Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-03-27
2004-08-17
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S015000, C435S091100, C536S023100, C536S024300, C536S024330, C536S025320
Reexamination Certificate
active
06777189
ABSTRACT:
FIELD OF THE INVENTION
The invention is in the field of molecular biology.
BACKGROUND
The determination of nucleic acid base sequences is important for both research and diagnostics. Many techniques for determining nucleic acid base sequences have been developed over the years, e.g., controlled chemical degradation (Maxim and Gilbert, Proceedings of the National Academy of Sciences USA 74: 560-564 (1977), 2′3′ dideoxy chain termination method (Sanger et al. Proceedings of the National Academy of Sciences USA 74: 5463-5467 (1977). A variation of the technique of chain termination sequencing is known as single base extension or “mini-sequencing” is performed when nucleic acid base sequence information is required for only a single base site adjacent to the 3′ terminus oligonucleotide primer. The technique of single base extension is described in U.S. Pat. No. 5,856,092 and Syvanen et al. Genomics 8, 684-692(1990).A problem with single base extension techniques is the difficulty associated with identifying the single base extension product, particularly in an electrophoresis . Variations in the signal produced by the single base extension product, e.g., as detected by an electrophoresis apparatus, maybe the result of variations in signal produced by differences between oligonucleotide primers. The problems associated with identifying single base extension products become particularly troublesome in multiplexed single base extension reactions. The inventors have provided methods, compositions, kits and software for ameliorating these problems associated with identifying single base extension reaction products.
SUMMARY OF CERTAIN EMBODIMENTS OF THE INVENTION
One embodiment of the invention is a method of producing an oligonucleotide extended by a single nucleotide base. An oligonucleotide and an extension terminating nucleotide are mixed with an enzyme having terminal transferase activity. The reaction produces an oligonucleotide extended by a single base. The extended oligonucleotide may be used as a size standard for single base extension reactions.
Another embodiment of the invention is a method of producing a mixture of oligonucleotides extended by different single bases. An oligonucleotide, a first extension terminating nucleotide, and a second extension terminating nucleotide are mixed with an enzyme having terminal transferase activity. The first and second extension terminating nucleotides comprise different nucleotide bases and are labeled with different labels. The identity of the different extension terminating nucleotides (and hence the extended oligonucleotides) may be ascertained by reference to the specific label incorporated. Another embodiment of the invention is a method of identifying the reaction products of single nucleotide base extension reactions on a detection instrument, e.g., an automated fluorescence detecting electrophoresis system, such as an Applied Biosystems PRISM® 377, PRISM® 3700 or PRISM® 3100. An oligonucleotide extension product is produced by mixing an oligonucleotide with an extension terminating nucleotide and an enzyme having terminal transferase activity, e.g., a terminal transferase. The single base oligonucleotide extension product may be used as a standard for comparison with the reaction products of single base extension reactions produced using a DNA polymerase, e.g., a mini-sequencing reaction product. Single base oligonucleotide extension products produced by the enzyme having terminal transferase activity may be resolved on a detection instrument, e.g. an electrophoresis apparatus, so as to produce a signal indicative of the single base extension product standard. The signal may be used as a standard for comparison with signals produced by the reaction products of template-dependent single base extension reaction products.
Other embodiments of the invention are kits for performing one or more methods of the invention. Embodiments of the subject kits include kits that comprise a terminal transferase and one or more extension terminating nucleotides. The extension terminating nucleotides may be labeled with the detectable moieties, such as fluorescent dyes.
DEFINITIONS
The term “terminal transferase” as used herein refers to an enzyme having terminal transferase activity, but not having significant DNA polymerase activity. The term “significant” as used in reference to DNA polymerase activity means DNA polymerase activity sufficient to perform a polynucleotide extension reaction that is template dependent at level sufficient to produce detectable amounts of template-dependent oligonucleotide extension product from an oligonucleotide primed template. Examples of terminal transferases include
E. coli
terminal transferase, calf thymus terminal transferase, and the like. Terminal transferases are commercially available from many companies such as Aphonix, Finnzyrnes, MBI Fernentas, New England Biolabs, Promega, Panvera, Sigma Biochemicals, and Roche Molecular Biochemicals .
The term “terminal transferase activity” as used herein refers to the enzymatic catalysis as of a reaction in which nucleotide triphosphates (including extension terminating nucleotides) are covalently attached to the 3′ terminus of an oligonucleotide primer in a template independent manner. Thus, by mixing an enzyme having terminal transferase activity within oligonucleotide having a free 3′-OH (or functional equivalent to) and with a nucleotide triphosphate, one or more nucleotides are added to the 3′ prime terminus of the oligonucleotide, irrespective of the presence or absence of a template complementary to the oligonucleotide.
The term “oligonucleotide” as used herein, unless clearly indicated otherwise by context, broadly refers to a polymer of natural or synthetic nucleobases, or a combination of both. The backbone of the capture polynucleotide can be composed entirely of “native” phosphodiester linkages, or it may contain one or modified linkages, such as one or more phosphorothioate, phosphoramidite or other modified linkages. As a specific example, a polynucleotide may be a peptide nucleic acid (PNA), which contains amide interlinkages. Additional examples of modified bases and backbones that can be used in conjunction with the invention, as well as methods for their synthesis can be found, for example, in U.S. Pat. No. 6,001,983; Uhlman & Peyman, 1990, Chemical Review 90(4):544-584; Goodchild, 1990, Bioconjugate Chem. 1(3):165-186; Egholm et al., 1992, J. Am. Chem. Soc. 114:1895-1897; Gryaznov et al., J. Am. Chem. Soc. 116:3143-3144, as well as the references cited in all of the above. Common modified or synthetic nucleobases of which polynucleotides may be composed include 3-methlyuracil, 5,6-dihydrouracil, 4-thiouracil, 5-bromouracil, 5thorouracil, 5-iodouracil, 6-dimethyl amino purine, 6-methyl amino purine, 2-amino purine, 2,6-diamino purine, 6-amino-8-bromo purine, inosine, 5-methyl cytosine, 7-deazaadenine, and 7-deaza guanosine. Additional non-limiting examples of modified or synthetic nucleobases of which the target nucleic acid may be composed can be found in Fasman, CRC PRACTICAL HANDBOOK OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, 1985, pp. 385-392; Beilstein's Handbuch der Organischen Chemie, Springer Verlag, Berlin and Chemical Abstracts, all of which provide references to publications describing the structures, properties and preparation of such nucleobases. The term “oligonucleotide” as used herein includes oligonucleotides that comprise additional molecules (or atoms) that have been joined, either covalently or non-covalently, to an oligonucleotide. These additional molecules (or atoms) maybe attached to virtually any site on the oligonucleotide, provided the attachment does not prevent the oligonucleotide from being used as a substrate for the enzyme having terminal transferase activity used in a given embodiment of the subject methods. Examples of such additional molecules include mobility modifier compounds such as those described as the subject of U.S. Pat. Nos. 5,514,543, 5,777,096, 5,703,096 and 5
Bishop Danielle
Wei Dong
Applera Corporation
Bortner Scott R.
Finn Andrew K.
Horlick Kenneth R.
Wilder Cynthia
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