Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-05-29
2001-07-10
Zitomer, Stephanie W. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091200, C435S252300, C435S325000, C536S025320
Reexamination Certificate
active
06258567
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to a method for uniformly labeling double-stranded DNA with stable isotopes of carbon and nitrogen and, more particularly, to the use of the polymerase chain reaction with labeled deoxynucleotide triphosphate precursors and Taq DNA polymerase to prepare uniformly labeled DNA duplexes.
BACKGROUND OF THE INVENTION
Since the advent of well-developed and standard techniques for the syntheses of uniformly
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N-labeled proteins and ribonucleic acids (RNA), multinuclear magnetic resonance (NMR) spectroscopy has been routinely applied for the determination of their structures. By comparison, the determination of DNA structures by multidimensional NMR is infrequent primarily due to the lack of economic and efficient techniques for the synthesis of uniformly
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N-labeled DNA molecules. The availability of
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N-labeling allows resolution and sequential assignment of spin-systems belonging to individual nucleotides in a long DNA duplex which, in turn, results in the generation of a large set of distance constraints that are essential for determining high-resolution structures. The generation of NMR spectra of uniformly labeled DNA enables the delineation of the structures of DNA complexes with specific proteins and the identification of conformational changes in DNA upon protein binding.
In principle, labeled DNA molecules can be chemically or enzymatically synthesized. However, the preparation of such oligodeoxynucleotides (DNA) by chemical synthesis is uncommon because the generation of uniformly
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N-labeled, chemically protected mononucleotides required for phosphoramidite polymerization is costly, inefficient and technically challenging, since such species are not available commercially. DNA labeling by enzymatic methods was introduced by D. P. Zimmer et al. in “NMR Of Enzymatically Synthesized Uniformly
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N-Labeled Oligonucleotides,” Proc. Natl. Acad. Sci., U.S.A. 92, 3091 (1995) and includes template-directed synthesis using Klenow DNA polymerase and a mixture of labeled deoxynucleoside triphosphate precursors (dNTPs). However, control of the extent of chain elongation is difficult; therefore, for preparation of labeled DNA having a desired length, hairpin templates must be designed such that the single-stranded overhang in the stem controls the length of the newly synthesized DNA strand. Alternatively, an RNA primer may be used in the enzymatic synthesis of labeled DNA having a defined length. In both procedures, uniformly
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N-labeled DNA duplexes are obtained by adding equimolar quantities of two complementary strands made from two different templates. Inherent in these methods, then, is a step in which the newly synthesized labeled strand is separated from an unlabeled template. This is achieved using denaturing gel electrophoresis, followed be elution of the DNA from the gel matrix. Poor yields of labeled DNA and residual polyacryamide contamination are concerns for NMR applications.
The Polymerase Chain Reaction (PCR) has recently been used to prepare uniformly isotope-labeled DNA oligonucleotides. In “Preparation Of Uniformly Isotope-Labeled DNA Oligonucleotides For NMR Spectroscopy,” by John M. Louis et al. J. Bio. Chem. 273, 2374 (1998), the chain extension is based upon self-priming which leads to synthesis of multiple copies of the sequence of interest having flanking restriction sites. Self-priming PCR works well only for GC-rich DNA strands, but not for AT-rich strands, since for AT-rich DNA strands, the melting temperature is significantly lowered and the accuracy of amplification deteriorates. Additionally, Louis et al. reports that some of the oligonucleotides cannot be cleaved to monomers, so that an additional chromatography step is necessary to remove the approximately 10% of unwanted dimeric material present after the DNA amplification.
Accordingly, it is an object of the present invention to provide a general method for preparing uniformly
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N-labeled single- or double-stranded oligodeoxynucleotides with high efficiency and accuracy.
Another object of the present invention is to provide a general method for preparing uniformly
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N-labeled single- or double-stranded oligodeoxynucleotides, without the necessity for complex purification steps.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
SUMMARY OF THE INVENTION
To achieve the foregoing and other objects, and in accordance with the purposes of the present invention, as embodied and broadly described herein, the method for the preparing
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N-labeled oligomers of this invention includes: attaching one Hinc II moiety to each end of the oligomer to be labeled; inserting the oligomer thus formed into a plasmid at a Hinc II site therein; amplifying the oligomer using PCR with the plasmid-oligomer as a template, chosen distinct upper and lower primers, and
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N-enriched dNTPs as precursors, such that a
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N-enriched oligomer strand is produced having an overall length determined by the primers selected and the length of the oligomer; and digesting the Hinc II moieties, whereby the desired
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N-labeled oligomer is generated.
It is preferred that the plasmid is inserted into cells which are grown to produce recombinant clones containing inserts of the oligomer before using PCR to amplify the plasmid-oligomer template.
Preferably also, the generated
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N-enriched oligomer produced is purified and then massively amplified using PCR with the chosen upper and lower primers and
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N-enriched dNTPs as precursors, before the Hinc II moieties are digested.
Benefits and advantages of the present invention include an efficient and general procedure for preparing labeled oligomers without the requirement of significant purification steps.
REFERENCES:
Zon et al. The Polymerase Chain Reaction Colony Miniprep, Bio Techniques, vol. 7(7), p. 696-8, 1989.*
Sambrook et al. Molecular Cloning: a laboratory manual, second edition, p. 14.14-14.17.*
Daniel P. Zimmer et al., “NMR of Enzymatically Synthesized Uniformly 13C15N-Labeled Oligonucleotides,” Proc. Natl. Acad. Sci., U.S.A. 92, 3091 (1995).
John M. Louis et al., “Preparation of Uniformly Isotope-Labeled DNA Oligonucleotides of NMR Spectroscopy,” J. Bio. Chem. 273, 2374 (1998).
Carmen C. Robinett et al., “In Vivo Localization of DNA Sequences and Visualization of Large-Scale Chromatin Organization Using Lac Operator/Repressor Recognition,” J. Cell Biol. 135, 1685 (1996).
Bradbury E. Morton
Chen Xian
Gupta Goutam
Freund Samuel M.
The Regents of the University of California
Tung Joyce
Zitomer Stephanie W.
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