Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Patent
1997-08-29
2000-03-28
Marschel, Ardin H.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
436501, 435 6, 435 912, 536 221, 536 2433, C12N 910, C07H 2104
Patent
active
060430702
ABSTRACT:
The invention relates to novel chimeric phosphoramidate oligonucleotides and their use in primer-extension methods such as DNA sequencing and nucleic acid amplification. The subject chimeric phosphoramidate oligonucleotides have both N3'-phosphoramidate linkages and phosphodiester linkages. The invention includes methods of primer extension using the subject chimeric oligonucleotides as primers. Primer extension methods of interest include nucleic acid amplification reactions, e.g. PCR, and polynucleotide sequencing reactions. In the primer extension methods of the invention, a chimeric phosphoramidate oligonucleotide primer is annealed to a polynucleotide template. After annealing, the chimeric oligonucleotide primer is extended by joining a nucleotide to the 3' end of the primer by a DNA polymerase catalyzed reaction. Other embodiments of the invention include methods of primer extension using phosphoramidate linkage containing polynucleotide templates. Such methods include annealing an oligonucleotide primer (chimeric or otherwise) to a polynucleotide template comprising at least one phosphoramidate linkage. Nucleotides are added to the primer in a DNA polymerase catalyzed reaction. Primer extension takes place across one or more of the phosphoramidate linkages in the template. The invention includes compositions comprising a chimeric phosphoramidate oligo-nucleotide of the invention and a divalent cation. Divalent cations serve to increase the binding affinity between the chimeric oligonucleotides of the invention and a polynucleotide template. Additionally, divalent cations may be used to stabilize the phosphoramidate linkages of the subject chimeric oligonucleotides against hydrolysis at elevated temperatures, such as the temperatures used in PCR and cycle sequencing. The subject compositions may further comprise a thermostable DNA polymerase such as Taq DNA polymerase.
REFERENCES:
patent: 5475925 (1995-12-01), Letsinger et al.
Uhlmann et al., Chemical Reviews, vol. 90, No. 4, pp. 544-584, 1990.
Sommer et al., Nucleic Acids Research, vol. 17, No. 16., p. 6749, 1989.
Peyrottes et al., "Oligodeoxynucleoside phosphoramidates (P-NH.sub.2): synthesis and thermal stability of duplexes with DNA and RNA targets," Nucelic Acids Research 24(10):1841-1848 (1996).
Ellis Nicole M.
Heiner Cheryl R.
Kuimelis Robert G.
Lazaruk Katherine D.
Walsh Patric Sean
Bartner Scott R.
Marschel Ardin H.
The Perkin-Elmer Corporation
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