Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-01-05
2002-03-19
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S091200, C435S091300, C435S091520, C435S006120, C536S023100
Reexamination Certificate
active
06358712
ABSTRACT:
BACKGROUND
The Molecular Biology revolution began with the discovery of enzymes that were capable of cleaving double stranded DNA, so that DNA fragments were produced that could be ligated to one another to generate new, so-called “recombinant” molecules (see, for example, Cohen et al.,
Proc. Natl. Acad. Sci. USA
70:1293, 1973; Cohen et al.,
Proc. Natl. Acad. Sci. USA
70:3274, 1973; see also U.S. Pat. Nos. 4,740,470; 4,468,464; 4,237,224). The revolution was extended by the discovery of the polymerase chain reaction (PCR), which allowed rapid amplification of particular DNA segments, producing large amounts of material that could subsequently be cleaved and ligated to other DNA molecules (see, for example, U.S. Patent Nos. 4,683,195; 4,683,202; 5,333,675).
Despite the power of these digestion and amplification techniques, however, there remains substantial room for improvement. Reliance on digesting enzymes, called “restriction enzymes”, can render molecular biological experiments quite expensive. Moreover, many of the enzymes are inefficient or are only available in crude preparations that may be contaminated with undesirable entities.
At first, it seemed that PCR amplification might itself avoid many of the difficulties associated with traditional cut-and-paste cloning methods since it was thought that PCR would generate DNA molecules that could be directly ligated to other molecules, without first being cleaved with a restriction enzyme. However, experience indicates that most PCR products are refractory to direct cloning. One possible explanation for this observation has come from research revealing that many thermophilic DNA polymerases (including Taq, the most commonly used enzyme) add terminal 3′-dAMP residues to the products they amplify. Invitrogen (Carlsbad, Calif.) has recently developed a system for direct cloning of such terminally-dAMP-tagged products (TA Cloning Kit®; see U.S. Pat. No. 5,487,993) if the molecule to which they are to be ligated is processed to contain a single unpaired 3′-dTMP residue. While the Invitrogen system has proven to be very useful, it is itself limited in application by being restricted to ligation of products with only a single nucleotide overhang (an A residue), and is further restricted in that the overhang must be present at the 3′ end of the DNA molecule to be ligated.
There is a need for the development of improved systems for nucleic acid cloning. Particularly desirable systems would allow DNA ligation with minimal reliance on restriction enzymes, would provide for efficient ligation, and would be generally useful for the ligation of DNAs having a wide variety of chemical structures. Optimal systems would even provide for directional ligation (i.e., ligation in which the DNA molecules to be linked together will only connect to one another in one orientation).
SUMMARY OF THE INVENTION
The present invention provides an improved system for linking nucleic acids to one another. In particular, the present invention provides techniques for producing DNA product molecules that may be easily and directly ligated to recipient molecules. The product molecules need not be cleaved with restriction enzymes in order to undergo such ligation. In preferred embodiments of the invention, the DNA product molecules are produced through iterative DNA synthesis reactions, so that the product molecules are amplified products.
The inventive system provides techniques and reagents for generating product molecules with 3′ overhangs, 5′ overhangs, or no overhangs, and further provides tools for ligating those product molecules with recipient molecules. Where overhangs are employed, the length and sequence of the overhang may be varied according to the desires of the practitioner.
The inventive system also further provides methods for directed ligation of product molecules (i.e., for selective ligation of certain molecules within a collection of molecules), and also for methods of exon shuffling, in which multiple different product molecules are produced in a single ligation reaction. Preferred embodiments of the invention involve ligation of product molecules encoding functional protein domains, particularly domains naturally found in conserved gene families. The inventive DNA manipulation system is readily integrated with other nucleic acid manipulation systems, such as ribozyme-mediated systems, and also is susceptible to automation.
Specifically, in one aspect, a double stranded DNA molecule with a single stranded overhang comprised of RNA is provided. Additionally, in another aspect, a library of nucleic acid molecules, wherein each member of the library comprises 1) at least one nucleic acid portion that is common to all members of the library; and 2) at least two nucleic acid portions that differ in different members of the library, is also provided by the present invention. In a preferred embodiment, each of the nucleic acid portions in the library comprises protein-coding sequence and each library member encodes a continuous polypeptide. In yet another particularly preferred embodiment, each of the variable nucleic acid portions encodes a functional domain of a protein. This functional domain is preferably one that is naturally found in a gene family selected from the group consisting of the tissue plasminogen activator gene family, the animal fatty acid synthase gene family, the polyketide synthase gene family, the peptide synthetase gene family, and the terpene synthase gene family.
In yet another aspect of the present invention, a method of generating a hybrid double-stranded DNA molecule is provided. This method comprises the steps of 1) providing a first double-stranded DNA molecule, which double-stranded DNA molecule contains at least one single stranded overhang comprised of RNA; 2) providing a second double-stranded DNA molecule containing at least one single-strand overhang that is complementary to the RNA overhang on the first double-stranded DNA molecule; and 3) ligating the first and second double-stranded DNA molecules to one another so that a hybrid double-stranded DNA molecule is produced.
A further aspect of the present invention includes a method of generating a hybrid double-stranded DNA molecule, the method comprising 1) generating a first double-stranded DNA molecule by extension of first and second primers, at least one of which includes at least one base that is not copied during the extension reaction so that the extension reaction produces a product molecule containing a first overhang; 2) providing a second double-stranded DNA molecule containing a second overhang complementary to the first overhang; and 3) ligating the first and second double-stranded DNA molecules to one another, so that a hybrid double-stranded DNA molecule is produced.
In still a further aspect of the present invention, a method of generating a hybrid double-stranded DNA molecule is provided, the method comprising: 1) generating a first double-stranded DNA molecule by extension of first and second primers, at least one of which includes at least one potential point of cleavage; 2) exposing the first double-stranded DNA molecule to conditions that result in cleavage of the cleavable primer at the potential point of cleavage, so that a first overhang is generated on the first DNA molecule; 3) providing a second double-stranded DNA molecule containing a second overhang complementary to the first overhang; and 4) ligating the first and second double-stranded DNA molecules to one another, so that a hybrid double-stranded DNA molecule is produced.
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Coljee Vincent W.
Donahue William
Jarrell Kevin A.
Mikheeva Svetlana
Choate, Hall and Stewart
Fredman Jeffrey
Goldberg Joanine
Herschbach Jarrell Brenda
Trustee of Boston University
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