Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-05-16
2002-11-12
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C536S025300, C536S025310, C536S025320, C536S025330, C536S025340, C536S024300, C536S026220, C436S501000, C424S094100
Reexamination Certificate
active
06479262
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to processes for the synthesis of polynucleotides, such as DNA and fragments of DNA, RNA and fragments of RNA, plasmids, genes, and chemically and/or structurally modified polynucleotides.
BACKGROUND OF THE INVENTION
Living cells can be “reprogrammed,” in vitro or in vivo, to produce useful amounts of desired proteins or other compounds by introducing the appropriate nucleic acids (DNA or RNA) into them; this concept is the keystone of modern biotechnology. The construction of recombinant DNA molecules necessary to achieve this “reprogramming” or to perform a varied and growing number of other functions is a frequent and necessary activity of molecular biology research and of biotechnological endeavors in industrial and academic settings. By improving the process by which DNA or RNA molecules of arbitrary sequence are made, a significant increase of productivity in biotechnology could be achieved, resulting in benefits in many fields including medical research, agriculture and the chemical industry. For example, numerous efforts to sequence the entire genomes of a variety of organisms (microbes, animals and plants) has generated many large databases of gene sequences. These genes can be made and studied experimentally through laborious and time-consuming techniques involving the isolation and subsequent manipulation (generally referred to as molecular cloning) of DNA from the organism in which the gene is found and/or expressed. Alternatively, inefficient DNA synthesis methods can be used, as described below.
The ability to synthesize large RNA or DNA molecules (e.g., entire genes) is of value to any endeavor that relies on recombinant DNA technology. As alluded to above, DNA molecules of arbitrary sequence can be synthesized in vitro. A solid phase method to synthesize oligonucleotides that is now widely used in commercial DNA synthesizers is reported in U.S. Pat. No. 4,458,066. Current DNA synthesizers, however, are limited to the production of relatively short single-stranded DNA oligonucleotide molecules of length typically less than 200 nucleotides (nt). In contrast, the average prokaryotic gene is 1000 basepairs (bp) in length, a eukaryotic cDNA is frequently longer than 2000 bp, and most plasmids are larger than 3000 bp. Although state-of-the-art oligonucleotide synthesizers relying on beta-cyanoethyl phosphoramidite chemistry (U.S. Pat. No. 5,935,527) can make and purify 48 oligonucleotides in less than 48 hours (25 nt/oligo×48 oligonucleotides=1200 nt, a typical bacterial gene), it is still very time consuming and labor-intensive to assemble these oligonucleotides together into a single gene.
Gene synthesis, a service frequently offered commercially by oligonucleotide manufacturers, is expensive (approximately $10 to $20/bp) and slow (frequently requiring several weeks) because current methods are labor-intensive. A method to make relatively large DNA molecules by mixing two long oligonucleotides (up to 400 nt) and amplifying the desired double-stranded DNA fragment from the mixture using the polymerase chain reaction (PCR) is reported in European Patent Application 90201671.6. This method becomes more complicated and requires extensive manipulations by a skilled technician when molecules larger than 400 bp must be synthesized. Similar statements can be made of the method of Khorana,
Science
, 1979, 203, 614-625.
A method to synthesize long nucleic acid molecules in which a ribo- or deoxyribo-oligonucleotide attached to a solid support is extended by the sequential addition of other “assembly” oligonucleotide is reported in U.S. Pat. No. 5,942,609. Of key importance to this process is the annealing of a partially complementary “bridging” oligonucleotide to the two oligonucleotides that will be covalently linked together by a ligase. Although this method will likely achieve its stated goal of synthesizing long polynucleotides, the need for the synthesis of a bridging oligonucleotide adds to the total number of oligonucleotides which must be synthesized and purified, with an attendant increase in costs and time of synthesis. In addition, the assembly of a complex mixture of oligonucleotides would greatly complicate this process because of the large number of different bridging oligonucleotides that would be needed to bring together the assembly oligonucleotides. Moreover, it would be advantageous to obviate the need for the annealing step required to productively bind the bridging oligonucleotide to its target assembly oligonucleotides. Such a step may introduce complications due to the need to avoid non-specific hybridization problems. Complications may include the need to carefully control hybridization temperatures over lengthy incubation periods as well as to carefully design each bridging oligonucleotides to bind specifically to the desired sequence.
International Publication WO83/02626 reports a method of assembling a polyribonucleotide using the enzyme T4 RNA ligase, including time-consuming purification steps, but does not include the use of solid phase methods which would facilitate automation and increase the reliability of the process. In contrast, Mudrakovskaia et al. (
Bioorg. Khim
., 1991, 17, 819-822) report a “solid-phase enzymic synthesis of oligoribonucleotides” but do not disclose how the method could be used to couple more than a few nucleotides to a tethered oligonucleotide. Neither International Publication WO83/02626 nor Mudrakovskaia et al. disclose how their methods could be used to synthesize large (>200 nt) DNA or RNA molecules without requiring numerous and laborious purification steps.
Harada et al. (
Proc. Natl. Acad. Sci. USA
, 1993, 90, 1576-1579) reports in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 RNA ligase. Tessier et al. (
Anal. Biochem
., 1986, 158, 171-178) reports a set of reaction conditions for ligation of DNA fragments up to 40 bases in length. Zhang et al. (
Nuc. Acids Res
., 1996, 24, 990-991) reports single-stranded DNA ligation by T4 RNA ligase for PCR cloning of 5′ noncoding fragments and coding sequence of a particular gene.
A method of synthesizing large polynucleotides (such as RNA or DNA molecules longer than 200 bp) of arbitrary or predefined sequence and in a manner that will more readily lend itself to automation is desired. In addition, an improved version of the enzyme T4 RNA ligase that would increase the ability of this enzyme to catalyze the ligation of two oligonucleotides is also desired. Ideally, the improved enzyme would catalyze efficiently the ligation of oligonucleotides. Also, the ability of the enzyme to carry out these reactions at an elevated temperature or to use ddATP instead of ATP would be valuable properties in an improved ligase. By increasing the productivity of gene synthesis in laboratories, the present invention would improve scientists' ability to find, for example, enzymes capable of catalyzing reactions necessary to synthesize a new drug.
SUMMARY OF THE INVENTION
The present invention provides methods of preparing large polynucleotides (such as RNA or DNA molecules longer than 200 bp) of arbitrary sequence and in a manner that will more readily lend itself to automation than existing methods.
One aspect of the present invention is directed to methods of preparing a polynucleotide having at least 200 nucleotides and a predetermined nucleotide sequence comprising: providing a solid support, providing a plurality of oligonucleotides, wherein the combination of the nucleotide sequences of the oligonucleotides comprises the nucleotide sequence of the polynucleotide, contacting the solid support with the 3′ terminus of a first oligonucleotide from the plurality of oligonucleotides to form a tethered oligonucleotide, ligating the 3′ terminus of another oligonucleotide from the plurality of oligonucleotides to the 5′ terminus of the tethered oligonucleotide, and repeating the ligation with other oligonucleotides until the polynucleotide i
Hercules Incorporated
Horlick Kenneth R.
Strzelecka Teresa
Woodcock & Washburn LLP
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