primers with backbone (phosphate and sugar) modifications at or near the 3'-end will be explored for: (1) preventing cleavage of 3'-convertide by proofreading polymerases in PCR reactions (Project 1) and (2) improving fidelity of ligase reactions by destabilization of ligase-DNA complex (Project 3).
Showing the most recent 10 out of 74 publications
