Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-12-19
2001-09-04
Ketter, James (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
Reexamination Certificate
active
06284883
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method for purifying DNA and RNA oligonucleotides and analogs thereof.
2. Background
DNA oligomers containing a few to on the of order 100 bases are commonly produced by automatic synthesizer machines and are available from many commercial suppliers. Oligomers are formed by adding the DNA bases one at a time by chemical modification of the ends of the growing oligomers. The steps in this process are not 100% efficient, and the resulting oligomeric products typically have errors of base omission of a fraction of a percent per base. There may also be errors of base substitution or addition that are dependent on chemical purity. Hence, significant impurities will be present in the form of shortened and base-substituted oligomers. While there may be a significant number of oligomers containing errors, the quantity of oligomers having any particular incorrect sequence is very small. The present state of the art requires purification using columns or electrophoresis gels to attain 95% purity.
Mosaic Technologies offers a gel supported DNA product that is capable of carrying out this type of purification. However, the gel operates by immobilizing the desired DNA by capture on DNA adhered to the gel having complementary sequences and subsequently removing the desired DNA from the gel as a purified product.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a method of separating target DNA oligonucleotides, RNA oligonucleotides and analogs thereof from impurity DNA oligonucleotides, RNA oligonucleotides and analogs thereof using affinity chromatography. The method uses a chromatographic material having a separation medium and separation oligomers with a subunit sequence complementary to the desired subunit sequence. The chromatographic separation which may include, for example, column chromatography or electrophoresis, is conducted under conditions wherein the rates of hybridization and dissociation of oligomers having the desired sequence and the separation oligomers is about the same. This may be accomplished by conducting the separation at about the melting temperature of the double stranded complex consisting of the oligomer having the desired sequence and its complement.
In various embodiments, the separation medium may be, for example, an electrophoretic gel or a chromatographic matrix such as beads or fibers that may be loaded into a chromatography column. The separation oligomers may be bound to the separation material or travel with the target oligomers through the separation medium. The method is particularly useful for purifying DNA and isolating DNA oligonucleotides having single nucleotide polymorphisms.
One advantage of some embodiments is a form of DNA affinity chromatography that provides a higher purity than prior art purification processes.
Further objectives and advantages will become apparent from a consideration of the description and examples.
REFERENCES:
Jarrett, J. Chromatography, vol. 618 (1993) pp. 315-339.
Ketter James
Lucent Technologies - Inc.
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