Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – At least one aryl ring which is part of a fused or bridged...
Reexamination Certificate
2000-02-18
2002-11-05
Woodward, Ana (Department: 1711)
Synthetic resins or natural rubbers -- part of the class 520 ser
Synthetic resins
At least one aryl ring which is part of a fused or bridged...
C204S451000, C204S601000, C525S218000
Reexamination Certificate
active
06476118
ABSTRACT:
BACKGROUND OF THE INVENTION
Capillary array electrophoresis is the method of choice for fast reliable DNA sequencing, or other DNA analysis, using automated, commercial sequencing instruments. However, the read length per capillary run that these instruments can deliver is typically no longer than 600-800 bases. To increase the read length further, the separation polymer matrix as well as the separation conditions need to be improved.
It is well known that an elevated column temperature helps to resolve compressions and to increase read length by relaxing stretched DNA in the applied field. An optimum separation of 1000 bases with 97% accuracy has been achieved at 150 V/cm and a column temperature of 50° C. with a matrix containing 2% w/w of a high molecular mass LPA (Carrilho et al., Anal. Chem. 68:3305-3313, 1996). If elevations in column temperature for a sequencing run beyond 50° C. were obtainable, even longer read DNA sequencing lengths should be achievable. However, the thermal stability of the polymer solution network puts limits on the maximum possible column temperature for sequencing runs.
BRIEF SUMMARY OF THE INVENTION
We have determined that a blend of two hydrophilic polymers (e.g., polymers having alkyl residue groups, particularly linear polyacrylamides (LPA) or substituted polyacrylamides), one with high weight-average molecular mass (HMM), 5,000,000 or higher, and another with low weight-average molecular mass (LMM), 200,000 or higher, each present at a low concentration but wherein the concentration of the HMM LPA is above its entanglement threshold, forms an excellent, thermally stable matrix for long read length DNA sequencing. The molecular mass of the low molecular mass polymer is preferably up to about 15% of the mass of the high molecular mass polymer. Polymer separations using the matrix of the invention can be carried out at elevated temperatures, e.g., 70° C., for improved selectivity in the long-base-fragment region.
In a preferred embodiment, the concentration of the low weight-average molecular mass LPA polymer is from 0.25% to 2%, the concentration of the high weight-average molecular mass LPA polymer is from 1.5% to 4% and the ratio of the concentration of the low-weight-average molecular mass LPA polymer to the concentration of the high-weight-average molecular mass LPA polymer is from 1:25 to 1:2.
Preferably, in the method of the invention, the device for conducting capillary electrophoresis is a coated capillary tube or integrated microchannel device. Preferred coatings are, e.g., covalent, adsorbed or polymer matrix adsorbed.
REFERENCES:
patent: 6156819 (2000-12-01), Lai et al.
CAPLUS Abstract of Baba et al. (“Separation of DNA Fragments by High-Performance Liquid Chromatography and Capillary Electrophoresis”, J. Liq. Chromatogr. (1993), 16(4), 945-53).*
Oscar Salas-Solano et al., “Routine DNA Sequencing of 1000 Bases in Less Than One Hour by Capillary Electrophoresis with Replaceable Linear Polyacrylamide Solutions”, Analytical Chemistry, vol. 70, No. 19, pp. 3996-4003 (1998).
Eliza N. Fung et al., “High-Speed DNA Sequencing by Using Mixed Poly(ethylene oxide) Solutions in Uncoated Capillary Columns”, Analytical Chemistry, vol. 67, No. 13, pp. 1913-1919 (1995).
Annelise E. Barron et al., “The effects of polymer properties on DNA separations by capillary electrophoresis in uncrosslinked polymer solutions”, Electrophoresis, vol. 17, pp. 744-757 (1996).
Chunhung Wu et al., “Polyacrylamide solutions for DNA sequencing by capillary electrophoresis: Mesh sizes, separation and dispersion”, Electrophoresis, vol. 17, pp. 1103-1109 (1996).
Yongseong Kim et al., “Separation of DNA sequencing fragments up to 1000 bases by using poly(ethlene oxide)-filled capillary electrophoresis”, Jounal of Chromatography A, vol. 781, pp. 315-325 (1997).
Yongseong Kim et al., “DNA sequencing with pulsed-field capillary electrophoresis in poly(ethylene oxide) matrix”, Electrophoresis, vol. 18, pp. 2901-2908 (1997).
Goetzinger Wolfgang
Karger Barry L.
Kotler Lev
Salas-Solano Oscar
Sosic Zoran
Northeastern University
Weingarten Schurgin, Gagnebin & Lebovici LLP
Woodward Ana
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