[One-step PCR sequencing]. Final report, July 1, 1994--August 31, 1997
The author explored the use of a novel class of boronated nucleic acids, the boranophosphates, as an alternative, but complementary method to dideoxysequencing. Boranophosphates can be used to directly amplify and sequence single- or double-stranded DNA. Fragments are derived not from truncations during polymerase synthesis, but from insertion and digestion back to a boronated marker or delimiter that was incorporated during exponential amplification. The method, which the author calls Boronated One-Step PCR Sequencing, is unique in that it employs a new class of {alpha}-P-boronated 2{prime}-deoxynucleoside 5{prime}-triphosphates first synthesized in the laboratory. These boronated triphosphates exhibit useful properties: (a) they are heat stable, (b) they can be base-specifically incorporated into DNA during the polymerase chain reaction, and (c) once incorporated, the boranophosphate nucleotide (marker) blocks the action of exonuclease. Thus, the positions of the stably-incorporated boronated markers can be revealed by a simple exonuclease digestion, producing a series of fragments--each of which is terminated base-specifically at the boronated markers--and thereby defining the sequence of the PCR product.
- Research Organization:
- Duke Univ., Durham, NC (United States)
- Sponsoring Organization:
- USDOE Office of Energy Research, Washington, DC (United States)
- DOE Contract Number:
- FG05-94ER61882
- OSTI ID:
- 353382
- Report Number(s):
- DOE/ER/61882-T1; ON: DE99002794; TRN: AHC29923%%215
- Resource Relation:
- Other Information: PBD: [1997]
- Country of Publication:
- United States
- Language:
- English
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