Cloning vectors and vector components

Details Cloning vectors and vector components Cloning vectors and vector components

2001-11-15

2004-03-23

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Vector, per se

C536S023100

Reexamination Certificate

active

06709861

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to systems, methods, and compositions for cloning and sequencing insert nucleic acid sequences. In particular, the present invention provides vectors and vector components configured for multiplex cloning, multiplex sequencing, and fixed orientation cloning. The present invention also provides vectors and vector components that allow insert sequences that are deleterious to a host cell to be successfully cloned.
BACKGROUND OF THE INVENTION
Prior to the 1990's, DNA sequencing was a time consuming, labor intensive, manual protocol by which individual researchers read 100's of bases per day from a single DNA template. It has since evolved into an automated, robotic process by which major genome sequencing centers read tens of millions of bases from tens of thousands of DNA templates per day. This vast increase in sequencing capacity has broadened the scope of DNA sequencing to entire genomes rather than individual genes. It has likewise created a need to increase the rate of throughput in all stages of the sequencing process.
The most prominent example of large scale sequencing to date is the Human Genome Initiative, an effort to sequence all 3.3 billion bases of the human genome. Begun in 1990, the Human Genome Initiative was declared “finished” on Jun. 26, 2000, by the major genome centers involved. The public draft genome released by the National Institutes of Health consortia was 85% assembled, with 97% of the genome covered by clones whose location is known. This project required reading some 25 million DNA sequences. In a completely independent effort, Celera Corporation claimed to have 99% of the genome sequence assembled at a 3× redundancy level, which required 27 million DNA sequencing reads.
The public effort for “complete and accurate” sequencing, typically defined as 5× coverage and an accuracy of not more than 1 mistake every 10,000 bases, will require sequencing millions of additional plasmid clones over several more years to obtain high quality data on the entire genome. Because so much of the human genome is not characterized, a more complete understanding of it will be facilitated by sequencing the genomes of other organisms for comparison, such as the mouse, rat, dog, and chimpanzee. In fact, Celera claims to have sequenced three mouse genomes during the year 2000, while the NIH consortia of university and international genome centers have begun work on the mouse and rat genome. The NIH has also initiated funding of pilot sequencing projects for the chicken, puffer fish, and zebra fish.
At the 12
th
International Genome Sequencing and Analysis Conference in Miami, Fla. (Sep. 12-15, 2000), Celera presented data showing that over 200,000 plasmid template purifications a day are required to sustain their ongoing sequencing efforts. The NIH consortia purify a similar number of templates on a daily basis. Genome sequencing facilities at other large corporations, overseas national genome projects, and smaller academic labs sequence an additional 500,000 plasmid templates per day. Thus, the worldwide rate of sequencing is rapidly approaching 1,000,000 templates per day.
The generation of clone banks, or libraries, of DNA is an important intermediate step in sequence analysis of whole genomes. In a process called shotgun cloning and sequencing, large molecules of DNA, often more than 100,000 bases (100 kb) in length, are fragmented and reduced to libraries of numerous sub-clones of approximately 1-4 kb for propagation and sequence analysis. Most large-scale DNA sequencing strategies depend on a multi-step process to randomly fragment the target molecule into these smaller pieces, which are then enzymatically joined (ligated) into a cloning vector in a reaction that inserts one or more DNA fragments into a single site in each vector molecule (Fitzgerald et al., Nucleic Acids Res. 14:3753 [1992]). This ligation mixture is introduced into specific strains of
Eschericia coli
(
E. coli
), with each bacterial cell propagating one vector along with any DNA fragments it carries. The vector DNA, which may or may not contain an insert, is purified from each cell line and used as a template in an enzymatic sequencing reaction (Sanger et al., Proc Natl Acad Sci USA 74:5463 [1977]; Prober et al., Science 238:336 [1987]; Tabor and Richarson,
Proc Natl Acad Sci U S A
92:6339 [1995], all of which are hereby incorporated by reference). The reaction product is analyzed by automated sequencing instruments to determine the linear sequence of the sub-cloned DNA fragments (Smith et al.,
Nature
321:674 [1986], hereby incorporated by reference). Computer algorithms are used to assemble the data from the library of sub-fragments, typically producing sequence information for 80-95% of the original DNA molecule. “Gap filling” techniques are used to determine the remaining 5-20% of the target DNA.
Although most DNA sequencing methods utilize one template or primer per sequencing reaction, there are exceptions to this pattern. In early examples, Church et al. (Science 240: 185 [1988]) and Creasey et al. (BioTechniques 11: 102 [1991]) performed multiple Sanger dideoxy sequencing reactions in a single set of four tubes, using vectors containing unique sequence tags. The reactions from each set of tubes were run on a sequencing gel and transferred to a nylon membrane. Each sequence reaction was then detected by sequentially probing the membrane with an oligonucleotide specific for the tag on each vector. Other variations on this theme have also been developed (Cherry et al., Genomics 20: 68 [1994]).
Subsequently, Wiemann et al. (Anal. Biochem. 224: 117 [1995]; Anal. Biochem. 234: 166 [1996]) showed that fluorescently labeled sequencing primers could be used to simultaneously sequence both strands of a dsDNA template. Recent examples have demonstrated multiplex co-sequencing using the four-color dye terminator reaction chemistry pioneered by Prober et al. (Science 238: 336 [1987]). At the 10th International Genome Sequencing and Analysis Conference, (Sep. 17-20, 1998, Miami Beach, Fla.), Uhlen (Royal Institute of Technology) and Chiesa (PE Biosystems) independently showed that biotinylated oligomers could be used to specifically capture an individual sequencing reaction from a pool of multiple reactions in a single tube.
Numerous vectors are available for cloning DNA into
E. coli.
Conventional plasmid vectors are normally double stranded circular DNA molecules containing restriction enzyme recognition sites suitable for inserting exogenous DNA sequences, an antibiotic selectable gene, an origin of replication for autonomous propagation in the host cell, and a gene for the discrimination or selection of clones that contain recombinant insert DNA.
One of the first recombinant DNA cloning systems used a dual antibiotic resistant plasmid such as pBR322 (Bolivar et al., Gene 2:95 [1977]). One of the resistance genes served to select for those cells taking up plasmid DNA. This gene was typically the beta-lactamase gene (Amp or ampR), which confers resistance to ampicillin (amp). The other resistance gene, Tet or tetR, encoding resistance to tetracycline (tet), was used indirectly as the indicator for recombinant clones. The foreign DNA fragment was inserted into any of a number of restriction sites within the Tet gene, resulting in inactivation of the Tet gene and sensitivity of the transformed cell to killing by tetracycline.
Thus, to find those clones that might have contained foreign insert DNA, the transformed cells were first spread onto ampicillin-containing plates. Those colonies that grew were replica plated onto tetracycline-containing plates. The colonies growing on the ampicillin but not on the tetracycline plates were likely candidates for further analysis. This screening method required additional labor and time compared to newer methods and is rarely used now.
The predominant cloning system in

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