Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-10-06
2004-11-16
Shukla, Ram R. (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S091100, C536S023100, C536S025300, C536S025320
Reexamination Certificate
active
06818760
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention pertains to the field of the purification of polynucleotides from mixtures that contain fluorescent dye-labeled molecules. The polynucleotides can include, for example, reaction products from nucleic acid sequencing reactions.
2. Background
The demands of the Human Genome Project and the commercial implications of polymorphism and gene discovery have driven the development of significant improvements in DNA sequencing technology. Contemporary approaches to DNA sequencing have imposed stringent demands on reliability and throughput for DNA sequencers. Recent reports have demonstrated the extraordinary potential of capillary electrophoresis (CE) for DNA sequencing given the inherent speed, resolving power and ease of automation associated with this method as compared to slab gel electrophoretic methods (Carrilho et al.,
Anal. Chem
. 1996, 68, 3305-3313; Tan and Yeung,
Anal. Chem
. 1997, 69, 664-674; Swerdlow et al.,
Anal. Chem
. 1997, 69, 848-855).
Relative to cross-linked gel capillary electrophoretic columns, the recent development of replaceable polymer solutions to achieve size separation of single-stranded DNA fragments has increased the lifetime of the columns and eliminated the requirements of gel pouring and casting (Ruiz-Martinez et al.,
Anal. Chem
. 1993, 65, 2851-2858). Additionally, improvements in the composition of the separation matrix have led to sequencing over 1000 bases per run (Carrilho et al.,
Anal. Chem
. 1996, 68, 3305-3313). Automated capillary electrophoresis systems for DNA sequencing have been introduced commercially by three major scientific instrument manufacturers (Beckman Coulter CEQ™ 2000 DNA Analysis System; Amersham Pharmacia MegaBACE 1000 DNA Sequencing System; and PE Biosystems ABI Prism 3700 DNA Analyzer).
Realizing the potential of this new generation of automated DNA sequencers is proving difficult, however, as problems in read length and accuracy remain, primarily due to the limitations associated with the methods currently available for purifying the products of cycle sequencing reactions. Indeed, the critical importance of sample preparation for the successful implementation of capillary electrophoresis has not been sufficiently emphasized.
In contrast to slab gel electrophoresis, primer extension products are introduced into the capillary column using electrokinetic injection, which provides focusing of the single-stranded DNA fragments at the head of the column (Swerdlow et al.,
Proc. Natl. Acad. Sci. U.S.A
. 1988, 85, 9660-966). However, electrokinetic injection is biased toward high electrophoretic mobility ions, such as chloride, deoxynucleotides and dideoxynucleotides, which, if present in the sequencing reaction solution, negatively affect the focusing of single-stranded DNA fragments. Consequently, to increase the amount of DNA injected into the capillary column, and to improve the focusing of the injected DNA, an effective removal of these small ionic species is required.
A further group of especially problematic high electrophoretic mobility ions are the dye-labeled fluorescent dideoxynucleotide terminators, and in particular the recently commercialized terminators having two fluorescent moieties configured as energy transfer pairs (e.g., ABI PRISM BigDye™ Terminators from PE Biosystems and DYEnamic ET™ Terminators from Amersham Pharmacia). These reagents, and the hydrolysis products derived therefrom, have been found to be particularly difficult to remove from primer extension reactions, resulting in the presence of fluorescent artifacts (routinely described as “dye blobs”) that negatively affect the automated analysis of sequencing data (Rosenblum, B.; Lee, L.; Spurgeon, S.; Khan, S.; Menchen, S.; Heiner, C. and Chen, S.,
Nucleic Acids Research
, 1997, 25, 4500-4504). Dye-labeled sequencing primers, when employed as an alternative to dye-labeled terminators, afford similar problems (Jingyue, J.; Ruan, C.; Fuller, C.; Glazer, A. and Mathies, R.,
Proc. Natl. Acad. Sci. USA
, 1995, 92, 4347-4351. Jingyue, J.; Kheterpal, I.; Scherer, J.; Ruan, C.; Fuller, C.; Glazer, A. and Mathies, R.,
Anal. Biochem
., 1995, 231, 131-140. Jingyue, J.; Glazer, A. and Mathies, R.,
Nucleic Acids Research
, 1996, 24, 1144-1148. Lee, L.; Spurgeon, S.; Heiner, C.; Benson, S.; Rosenblum, B.; Menchen, S.; Graham, R.; Constantinescu, A.; Upadhya, K. and Cassel, J.,
Nucleic Acids Research
, 1997, 25, 2816-2822).
