Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-06-11
2001-09-11
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C536S026600
Reexamination Certificate
active
06287821
ABSTRACT:
FIELD OF THE INVENTION
The present invention describes the design and synthesis of a class of internally quenched, fluorogenic substrates for nucleic acid polymerases. More specifically, the present invention describes the design and synthesis of nucleotide analogues with 3′-pro-fluorescent fluorophores (3′-PF-ddNTPs), which are useful as detectors of specific enzyme activity, as well as terminator and non-terminator substrates for nucleic acid polymerases. Methods of the present invention include the use of the nucleotides of the present invention in nucleic acid sequence analysis, as well as the kinetic analysis of polymerase activity.
BACKGROUND OF THE INVENTION
I. Sequence Analysis Using Dideoxynucleotides
Chain terminating 2′,3′-dideoxynucleotide triphosphates (ddNTPs) are widely used in nucleic acid sequencing technology (Sanger, et al.,
Proc. Nat. Acad. Sci.(USA)
, 74:5463 (1977)). Traditionally, Sanger sequencing involves the polymerase-mediated incorporation of a dideoxynucleotide onto the 3′ end of an elongating DNA chain. Incorporation of a dideoxynucleotide results in chain termination, as dideoxynucleotides lack a 3′-OH suitable for further elongation. Methods for Sanger sequencing are disclosed, for example, in J. Sambrook, et al.,
Molecular Cloning, A Laboratory Manual
, 2
nd Edition
, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and in J. W. Zyskind, et al.,
Recombinant DNA Laboratory Manual
, Academic Press, Inc., New York (1988)), both herein incorporated by reference.
In Sanger sequencing, four reactions are typically performed for each polynucleotide to be sequenced. Each reaction contains a DNA template, an oligonucleotide primer, and a mixture of all four deoxynucleotide triphosphates (dNTPs). In addition, each reaction contains one of the four ddNTPs. Either the oligonucleotide primer or the ddNTPs are radiolabeled. The reaction results in a series of radioactivly labeled, prematurely terminated extension reaction products, which are then separated on individual lanes of a high resolution polyacrylamide gel. The gel is then dried and exposed to photographic or x-ray film to record the results for analysis. The procedure is time-consuming, requires significant technical expertise, and involves the use of both radioactive and neurotoxic materials.
The original chain termination or Sanger method has been improved in several ways, and serves as the basis for all currently available automated DNA sequencing machines. See, e.g., Sanger et al.,
J. Mol. Biol
. 143:161-78 (1980); Schreier et al.,
J. Mol. Biol
. 129:169-72 (1979); Smith et al.,
Nucleic Acids Res
. 13:2399-2412 (1985); Smith et al.,
Nature
321:674-79 (1987); Smith et al., U.S. Pat. No. 5,171,534; Prober et al.,
Science
238:336-41 (1987); Section II,
Meth. Enzymol
. 155:51-334 (1987); Church et al.,
Science
240:185-88 (1988); Swerdlow et al.,
Nucleic Acids Res
. 18:1415-19 (1989); Ruiz-Martinez et al.,
Anal. Chem
. 65:2851-58 (1993); Studier,
Proc. Natl. Acad. Sci
. (
USA
) 86:6917-21 (1989); Kieleczawa et al.,
Science
258:1787-91; and Connell et al.,
BioTechnigues
5:342-348 (1987)).
II. Fluorescently Labeled Nucleotides
In an effort to overcome some of the obvious drawbacks and limitations of Sanger sequencing, fluorescently labeled dideoxynucleotides have been developed for use in nucleic acid sequencing (Bergstrom et al.,
J. Am. Chem. Soc
., 98:1587-(1976); Hobbs et al., (U.S. Pat. No. 5,047,519; Ward et al., U.S. Pat. No. 5,449,767). In these fluorescently labeled ddNTPs, the dye is conjugated to the base. The use of four distinguishably labeled ddNTPs has further allowed the process to be condensed to a single reaction, run on a single lane on a gel (L. G. Lee, et al.,
Nucleic Acids Res
., 20:2471-2483 (1992)). This has facilitated the automation of DNA sequencing.
