In order to meet the goals of the Human Genome Program, literally billions of bases of DNA must be sequenced. To accomplish this feat, technology must advance so that the amount of bases determined is vastly increased, the quality of the data is highly accurate and the cost per base is significantly decreased, ie faster, better, cheaper. Additionally, it would be advantageous if technological advances, in addition to impacting large sequencing projects, would benefit researchers in laboratory settings where researchers have access to cost effective equipment that serve a multitude of purposes. Two types of sequencing technologies are being developed and are related by their use of short oligonucleotides. One technology, Cyclic Ligation Sequencing (CLS), uses a thermal cycling procedure to anneal hexamers to a dsDNA template, ligate the hexamers using T4 DNA ligase, denature the ligated primers from the template, and repeatedly cycle this temperature regime. Subsequently, the ligated hexamers are used to prime a DNA sequencing reaction. Optimization of CLS is dependent upon optimization of the ligation reaction. Another technology uses octamers to efficiently prime a DNA sequencing reaction. Optimization of octamer sequencing depends on experimentally determining the rules important in designing octamers which produce high quality sequence data and constitute a reasonably sized primer library. Both CLS and octamer sequencing would be faster; the existence of a primer library eliminates the delay caused waiting for the next primer to be synthesized. Both yield sequencing results equivalent to or better than traditional primer walking due to pre-selection of optimal primers. Both technologies would be significantly cheaper; standard sequencing primers, the major cost in the reaction, would be replaced by a library composed of short oligonucleotides able to prime multiple reactions.
| Kraltcheva, A I; Hardin, S H (2001) Octamer-primed sequencing technology: effects of dNTP supplementation. Nucleic Acids Res 29:E48 |
| Posey, K L; Jones, L B; Cerda, R et al. (2001) Survey of transcripts in the adult Drosophila brain. Genome Biol 2:RESEARCH0008 |
| Mei, G; Hardin, S H (2000) Octamer-primed sequencing technology: development of primer identification software. Nucleic Acids Res 28:E22 |
| Ying, J; Bradley, R K; Jones, L B et al. (1999) Guanine-rich telomeric sequences stimulate DNA polymerase activity in vitro. Biochemistry 38:16461-8 |
| Jones, L B; Hardin, S H (1998) Octamer-primed cycle sequencing using dye-terminator chemistry. Nucleic Acids Res 26:2824-6 |
| Hardin, S H; Jones, L B; Homayouni, R et al. (1996) Octamer-primed cycle sequencing: design of an optimized primer library. Genome Res 6:545-50 |
