Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-02-04
2001-05-15
Fredman, Jeffrey (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S024300
Reexamination Certificate
active
06232076
ABSTRACT:
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention generally relates to methods and compositions for stabilizing labeled nucleic acids. More specifically, the invention relates to the stabilization of nucleic acids which are labeled with nonradioactive compounds such as fluorescent dyes for methods such as sequencing.
(2) Description of the Related Art
Nonradioactively labeled nucleic acids generally utilize various compounds covalently attached to a portion of the nucleotide base. These compounds may be detectable by treating the nucleic acid with a specific ligand of the compound, such as an antibody when the compound is a hapten such as digoxigenin. See, e.g., U.S. Pat. No. 6,198,537. Alternatively, the compounds may be detectable without additional treatment, such as when the compound is a fluorescent dye. Fluorescent dyes have been particularly useful in DNA sequencing applications because the dyes can be detected at very low concentration and they may be easily incorporated into the nucleic acid to be sequenced. See, e.g., U.S. Pat. No. 5,861,287.
Methods for the sequencing of nucleic acids have undergone numerous improvements, such that sequencing is now rapid, routine and available for automated throughput. See, e.g., U.S. Pat. No. 5,861,287 for a review of some available manual and automated sequencing methods.
Of the several approaches to DNA sequence determination, the dideoxy chain termination method of Sanger et al., 1977,
Proc. Natl. Acad. Sci. USA
74:560-564, is most commonly used and serves as the basis for all currently available automated DNA sequencing protocols. In the dideoxy method, a sequencing reaction mixture is prepared which generally comprises (a) a DNA template comprising a portion which is to be sequenced, (b) a primer which is complementary to a fragment of the DNA template at the 3′ end of the portion to be sequenced, (c) unlabeled deoxyribonucleoside triphosphates (dNTPs), (d) at least one dideoxyribonucleoside triphosphate (ddNTP), (e) a dNTP, a primer or ddNTP which is labeled with a detectable moiety such as a radioactive atom (e.g.,
35
S or
32
P) or a fluorescent dye, (f) a DNA polymerase and (g) an aqueous solution comprising a buffer such as Tris-HCl and other components required for polymerase activity such as Mg
+2
. The sequencing reaction mixture is subjected to conditions suitable for annealing of the primer to the 3′ end of the portion of the DNA template, followed by polymerase extension of the primer along the DNA template. Each sequencing reaction is stopped when a ddNTP is incorporated at the 3′ end of the growing polymerase extension product. The resulting polymerase extension products represent substantially all complementary extension products with a 5′ terminus complementary to the 3′ end of the portion of the DNA template, and with a 3′ terminal dideoxyribonucleotide at any position along the portion of the DNA template. The polymerase extension products are also labeled with the detectable moiety. The polymerase extension products are then subjected to electrophoresis to separate the various extension products by size, and the order of each of the four bases along the portion of the template is determined by determining which dideoxyribonucleotide terminates each sequential polymerase extension product.
Common variations in the basic dideoxy chain termination sequencing method described above result from variations in the source of the DNA used for the template, the source of the primer, the composition of the unlabeled dNTPs, the ratio of dNTPs to ddNTPs, the polymerase used, whether the polymerase extension products are synthesized in a cycled reaction, whether the polymerase extension reactions for all four bases are executed together or separately, the nature of the detectable moiety, whether the polymerase reaction products are purified before electrophoreses, and whether the electrophoresis is performed on a slab gel or in capillaries. See, e.g., Fredrick M. Ausubel et al. (1995), “Short Protocols in Molecular Biology”, John Wiley and Sons; Joseph Sambrook et al. (1989), “Molecular Cloning, A Laboratory Manual”, second ed., Cold Spring Harbor Laboratory Press; U.S. Pat. No. 5,861,287; and “Automated DNA Sequencing Chemistry Guide,” 1998, Perkin-Elmer Corporation.
