Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-08-27
2002-10-29
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025330, C536S025410, C536S025420
Reexamination Certificate
active
06472522
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to the field of biopolymers, and more particularly relates to the purification of oligomers such as oligonucleotides, oligopeptides, oligosaccharides, and the like.
BACKGROUND OF THE INVENTION
There is an increasing demand for oligonucleotides for use in nucleic acid hybridization assays, as polymerase chain reaction (“PCR”) primers or as sequencing primers in Sanger, or dideoxy, sequencing. Synthesis and purification of research-purpose quantities of oligonucleotides routinely yields product having purity of greater than 95%, but this high purity requires a lengthy, time-consuming, and labor intensive purification protocol. Typically, a 0.2 micromole-scale preparation requires a seven-step purification procedure: 1) preparing a purification gel; 2) loading the gel with the reaction mixture to be purified, iii) running the gel overnight; 4) visualizing and cutting the appropriate bands from the gel; 5) soaking the bands in elution buffer for two days to extract the desired product from the gel matrix; 6) manual desalting the extracted product on a reverse phase (“RP”) column and drying the solvent; and 7) manually precipitating the product from the solvent. The amount of the product obtained is quantitated using UV spectroscopy.
Any simplification of these lengthy, time consuming, and labor intensive purification protocols would be very valuable. Further, a purification scheme that could be automated and applied to oligomers other than oligonucleotides would be desirable as well.
EARLIER APPROACHES TO SIMPLIFY THE PURIFICATION OF DNA OLIGOMERS:
During the process of oligonucleotide synthesis, depurinated sites can be introduced at random sites caused by prolonged exposure to acid; the final ammonium hydroxide deprotection step cleaves the oligonucleotide chain at the depurinated sites. McHugh et al. (1995)
Nucleic Acids Research
23:1664-1670. Methods that were devised to simplify DNA purification by, e.g., Efcavitch et al. (1985)
Nucleosides
&
Nucleotides
4:267 and McBride et al. (1988)
BioTechniques
66:362-367, were only capable of purifying shorter DNA oligomers because they did not fully account for the complicating nature of the ammonium hydroxide cleavage products.
An enzymatic purification scheme has been reported in which an oligomer is first synthesized on a solid support. Urdea et al. (1986)
Tetrahedron Lett
. 27:2933-2936. Subsequent to preparation of the desired-length solid support-bound oligonucleotide, exocyclic amines and phosphate groups in the oligomer were deprotected without cleavage of the linkage to the support. The purification used spleen phosphodiesterase to digest failure sequences that did not contain a terminal 5′-benzoyl group of the full-length oligomeric product. The process resulted in oligomers of improved purity, but abasic sites in the product oligomer remained.
A rapid cartridge purification method has also been described by Horn et al. (1988) in
Nucleic Acids Res
. 16:11559-11571. The key step in this procedure is the cleavage of all apurinic sites in the oligomer with a solution of aqueous lysine prior to removal of the crude product from the solid support. As a result, essentially all of the truncated 5′—O-dimethoxytrityl (“DMT”)-containing oligomers are eliminated from the mixture of cleaved oligomers. The authors report that DNA oligomers of up to 118 bases in length were purified to near homogeneity using the procedure.
An approach related to that described in Horn et al. (1988) made use of a solid support with a disilyloxy linkage. Cleavage of abasic sites in the oligomer under very mild conditions, while the oligomer was still attached to the support, ensured that all 5′—O-DMT-containing molecules, when cleaved from the support, had correct 3′- and 5′-ends. Kwiatkowski et al. (1996)
Nucleic Acids Res
. 24:4632-4638.
