Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2002-05-24
2003-07-01
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023100, C435S091200
Reexamination Certificate
active
06586237
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to cloning nucleic acid molecules that have been exposed to an enzyme, or other substance, which produces an overhanging nucleotide(s) at one or more 3′ or 5′ ends of the molecules.
BACKGROUND
The polymerase chain reaction (PCR) has become an invaluable tool for molecular biologists. Numerous applications have been discovered for PCR. Examples include DNA fingerprinting, DNA sequencing, site directed mutagenesis and cloning applications.
PCR is based on three discrete, multiply repeated steps: denaturation of a DNA template, annealing of a primer to the denatured DNA, and extension of the primer with a polymerase to create a nucleic acid complementary to the template. Each primer extension product specifically anneals with a complementary primer and the resulting primed template DNA acts as a substrate for a further extension reaction. These steps are repeated many times, for example, by using an automated cycling procedure, thereby exponentially amplifying the initial nucleic acid material. The conditions under which these steps are performed are well established in the art. Procedures for conducting PCR have been extensively described. See, for example, U.S. Pat. Nos. 4,683,195 and 4,683,202, which are incorporated in their entirety herein by reference.
It is often desirable to obtain clones of a PCR amplified DNA product. A common method for cloning PCR products involves incorporation of flanking restriction sites onto the ends of primer molecules. In this method the PCR cycling is carried out and the amplified DNA is purified, restricted with an appropriate endonuclease(s) and ligated to a compatible vector preparation. Thus, this PCR cloning method requires the step of preparing PCR primer molecules which include base sequences having a preferred restriction recognition sequence. Also, this method can result in unintended internal restriction of uncharacterized restriction site sequences. In addition, cleavage of the PCR product by the preferred restriction enzyme may often be inefficient because the restriction site is close to the end of the DNA molecule to be cleaved. Such limitations add to the cost and complexity of cloning PCR products on a routine basis.
Many DNA polymerases, including Taq DNA polymerase and other thermostable DNA polymerases may catalyze a non-template directed addition of single deoxynucleotide monophosphate (dNMP) residue to the 3′ termini of blunt-ended DNA duplexes. Thus, DNA polymerase may naturally create a single base, cohesive termini on otherwise blunt end DNA fragments. For example, during PCR, a thermostable DNA polymerase used may add a single nucleotide to the 3′ end of an otherwise blunt end DNA PCR product. These overhanging residues are widely viewed as incompatible with most molecular cloning schemes. The overhanging residues may be removed with enzymes such as S1 nuclease and Klenow fragment to create blunt ends. The blunted PCR products can then be cloned into a vector which has been digested with a restriction enzyme that will leave a blunt end compatible with the blunted PCR fragment.
Many problems exist with blunt end cloning of PCR products. For example, enzymatic blunting of the PCR product may often be inefficient. Also, recircularization of the blunt ended linear vector with no target sequence inserted is common leading to a low cloning efficiency. To help overcome this last problem dephosphorylation is sometimes used.
Dephosphorylation of the vector can help prevent self-ligation thereby increasing the efficiency of target sequence insertion. However, this procedure can be problematic because of, for example, the difficulty that may be involved in inactivating the phosphorylase.
One technique that has been developed to directly clone PCR products takes advantage of the Taq polymerase catalyzed non-template directed addition of dAMP residue to the 3′ termini of some blunt-ended DNA PCR products (described in U.S. Pat. Nos. 5,487,993 and 5,827,657). This technique uses a linearized vector with compatible, cohesive, single nucleotide dTMP residues overhanging each end of the linearized vector. PCR DNA products which have a dAMP nucleotide overhang at both 3′ ends may be cloned into these vectors. This method is based on the publication of Clark (Nucleic Acids Res. Vol. 16 pg. 9677-9686). In Clark, the data showed that a dAMP nucleotide was preferentially added to an 18 base primer in a non-template directed fashion by a variety of DNA polymerases, including Taq DNA polymerase.
It has become apparent that nucleotides other than DAMP may be preferentially added to a 3′ end of certain PCR products in a non-template dependent manner. In particular, dGMP may often be the preferred nucleotide added. dCMP and dTMP may also be preferentially added but possibly to a lesser degree than dAMP and dGMP. Variables that may effect which nucleotide is added to the 3′ end of the PCR product include the primer sequences, the sequence of the target DNA and the DNA polymerase used.
The above-described cloning techniques are ineffective to directly clone PCR products that have a non-dAMP nucleotide added to one or both of the PCR product's 3′ end(s).
In view of the above considerations, what is needed are compositions and methods for directly cloning PCR products which have been extended at each 3′ end by any nucleotide, for example, dGMP, dAMP, dCMP or dTMP.
SUMMARY
The present invention meets this need and provides for compositions and methods that allow for the cloning of PCR products which include PCR products with any combination of the nucleotides dAMP, dTMP, dCMP or dGMP overhanging the PCR product's 3′ ends.
In accordance with the present invention there are provided compositions which include one or more or two or more linear DNA molecules. These DNA molecules include a single nucleotide overhanging each 3′ end. The compositions may include one or more DNA molecules selected from: a linear DNA molecule with an overhanging dTMP residue at each 3′ end, a linear DNA molecule with an overhanging dGMP at each 3′ end, a linear DNA molecule with an overhanging dCMP at each 3′ end, a linear DNA molecule with an overhanging dAMP at each 3′ end, a linear DNA molecule with an overhanging dTMP at one 3′ end and an overhanging dAMP at another 3′ end, a linear DNA molecule with an overhanging dTMP at one 3′ end and an overhanging dGMP at another 3′ end, a linear DNA molecule with an overhanging dTMP at one 3′ end and an overhanging dCMP at another 3′ end, a linear DNA molecule with an overhanging dAMP at one 3′ end and an overhanging dGMP at another 3′ end, a linear DNA molecule with an overhanging dAMP at one 3′ end and an overhanging dCMP at another 3′ end and/or a linear DNA molecule with an overhanging dGMP at one 3′ end and an overhanging dCMP at another 3′ end. This embodiment allows for the cloning of PCR products with any combination of dNMPs non-specifically attached to each 3′ end of the PCR product.
In one embodiment, the one or more or two or more DNA molecules, which comprise a composition of the invention, are the same or substantially the same nucleotide sequence except for the single nucleotide overhanging each 3′ end of the DNA molecule or molecules.
In one embodiment, the one or more or two or more DNA molecules which comprise a composition of the invention are a linearized DNA molecule, for example, a linearized vector. Examples of vectors include a plasmid, a phagemid, a bacteriophage, a prokaryotic expression vector, a eukaryotic vector, a eukaryotic expression vector or a virus. In certain embodiments, the vectors may include a prokaryotic replicon, a transcription start site and/or antibiotic resistance.
Compositions of the invention may include a nucleic acid PCR product. The compositions of the invention may include being transformed into a host cell. The host cell may be eukaryotic or
Ketter James
Sullivan Daniel M.
Yesland Kyle D.
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