The objective of the Phase II study is to develop specifications for DNA sequencing kits based on Genomic Coupled Amplification and Sequencing (Genomic CAS). During Phase I, the original CAS procedure was modified to sequence genomic DNA in a one-step process without sample handling. Incompletely extended amplification products were eliminated by adding single- stranded binding protein (SSBP) to the reaction mixture and by 100 percent substitution of dGTP by 7-deaza-dGTP. The key variables to be optimized for genomic CAS for any locus/template combinations were identified. Co-investigators at Yale University broadened the use of the original CAS by constructing phylogenetic trees with CAS-derived sequences, and by complementing CAS with competitive oligo priming to derive haplotypes. The proposed effort will entail genomic CAS of eight loci on each of five genomic templates provided by collaborators at Yale and the Medical College of Virginia. The plans are to update and refine the optimization protocols, sequence the eight loci using manual and automated formats, and to develop specifications for final kit configuration. After the two year period, the technology will be formatted into simple robust manual kits for sequencing any DNA flanked by a set of amplification primers, into at least two """"""""locus- specific"""""""" test kits--one in the area of individual identification and one in the area of diagnosis of parasitic disease, and into fluorescent-based kits compatible with automated DNA sequencing.