Nevertheless, the use of fluorescent labels in nucleic acid sequence analysis also has its drawbacks. Fluorescent labels are less sensitive than radioactive labels. Consequently, either a greater amount of material must be used to achieve a detectable signal or the template DNA must be amplified by a method such as the Polymerase Chain Reaction (PCR). In addition, Sanger sequencing with fluorescently labeled ddNTPs retains the drawbacks inherent in polyacrylamide gel use. That is, it is time-consuming, requires significant technical expertise, and utilizes acrylamide (a neurotoxin).
III. 3′-Substituted Nucleotides
Nucleotides substituted at the 3′ position of the sugar have also been reported to function as terminators for polymerases. Substitutions reported to efficiently terminate DNA synthesis include azido (H. Mitsuya, et al.,
Proc. Natl. Acad. Sci USA
, 83:1191-1915 (1986)), mercapto (A.A. Yuzhakov, et al.,
FEBS Lett
., 306:185-188 (1992)), amino (M. Herrlein, et al.,
Helvetica Chimca Acta
, 77:586-596 (1994)), and fluoro groups (Z. G. Chidgeavadze, et al.,
FEBS Lett
., 183:275-278 (1985)). In addition, a class of nucleotides with 3′-conjugated reporters has been shown to function as dye terminators (B. Canard et al.,
Gene
, 148:1-6 (1994); M. L. Metzker, et al.,
Nucleic Acids Res
., 22:4259-4267 (1994); A. V. Azhayev et al.,
Nucleic Acids Res
., 6:625-643 (1979); K. Faulstich et al.,
Bioorganic & Med. Chem. Lett
., 4:1975-1978 (1994)).
IV. Fluorescence Quenching
Fluorescent dyes are sensitive to their environment. Free dyes give the maximum quantum yield, whereas conjugated dyes are typically quenched to some degree. Extrinsic quenchers, the most ubiquitous of which are paramagnetic species (such as O
2
) and heavy atoms (such as iodide), reduce fluorescence quantum yields in a concentration-dependent manner. Many fluorophores are also quenched by protein. Examples are NBD, fluorescein and BODIPY dyes, in which the effect is apparently due to charge-transfer interactions with aromatic amino acid residues. (
Biophys. J
. 69:716 (1995);
Biochemistry
16:5150 (1977);
Immunochemistry
14:533 (1977)). Consequently, antibodies raised against these fluorophores are effective and highly specific fluorescence quenchers.
Besides extrinsic quenching mechanisms, many other environmental factors influence fluorescence properties. The four most common are solvent polarity, the proximity and concentrations of quenching species, the pH of the aqueous medium, and the binding of a probe to its target (such as DNA stain dyes). However, these quenching mechanisms are not readily applicable to the design of dye terminators. Common causes of the fluorescence quenching seen in dye conjugates includes dipole-dipole interactions, hydrophobic interactions and charge/energy transfer.
V. Conclusions
It would be desirable to develop a fluorogenic system for detecting and measuring polymerase activity, where production of a fluorescent species correlates with the activity of a polymerase or other enzyme. The present invention meets this need and more, by providing a class of pro-fluorescent nucleotides that release a free fluorophore upon polymerase-mediated incorporation of the nucleotide. Thus, the invention allows for determination of base incorporation in the presence of unincorporated nucleotides. In doing so, in certain embodiments the present invention also provides a novel and highly sensitive means for amplifying the signal produced as a result of that incorporation. Thus, the present invention provides a class of reagents useful in a variety of applications, including kinetic studies, polymerase reaction monitoring, and non-gel-based sequence determination methods.
SUMMARY OF THE INVENTION
The present invention provides a novel class of fluorogenic, pro-fluorescent nucleotides (3′-PF-ddNTPs) that may act as either terminator or non-terminator substrates in nucleic acid polymerase reactions or other enzymatic reactions, depending upon the particular combination of polymerase, other enzymes, and 3′-pro-fluorescent nucleotide (3′-PF-ddNTP) species utilized. Owing to the fluorogenic nature of 3′-PF-ddNTPs, reporter release corre
Boyce-Jacino Michael T.
Goelet Phillip
Shi Jufang
Houtteman Scott W.
Kalow & Springut LLP
Kalow, Esq. David A.
Orchid BioSciences, Inc.
Schmidt, Esq. William D.
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