In some variations of the dideoxy sequencing method, fluorescent dyes are used. These methods are advantageous because the fluorescent dyes are generally highly sensitive yet are not hazardous like radioactive detection moieties. Examples include 5′-tetramethylrhodamine, fluorescein dyes, aromatic-substituted xanthine dyes, 4,7-dichlororhodamine dyes, asymmetric benzoxanthene dyes and BODIPY dyes. See, e.g., U.S. Pat. Nos. 5,840,999; 5,847,162; 6,008,379; Metzket et al., 1996,
Science
8:1420-1422. Additionally, various fluorescent labels have been developed which vary in emission wavelength maxima and which can be distinguished on that basis. This allows the practitioner to label each ddNTP with a different label in one reaction mixture. Alternatively, the primer for each reaction can be labeled with a different fluorescent dye in a separate reaction mixture for each ddNTP. All four sequencing reactions can then be electrophoresed together and the various terminal fluorescent-labeled ddNTP can be distinguished on the basis of the absorption and emission maxima. An example of dye sets which can be distinguished from each other are the dichlororhodamine dyes ROX, R6G, R110 and TAMRA (TMR), as discussed in Rosenblum et al., 1997,
Nucleic Acids Res.
25:4500-4504. See Table 1.
Energy transfer fluorescent dyes are dyes which include a donor fluorophore covalently conjugated to an acceptor fluorophore. When the donor fluorophore is excited, the energy emission from the donor is transferred to the acceptor fluorophore causing the acceptor to fluoresce. See, e.g., U.S. Pat. No. 5,945,526; Hung et al., 1996,
Anal. Biochem.
243:15-27; and Rosenblum et al., supra. See also “Automated DNA Sequencing Chemistry Guide,” 1998, Perkin-Elmer Corporation, which discloses energy transfer fluorescent dyes which include BigDye™ dyes. The most commonly used BigDye™ energy transfer dyes comprise one of the dichlororhodamine dyes R110, R6G, TAMRA or ROX as the acceptor dye, and 6-carboxyfluorescein (6-FAM or 6CFB) or 5-carboxyfluorescein (5-FAM or 5CFB) as the donor dye. When used as a label for ddNTPs or primers, the BigDye™ dyes emit the fluorescence at the same wavelength as acceptor dichlororhodamine dyes (530 nm, 565 nm, 595 nm, and 625 nm for R110, R6G, TAMRA, and ROX, respectively) but fluoresce 2-3 times brighter than the dichlororhodamine dyes.
The BigDye™ dyes are used in the Perkin-Elmer® Automated DNA sequencing system. In that system, where the dye is conjugated to the dideoxy terminator nucleotide, the terminator dye combinations are ddT-EO-6CFB-dTMR, ddC-EO-6CFB-dROX-2, ddA-PA-6CFB-dR6G, and ddG-EO-5CFB-dR110, where EO is propargyl ethoxyamino and PA is propargylamino, which are linkers between the ddNTP and the donor 6CFB. See Table 1. Similar reagents are used when the primers rather than the ddNTP terminator has the BigDye™ label.
After the polymerase extension products are synthesized in the sequencing reaction mixture using BigDye labels, Perkin-Elmer® recommends that the primer extension products are purified, e.g., by Sephadex G-50 chromatography and/or ethanol precipitation. The samples are then resuspended in deionized formamide with alkaline EDTA. See “Automated DNA Sequencing Chemistry Guide” supra and “DRAFT—ABI Prism® 3700 DNA Analyzer Chemistry Guide,” 1999, Perkin-Elmer Corporation. The formamide keeps the purified polymerase extension products denatured to prevent secondary structures in the primer extension products from affecting the results of the electrophoresis. If water is used instead of formamide, the electrophoretic runs are not as reproducible and random injection failures can occur when capillary electrophoresis is used.
However, Perkin-Elmer has reported that
Chakrabarti Arun
Fredman Jeffrey
Genaissance Pharmaceuticals, Inc.
Howell & Haferkamap, LC
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