Natt et al. (1997)
Tetrahedron
53:9629-9636 describe an approach to oligomer purification that used a lipophilic capping reagent to cap failure sequences during synthesis. The lipophilic nature of the failure sequences made it possible to separate capped failure sequences from detritylated full-length oligomers chromatographically. However, the method was inefficient with respect to depurinated/cleaved sequences since the two families of species, i.e., the detritylated 5′ segment and the detritylated 3′ segment, do not contain the lipophilic capping group. The use of trityl groups with enhanced lipophilic properties as 5′—O protecting groups has been advocated to facilitate RP-high performance liquid chromatography (“HPLC”) purification. Ramage (1993)
Tetrahedron Lett
. 34:7133-7136. As with the approaches discussed above, this process is also limited with regard to cleaved abasic sites.
Purification approaches that involve a “capture” step have been proposed. In each case, the 5′ end of the oligomer to be purified carries a moiety by which capture can be effected. For example, Bannwarth et al. (1990), in
Helv. Chim. Acta
73: 1139-114, described a combined purification/phosphorylation procedure for oligodeoxynucleotides that included a capture step. A special ribonucleotide, N
1
—(MMT—S—(CH
2
)
10
)—2′,3′Bz
2
-rU-5′-&bgr;-cyanoethyl (wherein “MMT” represents monomethoxytrityl and “Bz” represents benzyl), containing a protected thiol and a diol system, was incorporated into the oligonucleotide during the final DNA synthesis cycle. After complete deprotection and removal of the MMTr protecting group, the oligomer with a 5′—SH group could be captured on a controlled pore glass (“CPG”) support having surface-bound-S—S-pyridine groups; contaminating oligomers were removed by washing. The purified oligomer was released from the capture support after oxidative cleavage of the ribo-diol system and beta-elimination under basic conditions.
A purification procedure using a photolabile 5′-biotin reagent to capture oligomers on a avidin capture support has also been described. Olejnik et al. (1996)
Nucleic Acids Research
24:361-366. The linking groups could be cleaved by photolysis to release the product oligomer in the 5′-phosphate form.
Synthesis and purification of 5′-mercaptoalkylated oligonucleotides has been described in which thiolated oligomers were purified by a single-step covalent chromatography procedure using an activated sulfhydryl support. Kumar et al. (1996)
Bioorg. Med. Chem. Lett
. 6:683-688.
In addition, purification of proteins by taking advantage of the selectivity of unique nickel-nitrilotriacetic acid (“Ni—NTA”) solid supports with an affinity tag consisting of six consecutive histidine residues has been known for years. This type of immobilized metal affinity chromatography (“IMAC”) has been used for sequence-specific isolation of nucleic acids by peptide nucleic acids (“PNA”)-controlled hybrid selection using oligohistidine-PNA chimera (the chemistry of PNA and peptide assembly are essentially identical). Orum et al. (1995)
BioTechniques
19:472-480. The system has been extended to synthetic DNA oligomers containing six consecutive 6-histaminylpurine (“His”) nucleotides, introduced using a convertible nucleotide phosphoramidite and further derivatized to form the His nucleotides. Min et al (1996)
Nucleic Acids Research
24:3806-3810. The His
6
-tagged strand was selectively retained by a Ni—NTA-agarose chromatography matrix and the captured DNA thereafter eluted from the resin.
OVERVIEW OF THE ART:
Background references that relate generally to methods for synthesizing oligonucleotides include those related to 5′- to -3′ syntheses based on the use of &bgr;-cyanoethyl phosphate protecting groups, e.g., de Napoli et al. (1984)
Gazz. Chim. Ital
. 114:65, Rosenthal et al. (1983)
Tetrahedron Lett
. 24:1691, Belagaje et al. (1977)
Nucl. Acids Res
. 10:6295, and those references that describe solution-phase 5′- to -3′ syntheses, such as Hayatsu et al. (1957)
J. Am. Chem. Soc
. 89:3880, Gait et al. (1977)
Nucl. Acids Res
. 4:1
Horn Thomas
Urdea Michael S.
Bayer Corporation
Hartrum J. Elin
Reed Dianne E.
Wilson James O.
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