Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-05-10
2003-10-21
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120
Reexamination Certificate
active
06635453
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. The present invention also relates to the generation of libraries of polynucleotides, library screening and identification of library members having desired characteristics.
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 modem 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) have 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” oligonucleotides is reported in U.S. Pat. No. 5,942,609 and Chen, et al.,
Nucleic Acids Res.,
1990, 18, 871. 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 WO 83/02626 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. Similarly, Schmitz, et al., (
Org. Lett.,
1999, 1, 1729) describes the synthesis of short oligonucleotides from mononucleotide building blocks using T4 RNA ligase, but reports exceedingly long reaction times, militating against the formation of longer sequences. Neither International Publication WO 83/02626, Mudrakovskaia et al., nor Schmitz, 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. Ligation of oligonucleotides using T4 RNA ligase has also been reported in Walker, et al.,
Proc. Natl. Acad. Sci. USA,
1975, 72, 122 and Ohtsuka, et al,
Nucleic Acids Res.,
1976, 3, 1613, but the technique was recognized as problematic due to the accumulation of unwanted by-products (Krug, et al.,
Biochemistry,
1982, 21, 1858).
The enhanced ability for de novo synthesis of large polynucleotides or genes may greatly facilitate the preparation of combinatorial libraries of polynucleotides because it would be much more efficient than existing methods. For example, combinatorial libraries of genes can be made by cassette mutagenesis (Oliphant, et al.,
Gene,
1986, 44, 177 and Oliphant, et al.,
Proc. Natl. Acad. Sci. USA,
1989, 86, 9094) whereby genes with random combinations of nucleotides are created. Similarly, U.S. Pat. Nos. 5,723,323; 5,763,192; 5,814,476; and 5,817,483 describe libraries of expression vectors having stochastic DNA regions. By simultaneously randomly mutating fifteen nucleotides of a gene, a billion different sequences can be generated. Current methods of screening and molecular cloning often limit the number of sequences that can be screened to a much smaller number. Although there are examples of libraries with 10
8
individual mutants (Cwirla, et al.,
Proc. Natl. Acad. Sci USA,
1990, 87, 6378), certain screening methods to identify u
Delagrave Simon
Marrs Barry
Hercules Incorporated
Horlick Kenneth R.
Strzelecka Teresa
Woodcock & Washburn LLP
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