Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-12-14
2001-03-06
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S087000, C435S088000, C435S089000, C435S091200, C536S022100, C536S024300, C536S025300, C536S025320, C536S025330, C536S025340, C536S026100, C536S026600, C536S026800
Reexamination Certificate
active
06197555
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to nucleosite/tide compounds useful as substrates for polymerase enzymes, methods for using such compounds in a primer extension reaction, and polynucleotides containing such nucleotide compounds.
BACKGROUND
Nucleic acid sequencing has become a vitally important technique in modern biology and biotechnology, providing information relevant to fields ranging from basic biological research to drug discovery to clinical medicine. Because of the large volume of DNA sequence data to be collected, automated techniques have been developed to increase the throughput and decrease the cost of nucleic acid sequencing methods, e.g., U.S. Pat. No. 5,171,534; Connell et al.,
Biotechniques
, 5(4): 342-348 (1987); and Trainor,
Anal. Chem
., 62: 418-426 (1990).
A preferred automated nucleic acid sequencing method is based on the enzymatic replication technique developed by Sanger, et al.,
Proc. Natl. Acad. Sci
., 74: 5463-5467 (1977). In Sanger's technique, the sequence of a single-stranded template nucleic acid is determined using a nucleic acid polymerase to synthesize a set of polynucleotide fragments wherein the fragments (i) have a sequence complementary to the nucleic acid sequence, (ii) differ in length by a single nucleotide, and (iii) have a 5′-end terminating in a known nucleotide, e.g., A, C, G, or T. In the method, an oligonucleotide primer is hybridized to a 3′-end of the template nucleic acid to be sequenced, the 3′-end of the primer serving as an initiation site for polymerase-mediated polymerization of a complementary polynucleotide fragment. The enzymatic polymerization step, or primer extension reaction, is carried out by combining the template-primer hybrid with the four extendible nucleotides, e.g., deoxynucleotides (“dNTPs”), a nucleic acid polymerase enzyme, and a nucleotide “terminator”, e.g., 2′,3′-dideoxynucleotide triphosphate (“ddNTP”). The incorporation of the terminator forms a primer extension product which lacks a hydroxy group at the 3′-terminus and thus can not be further extended by the polymerase, i.e., the extension product is “terminated”. The competition between the ddNTP and its corresponding terminator for incorporation results in a distribution of different-sized extension products, each extension product terminating with the particular terminator used in the reaction. To discover the complete sequence of the template nucleic acid, four parallel reactions are run, each reaction using a different terminator. To determine the size distribution of the extension products, the extension products are separated by electrophoresis such that products differing in size by a single nucleotide are resolved.
In a modern variant of the classical Sanger technique, each nucleotide terminator is labeled with a fluorescent dye, e.g., Prober et al.,
Science
, 238: 336-341 (1987); and U.S. Pat. No. 5,151,507, and a thermostable DNA polymerase enzyme is used, e.g., Murray,
Nucleic Acids Research
, 17(21): 8889 (1989). Several advantages are realized by utilizing dye-labeled terminators, e.g., (i) problems associated with the storage, use and disposal of radioactive isotopes are eliminated, (ii) the requirement to synthesize dye-labeled primers is eliminated, and, (iii) when using a different dye label for each A,G,C, or T terminator, all four primer extension reactions can be performed simultaneously in a single tube. Using a thermostable polymerase enzyme provides several additional advantages, e.g., (i) the polymerization reaction can be run at elevated temperature thereby disrupting any secondary structure of the template, resulting in fewer sequence-dependent artifacts, and (ii) the sequencing reaction can be thermocycled, thereby serving to linearly amplify the amount of extension product produced, thus reducing the amount of template nucleic acid required to obtain a reliable sequence.
While these modern variants on Sanger sequencing methods have proven effective, several problems remain with respect to optimizing their performance and economy. One problem encountered when using presently available dye-labeled terminators in combination with thermostable polymerase enzymes in a Sanger-type nucleic acid sequencing process, particularly in the case of fluorescein-type dye labels, is that a large excess of dye-labeled terminator over the unlabeled extendible nucleotides is required, e.g., up to a ratio of 50:1. This large excess of labeled terminator makes it necessary to purify the sequencing reaction products prior to performing the electrophoretic separation step in order to avoid interference caused by the comigration of unincorporated labeled terminator species and bona fide labeled sequencing fragments. A typical clean-up method includes an ethanol precipitation or a chromatographic separation as described in ABI PRISM™
Dye Terminator Cycle Sequencing Core Kit Protocol
, PE Applied Biosystems, Revision A, p
402116 (August 1995). Such a clean-up step greatly complicates the task of developing totally automated sequencing systems wherein the sequencing reaction products are transferred directly into an electrophoretic separation process.
A second problem encountered when using presently available dye-labeled terminators in combination with a thermostable polymerase in a Sanger-type nucleic acid sequencing process is that the extent of incorporation of labeled terminators into a primer extension product is variable and therefore results in an uneven distribution of peak heights when the primer extension products are separated by electrophoresis and detected using fluorescence detection. Such uneven peak heights are disadvantageous because they make automated sequence determination and heterozygote detection substantially less reliable.
Thus, there remains a continuing need for labeled nucleotide terminator compounds which do not require a large excess over unlabeled extendable nucleotides in a primer extension reaction and, which produce an even peak height distribution in a Sanger-type sequencing reaction.
SUMMARY
The present invention is directed towards our discovery of a novel class of nucleosideltide compounds including a rigid linker portion and methods for using such compounds. These compounds are particularly useful as labeled terminators and as labeled chain-extending nucleotides in a primer extension reaction, e.g., in a Sanger-type DNA sequencing reaction or in a PCR reaction.
It is an object of the invention to provide a nucleotide which can be used to form a labeled chain-terminating or chain-extending nucleotide.
It is a further object of the invention to provide a labeled chain-terminating or chain-extending nucleotide.
It is yet an additional object of the invention to provide a chain-terminating nucleotide which includes a fluorescent label wherein a reduced excess concentration of such labeled chain-terminating nucleotide over an unlabeled chain-terminating nucleotide is required in a Sanger-type DNA sequencing process.
It is another object of the invention to provide a labeled chain-terminating nucleotide which results in a more even distribution of peak heights in a Sanger-type DNA sequencing process.
It is another object of the invention to provide labeled polynucleotides.
It is an additional object of the invention to provide methods including a primer extension reaction utilizing the nucleotide compounds of the invention.
In a first aspect, the foregoing and other objects of the invention are achieved by a nucleoside/tide compound having the structure
NUC-L-S-LB/LG
wherein NUC is a nucleoside/tide having a nucleobase portion B, L is a rigid linkage, S is a spacer, and LB/LG is a member of a linkage pair or a label. NUC is attached to L through B such that when B is a purine, L is attached to the 8-position of the purine, when B is 7-deazapurine, L is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, L is attached to the 5-position of the pyrimidine. In an important feature of the present invention, L has the structure
Khan Shaheer H.
Menchen Steven M.
Rosenblum Barnett B.
Zhen Weiguo
Grossman Paul D.
Riley Jezia
The Perkin-Elmer Corporation
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