Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-05-12
2003-02-18
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091400, C536S023100, C536S024200, C514S675000, C514S690000, C800S025000, C800S281000
Reexamination Certificate
active
06521427
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of oligonucleotide synthesis. More particularly, it concerns the assembly of genes and genomes of completely synthetic artificial organisms.
2. Description of Related Art
Present research and commercial applications in molecular biology are based upon recombinant DNA developed in the 1970's. A critical facet of recombinant DNA is molecular cloning in plasmids, covered under seminal patent of Cohen and Boyer (U.S. Pat. No. 4,740,470 “Biologically functional molecular chimeras”). This patent teaches a method for the “cutting and splicing” of DNA molecules based upon restriction endonucleases, the introduction of these “recombinant” molecules into host cells, and their replication in the bacterial hosts. This technique is the basis of all molecular cloning for research and commercial purposes carried out for the past 20 years and the basis of the field of molecular biology and genetics.
Recombinant DNA technology is a powerfull technology, but is limited in utility to modifications of existing DNA sequences which are modified through 1) restriction enzyme cleavage sites, 2) PAC primers for amplification, 3) site-specific mutagenesis, and other techniques. The creation of an entirely new molecule, or the substantial modification of existing molecules, is extremely time consuming, expensive, requires complex and multiple steps, and in some cases is impossible. Recombinant DNA technology does not permit the creation of entirely artificial molecules, genes, genomes or organisms, but only modifications of naturally-occurring organisms.
Current biotechnology for industrial production, for drug design and development, for potential applications of vaccine development and genetic therapy, and for agricultural and environmental use of recombinant DNA, depends on naturally-occurring organisms and DNA molecules. To create or engineer new or novel functions, or to modify organisms for specialized use (such as producing a human hormone), requires substantially complex, time consuming and difficult manipulations of naturally-occurring DNA molecules. In some cases, changes to naturally-occurring DNA are so complex that they are not possible in practice. Thus, there is a need for technology that allows the creation of novel DNA molecules in a single step without requiring the use of any existing recombinant or naturally-occurring DNA.
SUMMARY OF THE INVENTION
The present invention addresses the limitations in present recombinant nucleic acid manipulations by providing a fast, efficient means for generating practically any nucleic acid sequence, including entire genes, chromosomal segments, chromosomes and genomes. Because this approach is based on an completely synthetic approach, there are no limitations, such as the availability of existing nucleic acids, to hinder the construction of even very large segments of nucleic acid.
Thus, in a first embodiment, there is provided a method for the construction of a double-stranded DNA segment comprising the steps of (i) providing two sets of single-stranded oligonucleotides, wherein (a) the first set comprises the entire plus strand of said DNA segment, (b) the second set comprises the entire minus strand of said DNA segment, and (c) each of said first set of oligonucleotides being complementary to two oligonucleotides of said second set of oligonucleotides, (ii) annealing said first and said second set of oligonucleotides, and (iii) treating said annealed oligonucleotides with a ligating enzyme. Optional steps provide for the synthesis of the oligonucleotide sets and the transformation of host cells with the resulting DNA segment.
In particular embodiments, the DNA segment is 100, 200, 300, 40,, 800, 100, 1500, 200, 4000, 8000, 10000, 12000, 18,000, 20000, 40,000, 80,000; 100,000, 10
6
, 10
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, 10
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or more base pairs in length. Indeed, it is contemplated that the methods of the present invention will be able to create entire artificial genomes of lengths comparable to known bacterial, yeast, viral, mammalian, amphibian, reptilian, avian genomes. In more particular embodiments, the DNA segment is a gene encoding a protein of interest. The DNA segment further may include non-coding elements such as origins of replication, telomeres, promoters, enhancers, transcription and translation start and stop signals, introns, exon splice sites, chromatin scaffold components and other regulatory sequences. The DNA segment may comprises multiple genes, chromosomal segments, chromosomes and even entire genomes. The DNA segments may be derived from prokaryotic or eukaryotic sequences including bacterial, yeast, viral, mammalian, amphibian, reptilian, avian, plants, archebacteria and other DNA containing living organisms.
The oligonucleotide sets preferably are comprised oligonucleotides of between about 15 and 100 bases and more preferably between about 20 and 50 bases. Specific lengths include, but are not limited to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64.65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100. Depending on the size, the overlap between the oligonucleotides of the two sets may be designed to be between 5 and 75 bases per oligonucleotide pair.
The oligonucleotides preferably are treated with polynucleotide kinase, for example, T4 polynucleotide kinase. The kinasing can be performed prior to mixing of the oligonucleotides set or after, but before annealing. After annealing, the oligonucleotides are treated with an enzyme having a ligating function. For example, a DNA ligase typically will be employed for this function. However, topoisomerase, which does not require 5′ phosphorylation, is rapid and operates at room temperature, and may be used instead of ligase.
In a second embodiment, there is provided a method for construction of a double-stranded DNA segment comprising the steps of (i) providing two sets of single-stranded oligonucleotides, wherein (a) the first set comprises the entire plus strand of said DNA segment, (b) the second set comprises the entire minus strand of said DNA segment, and (c) each of said first set of oligonucleotides being complementary to two oligonucleotides of said second set of oligonucleotides, (ii) annealing pairs of complementary oligonucleotides to produce a set of first annealed products, wherein each pair comprises an oligonucleotide from each of said first and said second sets of oligonucleotides, (iii) annealing pairs of first annealed products having complementary sequences to produce a set of second annealed products, (iv) repeating the process until all annealed products have been annealed into a single DNA segment, and (v) treating said annealed products with ligating enzyme.
In a third embodiment, there is provided a method for the construction of a double-stranded DNA segment comprising the steps of (i) providing two sets of single-stranded oligonucleotides, wherein (a) the first set comprises the entire plus strand of sand DNA segment, (b) the second set comprises the entire minus strand of said DNA segment, and (c) each of said first set of oligonucleotides being complementary to two oligonucleotides of said second set of oligonucleotides, (ii) annealing said the 5′ terminal oligonucleotide of said first set of oligonucleotide with the 3′ terminal oligonucleotide of said second set of oligonucleotides, (iii) annealing the next most 5′ terminal oligonucleotide of said first set of oligonucleotides with the product of step (ii), (iv) annealing the next most 3′ terminal oligonucleotide of said second set of oligonucleotides with the product of step (iii), (v) repeating the process until all oligonucleotides of said first and said second sets have been annealed, and (vi) treating said annealed oligonucleotides wi
Campbell & Flores LLP
Chakrabarti Arun K.
Egea Biosciences, Inc.
Jones W. Gary
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