Fluorescent energy transfer dye-labeled dideoxynucleotide triphosphate terminators suitable for use in DNA sequencing are described in U.S. Pat. No. 5,800,996. Fluorescent energy transfer dye-labeled primers suitable for use in DNA sequencing are described in U.S. Pat. Nos. 5,688,648, 5,707,804 and 5,728,528.
During the course of cycle-sequencing reactions, deoxynucleotide triphosphates (dNTPs) and dye-labeled dideoxynucleotide triphosphates (ddNTPs) undergo hydrolysis of the phosphate ester bonds during the denaturation step that proceeds each amplification cycle when the temperature is elevated to from 95° C. to 99° C. This results in the generation of dye-labeled artifacts including dideoxynucleotide diphosphates (ddNDPs), dideoxynucleotide monophosphates (ddNMPs) and dideoxynucleosides. The ddNTPs derived from the pyrimidine bases, dideoxythymidine (ddTTP) and dideoxycytidine (ddCTP), are particularly labile in this regard. The dye-labeled ddNTPs, ddNDPs and ddNMPs elute from capillary electrophoretic columns prior to the dye-labeled primer extension products, and consequently do not directly interfere with the interpretation of the sequencing data. However, the intensity of the signals associated with the dye-labeled ddNTPs, ddNDPs and ddNMPs may exceed those of the primer extension products by many orders of magnitude. This discontinuity in signal intensity has proven problematic with respect to automated base-calling software, resulting, in a worse-case scenario, in the software interpreting the fluorescent artifact peaks as primer extension products and the primer extension products as baseline noise. A further significant complication is associated with the presence of dye-labeled dideoxynucleosides, in that they are found to co-elute from both capillary electrophoretic columns and slab gels with the primer extension products. Not only is the intensity of the artifact signal disproportionately high as compared to the signals associated with the primer extension products, but the width of the peak is sufficiently broad to obscure the analysis of from 5 to 20 bases.
The sample preparation scheme now routinely employed for both slab gel electrophoresis and CE consists of desalting DNA sequencing samples by ethanol or isopropanol precipitation, followed by reconstitution of the DNA fragments and template in a mixture of formamide-0.5 M EDTA (49:1) prior to loading or injection (Figeys et al., 1996, 744, 325-331; Sambrook, J.; Fritsch, E. F.; Maniatis, T.
Molecular Cloning: A Laboratory Manual
; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 1989; section 9.49). Although widely utilized, this method has been found to exhibit variable reproducibility in terms of DNA recovery, to provide marginal performance with respect to the quantitative removal of dye-labeled artifacts, and is not easily automated (Tan, H.; Yeung, E. S.
Anal. Chem
. 1997, 69, 664-674, and Hilderman, D.; Muller, D.
Biotechniques
1997, 22, 878-879).
High electrophoretic mobility ionic species in DNA sequencing samples are not the only contaminants that cause degradation in sequencing read length. Template DNA also has been shown to interfere with the analysis of primer extension products in both thin slab gels (Tong et al.,
Biotechniques
1994, 16, 684-693), and capillary columns (Swerdlow et al.,
Electrophoresis
1996, 17, 475-483). Upon injection of the sequencing reaction solution, a current drop and significant deterioration in the resolving power of the capillary column is observed when template DNA is present in the sample (Salas-Solano et al.,
Anal. Chem
. 1998, 70, 1
Hughes Karin A.
Kaiser Robert J.
Mahoney James E.
Spicer Douglas A.
Springer Amy L.
Prolinx Incorporated
Shukla Ram R.
Townsend and Townsend / and Crew LLP
Zara Jane
LandOfFree
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