Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-07-20
2001-02-27
Sisson, Bradley L. (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091100, C436S094000, C536S023100, C536S024330
Reexamination Certificate
active
06194179
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for the preparation of quantities of substantially pure polynucleotide sequences, which may be isotopically enriched. Such sequences may be used for therapeutic purposes, for the identification of interactions between the polynucleotide sequences and proteins or other binding partners, and to identify agents which modulate these interactions.
BACKGROUND OF THE INVENTION
Since the advent of DNA thermal amplification technology, numerous procedures have been developed to amplify polynucleotide sequences on a preparative scale. Of these, the concatamer chain reaction developed independently by Rudert et al. (17a) and White et al. (17b) employ a DNA polymerase-catalyzed thermal amplification system for the generation of DNA concatamers. In this procedure, the primer and template for the amplification reaction are the identical molecule, producing large sequences comprising tandem repeats of the target DNA sequence. Following similar procedures, Louis et al. (17c) prepared isotopically-labeled DNA oligonucleotides for NMR spectroscopy, utilizing labeled deoxynucleotide triphosphates. Although the lafter procedure produced oligonucleotides, amounts of product are still limited and restrict the utility of subsequent studies requiring larger quantities of oligonucleotides. Furthermore, these procedures introduce a degree of heterogeneity in the product, making it unsuitable for certain uses, in particular, high resolution heteronuclear NMR spectroscopy.
Multi-dimensional heteronuclear NMR has become a standard technique to determine the three-dimensional structure of proteins and RNA in solution (1,2). One of the most important advances in the application of NMR spectroscopy to the study of biological systems has been the ease of incorporation of
13
C and/or
15
N into proteins (2-5) and RNA.(6-8). The enrichment of macromolecules in these stable isotopes allows for the dispersion of
1
H,
13
C and
15
N chemical shifts into multiple spectral dimensions in a manner that preserves the chemical and/or spatial relationship between atoms within a molecule of interest (2-5). The resulting enhancement of spectral sensitivity and resolution has had a tremendous impact on the study of chemical and biological phenomena of proteins and RNA. (2-8). In contrast, both the detailed analysis of structure and dynamics of DNA in solution have remained largely inaccessible to the NMR spectroscopist despite the ease of preparation of oligonucleotide duplexes of biological interest. Poor proton density and narrow chemical shift dispersion limits the detailed analysis of structural parameters by homonuclear
1
H-NMR to very small oligonucleotides. While it is desirable to apply heteronuclear NMR to the study of DNA in solution, it has required de novo synthesis of DNA precursors for solid-phase synthesis (9-11). These methods require a certain level of synthetic expertise and are both cost and labor intensive. In this regard, an enzymatic approach would be advantageous and a few such methods have been proposed in recent years (12-17).
It is towards the improvement in the quantity and quality of the large scale preparation of polynucleotide sequences, and in particular, isotopically enriched polynucleotide sequences, and uses thereof, that the present invention is directed.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.
SUMMARY OF THE INVENTION
In its broadest aspect, the present invention is directed to a method for preparing a quantity of a preselected polynucleotide sequence which is flanked by half sequence-specific endonuclease sites, following the steps of (1) preparing an amplification template/primer comprising at least one tandem repeat of the preselected polynucleotide sequence separated by a full sequence-specific endonuclease site and flanked by half endonuclease sites; (2) amplifying the template/primer by a DNA polymerase-catalyzed thermal amplification reaction in a first step utilizing an optimized annealing temperature; (3) further amplifying the product of the previous step by a DNA polymerase-catalyzed thermal amplification reaction utilizing an optimized annealing temperature; and (4) cleaving the product of the previous reaction using the endonuclease, producing the preselected polynucleotide sequence. To enhance the quantity of product produced by the above reaction, a quantity of the preselected polynucleotide sequence may be included in the second amplification step.
The template/primer for the above-described procedures comprises at least one tandem repeat of the preselected polynucleotide sequence. It may be prepared by solid-phase phosphoramidite chemistry. The preselected polynucleotide sequence may be, by way of non-limiting example, duplex DNA, single-stranded DNA, triplex DNA, quadruplex DNA, 3-way junction DNA, or 4-way junction DNA. The half endonuclease sites on the template/primer may be blunt-ended or overhanging. Additional endonuclease sites may be included in the template/primer such that selective cleavage after amplification may be used to generate quantities of particular sequences.
To prepare a quantity of an isotopically enriched polynucleotide sequence, the deoxynucleotide triphosphates used in the amplification steps are isotopically enriched. To enhance the quantity of isotopically enriched product, a quantity of the preselected polynucleotide sequence may be included in the second amplification step. This added preselected polynucleotide sequence may or may not be isotopically enriched. Non-limiting examples of isotopically enriched atoms of the deoxynucleotide triphosphates include
13
C,
15
N,
2
H, and any combination thereof.
The optimal annealing temperature of the first amplification step of the above process is selected by determining an annealing temperature which yields a maximal amount of endonuclease-cleaved preselected polynucleotide sequence as a product of the first amplification step. Likewise, the optimized annealing temperature of the second amplification step is selected by determining an annealing temperature which yields a maximal amount of endonuclease-cleaved preselected polynucleotide sequence as a product of the second amplification step.
To prepare isotopically enriched deoxynucleotide triphosphates, enzymatic phosphorylation of deoxynucleotide phosphates may be obtained for example by digestion of nucleic acid isolated from an organism grown on an isotopically enriched carbon source, an isotopically enriched nitrogen source, or the combination of the two. Other isotopically enriched sources may be utilized to provide other isotopically enriched atoms. In one non-limiting example, nucleotide phosphates isolated from an organism may be enzymatically phosphorylated using suitable enzymes in combination with thymidylate monophosphate kinase (TMPK) and cytidylate monophosphate kinase (CMPK).
In a second aspect of the present invention, a method is provided for preparing a quantity of a substantially pure, preselected polynucleotide sequence which is flanked by half sequence-specific endonuclease sites, following the steps of (1) preparing an amplification template/primer comprising at least one tandem repeat of the preselected polynucleotide sequence separated by a full sequence-specific endonuclease site and flanked by half endonuclease sites; (2) amplifying the template/primer by a DNA polymerase-catalyzed thermal amplification reaction in a first step utilizing an optimized annealing temperature; (3) further amplifying the product of the previous step by a DNA polymerase-catalyzed thermal amplification reaction utilizing an optimized annealing temperature; (4) cleaving the product of the previous reaction using the endonuclease, producing the preselected polynucleotide sequence; and (5) isolating the preselected polynucleotide sequence from the previous step. Isolation may be carrier out in a single-step chromatographic procedure, for example, using DEAE ion-exchan
Gupta Vineet
Werner Milton H.
Klauber & Jackson
Lu Frank
Sisson Bradley L.
The Rockefeller University
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