Chemistry: analytical and immunological testing – Heterocyclic carbon compound – Hetero-o
Reexamination Certificate
2001-06-01
2003-12-16
Whisenant, Ethan (Department: 1634)
Chemistry: analytical and immunological testing
Heterocyclic carbon compound
Hetero-o
C435S006120, C435S091100, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06664112
ABSTRACT:
TECHNICAL FIELD
The present invention is generally directed toward improving the sequence fidelity of synthetic double-stranded oligonucleotides. It is more particularly related to the removal of synthetic failures (including side products and truncated products) created in the synthesis of oligonucleotides, such as double-stranded DNA.
BACKGROUND OF THE INVENTION
Much of the discovery research in pharmaceutical companies is focused on genes, either as targets for drug development or as therapeutics in the form of their protein expression products. These companies have access to a majority of the human genes. Pharmaceutical companies are overwhelmed with potential opportunities, acutely aware that their competitors are looking at the same set of possibilities, and currently unable to work on more than a fraction of the genes that have been identified. One of the major bottlenecks in this research is the time and effort required to prepare genes for detailed analysis.
Gene synthesis, the production of cloned genes partially or entirely from chemically synthesized DNA, is one method of overcoming this bottleneck. In principle, gene synthesis can provide rapid access to any gene for which the sequence is known and to any variation on a gene. Reliable, cost-effective automated gene synthesis would have a revolutionary effect on the process of biomedical research by speeding up the manipulation and analysis of new genes.
One principal factor limiting the automation of gene synthesis is the low sequence fidelity of the process: gene clones created from chemically synthesized DNA often contain sequence errors. These errors can be introduced at many stages of the process: during chemical synthesis of the component oligonucleotides, during enzymatic assembly of the double-stranded oligonucleotides, and by chemical damage occurring during the manipulation and isolation of the DNA or during the cloning process.
Four types of base modifications are commonly produced when an oligonucleotide is synthesized using the phosphoramidite method: (1) Transamination of the O6-oxygen of deoxyguanosine to form a 2,6-diaminopurine residue; (2) Deamination of the N4-amine of deoxycytidine to form a uridine residue (Eadie, J. S. and Davidson, D. S., Nucleic Acids Res. 15:8333, 1987); (3) Depurination of N6-benzoyldeoxyadenosine yielding an apurinic site (Shaller, H. and Khorana, H. G., J. Am. Chem. Soc. 85:3828, 1963; Matteucci, M. D. and Caruthers, M. H., J. Am. Chem. Soc. 103:3185, 1981); (4) Incomplete removal of the N2-isobutyrlamide protecting group on deoxyguanosine. Each of these side products (byproducts) can contribute to sequence errors in cloned synthetic DNA.
Another synthetic failure of oligonucleotide synthesis is the formation of truncated products that are less than the full length of the desired oligonucleotide. The solid phase approach to oligonucleotide synthesis involves building an oligomer chain that is anchored to a solid support through its 3′-hydroxyl group, and is elongated by coupling to its 5′-hydroxyl group. The yield of each coupling step in a given chain-elongation cycle will generally be<100%. For an oligonucleotide of length ‘n’, there are n−1 linkages and the maximum yield of a desired coupling will be [coupling efficiency]
n−1
. For a 25-mer, assuming a coupling efficiency of 98%, the calculated yield of full-length product will be 61%. The other 39% consists of all possible shorter length oligonucleotides (truncated products) resulting from inefficient monomer coupling. The desired oligonucleotide can be partially purified from this mixture by purification steps using ion exchange or reverse phase chromatography. These purification procedures are not 100% effective and do not completely eliminate these populations. The final product therefore contains n−1 and to some extent n−2 and n−3 failure sequences. This type of undesired product of the oligonucleotide synthesis process can also contribute to sequence errors in synthetic genes.
Another class of synthetic failures is the formation of “n+” products that are longer than the full length of the desired oligonucleotide (User Bulletin 13, 1987, Applied Biosystems). The primary source of these products is branching of the growing oligonucleotide, in which a phosphoramidite monomer reacts through the bases, especially the N-6 of adenosine and the O-6 of guanosine. Another source of n+ products is the initiation and propagation from unwanted reactive sites on the solid support. Finally, these products also form if the 5′-trityl protecting group is inadvertently deprotected during the coupling step. This premature exposure of the 5′-hydroxyl allows for a double addition of a phosphoramidite. This type of synthetic failure of the oligonucleotide synthesis process can also contribute to sequence errors in synthetic genes.
Another process common to the preparation of synthetic genes is the ligation of synthetic double-stranded oligonucleotides to other synthetic double-stranded oligonucleotides to form larger synthetic double-stranded oligonucleotides. In vitro experiments have shown that T4 DNA ligase exhibits poor fidelity, sealing nicks with 3′ and 5′ A/A or T/T mismatches (Wu, D. Y., and Wallace, R. B., Gene 76:245-54, 1989), 5′ G/T mismatches (Harada, K. and Orgel, L. Nucleic Acids Res. 21:2287-91, 1993) or 3′ C/A, C/T, T/G, T/T, T/C, A/C, G/G or G/T mismatches (Landegren, U., Kaiser, R., Sanders, J., and Hood, L., Science 241:1077-80, 1988). These types of mismatches may occur during ligation of double-stranded nucleic acids into larger double-stranded nucleic acids.
Due to the difficulties in the current approaches to the preparation of oligonucleotides, such as genes, there is a need in the art for methods for improving the sequence fidelity of synthetic oligonucleotides. The present invention fills this need, and further provides other related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides a variety of methods for improving the sequence fidelity of synthetic double-stranded oligonucleotides. The methods comprise subjecting synthetic double-stranded oligonucleotides to preparative column chromatography or preparative gel chromatography under denaturing conditions sufficient to separate the synthetic double-stranded oligonucleotides into two populations, wherein one population is enriched for synthetic failures and the other population is depleted of synthetic failures. In one embodiment, the column chromatography is HPLC. A preferred embodiment is DHPLC. In another embodiment, the gel chromatography is gradient gel chromatography. In any of the embodiments, the oligonucleotides may comprise synthetic double-stranded DNA. Preferred synthetic double-stranded DNA comprises one or more fragments of a larger DNA molecule.
These and other aspects of the present invention will become evident upon reference to the following detailed description. In addition, various references are set forth herein. Each of these references is incorporated herein by reference in its entirety as if each was individually noted for incorporation.
DETAILED DESCRIPTION OF THE INVENTION
Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms to be used hereinafter.
Natural bases of DNA—adenine (A), guanine (G), cytosine (C) and thymine (T). In RNA, thymine is replaced by uracil (U).
Synthetic double-stranded oligonucleotides—substantially double-stranded DNA composed of single strands of oligonucleotides produced by chemical synthesis or by the ligation of synthetic double-stranded oligonucleotides to other synthetic double-stranded oligonucleotides to form larger synthetic double-stranded oligonucleotides.
Synthetic failures—undesired products of oligonucleotide synthesis; such as side products, truncated products or products from incorrect ligation.
Side products—chemical byproducts of oligonucleotide synthesis.
Truncated products—all possible
Mulligan John T.
Tabone John C.
Blue Heron Biotechnology, Inc.
Lu Frank Wei Min
Seed Intellectual Property Law Group PLLC
Whisenant Ethan
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