Sequencing

DNA Sequencing Technologies

We are currently investigating several different approaches to DNA sequencing. Examples include sequencing by denaturation (SBD), sequencing by ligation (SBL), and sequencing by synthesis (SBS):

• SBD is a novel approach to DNA sequencing that involves real-time monitoring of the melting profiles of dideoxy-terminated oligonucleotides on surface-bound clones. A Sanger synthesis reaction produces strands of various lengths, with each strand labeled with a fluorescent molecule that corresponds to the dideoxy nucleotide with which the strand was terminated. A gradual increase in temperature or other denaturants will cause the strands to melt in a length-dependent manner. The corresponding decrease in fluorescent intensity is used to decode the sequence information.
• SBL involves the use of a degenerate population of short oligonucleotides that query one or more positions along a template. Once the complimentary probe hybridizes to the immobilized template, it is enzymatically ligated to an adjacent primer and the query position is determined using epifluoresence or total internal reflection fluorescence (TIRF) microscopy. Multiple rounds of hybridization, ligation, and detection are used to read the DNA sequence.
• SBS utilizes the action of a polymerase to detect the incorporation of labeled nucleotides during DNA synthesis. The templates are typically immobolized on a chip and complimentary strands are generated one nucleotide at a time using fluorescent, terminating nucleotides. After detection, the termination is reversed and synthesis continues on to the next base.