Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
1999-08-12
2002-12-03
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
C204S452000, C204S461000, C204S612000
Reexamination Certificate
active
06488832
ABSTRACT:
TECHNICAL FIELD
This invention relates to methodology for reducing the linear dimension necessary to carry out electrophoretic separations of nucleic acid fragments. Separations normally requiring many centimeters of running distance, can now be resolved in several millimeters. This novel methodology provides for microelectrophoresis, with the inherent advantage of significantly reduced separation times.
BACKGROUND
Electrophoretic analysis is a powerful and widely used technique in the fields of biochemistry and molecular biology. The advent of recombinant DNA technology, the rapid growth of the polymerase chain reaction technology, and the initiative to sequence the human genome have further stimulated its useful development, particularly for the separation of nucleic acids. Nucleic acid analyses are carried out on sample mixtures which range in size from oligonucleotides several nucleotides in length to large DNA fragments millions of base pairs in length. Agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) are the two main types of electrophoresis used for analysis of nucleic acids. In most cases the short to intermediate sized nucleic acid fragments [10 to 1000 base pairs (bp)] are separated in polyacrylamide slab gels arranged in vertical formats, and the intermediate to high molecular weight nucleic acid fragments (100 to 100,000 nucleotides) are separated in agarose slab gels arranged in a horizontal formats. A special technique called pulse field electrophoresis is used to resolve very large DNA fragments up to 5 megabases. In general, slab gels used in these procedures range from so-called mini-gels which are approximately 5 cm×5 cm to the more standard sized gels which are 20 cm×20 cm formats. These formats provide running distances of 5 to 20 centimeters in which the separations occur.
In the case of DNA sequencing, where resolution of DNA fragments varying by one nucleotide is required, polyacrylamide gels from 20 cm to 40 cm are commonly used, and in some cases gels as long as 100 cm have been used. Improvements in DNA sequencing using polyacrylamide slab gels has been achieved mainly by using thinner and lower percentage polyacrylamide gels. Presently, using the most optimal techniques and equipment (Applied Biosystems, Inc., Automated Fluorescent DNA Sequencer) a 300-400 base sequence determination on slab gel with a 25 cm running distance takes four to six hours to complete. More recently, a newer technique called capillary electrophoresis is being developed for DNA sequencing applications. This technique uses very narrow diameter capillary tubes (50 um to 100 um) containing low percentage polyacrylamide gels. Separations comparable to the slab gel formats can be carried out in 30 to 40 minutes. The improved speed with the capillary format comes primarily from being able to apply higher voltages to the very thin, low percentage polyacrylamide gel; however, gel lengths of 40 to 70 centimeters are still required to achieve the resolution. Thus, in the almost twenty years of development in electrophoretic techniques, the linear dimension or length of gel required for the high resolution separation of nucleic acid fragments has stayed approximately the same.
A few early attempts were made to investigate the potential for microelectrophoresis. Edstrom, (
Biochem. Biophys. Acta,
22:378, 1956) first described a microtechnique for the electrophoretic separation of purine and pyrimidine bases along a silk thread. Matioli et al., (
Science,
150:1824, 1965) have separated hemoglobin variants on polyacrylamide fibers. In this work, 20% acrylamide gels were used to separate hemoglobin variants from single cells. Hemoglobin molecules (MW
~
64,000) with molecular radii of 2.66 nm are not pore size limited in 20% polyacrylamide gels, thus the separation occurs by the “normal gel sieving process”. See Andrews, A. T. in “Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications”, Oxford University Press, New York, chapter 2, pp. 5-74 (1986). As will be shown, the present invention is concerned with the microelectrophoretic separation of molecules whose molecular radius, stokes radius, or radius of gyrations are significantly greater than gel pore size.
Other separation systems have been described that were referred to as mini-gel electrophoresis, that emphasize separation of proteins. Grossbach, U. in “Electrophoresis and Isoelectrofocusing in Polyacrylamide Gels”, eds. Allen et al, Walter de Gruyter, New York, p.207 (1974), describes separation of proteins in 50 um to 100 um diameter capillaries tubes which were 2.5 centimeters long, using standard gels and buffers. Neuhoff et al.,
Biochem J.,
117:623 (1970) and Bispink et al, in “Electrofocusing and Isotachophoresis”, eds. Radola et al, Walter de Gruyter, New York, p.125 (1977) describe analytical PAGE on proteins carried out using 5 centimeter capillary tubes. “Micro versions” of polyacrylamide slab gels have been prepared on 75 mm×25 mm microscope slides [Maurer et al,
Anal. Biochem.,
46: 19 (1972)] and on 82 mm×102 mm glass microscope slides [Matsudaira et al,
Anal. Biochem.,
87:386 (1978)]. In all of the above work, gel lengths or running distances are at least five to forty times longer than described by the present invention; the above described systems are more accurately described as mini-gels or scaled down version of large gels. In the above work, standard gel formulations were used. That is, gel formulations in the above systems do not significantly deviate from what is used for the corresponding large scale separation. More importantly, in the above systems the molecules (proteins) are being separated by the “normal sieving process” which occurs for molecules which have a molecular radius that is smaller than the gel pore size of the separation medium. Finally, the term “micro” refers more accurately to the width, diameter, or thickness of the gels, rather than to the length or linear dimension.
Gradient gel electrophoresis is a technique in which a gel matrix having an increasing concentration of polyacrylamide (3% to 40%) along the separation axis is used to separate macromolecules in a wide range of sizes. In gradient gel electrophoresis the rate of migration of the components through a gel gradient varies inversely with time. After a sufficient electrophoresis time a stable pattern develops in which the different components continue to move slowly but their relative positions remain constant. That is, as the components reach the gel pore size that is close to their own size (molecular radius), their terminal velocity approaches zero. See, for example, Andrews, A. T., in “Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications”, Oxford University Press, New York, chapter 4, pp. 93-116 (1986). A great deal of theoretical work and application of techniques for determining molecular weights and molecular radii of proteins has been described by Rodbard et al,
Anal. Biochem.,
40:95-134 (1971); Manwell,
Biochem. J.,
165:487-495 (1977); and Campbell et al,
Anal. Biochem.,
129:31-36 (1983).
DNA separation by using gradient gel electrophoresis has been described by Jeppesen,
Anal. Biochem.,
58:195-207 (1974). DNA fragments of molecular weights from about 7×10
4
daltons (114 bp) to about 14×10
6
daltons (21,226 bp) were separated on linear gradient polyacrylamide gels having concentrations from 3.5% to 7.5% or from 2.5% to 7.5% with a crosslinker (C) concentration ranging from 2.5% to 5%. The gels described by Jeppeson were 14 cm long×14 cm wide×0.3 cm in thickness. Electrophoresis was carried out at 10 volts/cm from 16 to 20 hours, until the DNA fragments reached terminal velocities that approached zero. According to Jeppesen, maximum separation is achieved as the fragments approach zero velocity (gel pore limit), and further increase in running time results in little change in band position. Table 1 below shows the approximate acrylamide concentration (%T) or
Lyon & Lyon LLP
Nanogen Inc.
Warden Jill
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