Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-11-20
2004-12-07
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S320100, C536S023100, C536S024200
Reexamination Certificate
active
06828102
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to plasmids, either in circular or linear form, useful in monitoring the efficiency of a restriction endonuclease digestion reaction.
BACKGROUND OF THE INVENTION
Restriction endonucleases are a class of enzymes that occur naturally in bacteria and when they are purified away from other contaminating bacterial components, they can be used in the laboratory to break DNA molecules into precise fragments. They are the biochemical scissors by which genetic engineering and analysis is performed and, therefore, have proved to be indispensable tools in modern genetic research.
Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the “recognition sequence”) along the DNA molecule. Once bound, they cleave the molecule within, or to one side of, the sequence. Different restriction endonucleases have affinity for different recognition sequences. Close to one hundred different restriction endonucleases have been identified among the many hundreds of baterial species that have been examined to date.
Plasmid construction is an essential technique in the cloning and manipulation of genes. Most plasmid construction strategies are designed to ensure that insertion of a DNA fragment into a vector predominates over the default event in which the vector recircularizes without incorporating an insert. There are four major ways of achieving high insertion ratios. They all rely, in one way or another, on rendering the vector incapable of circularizing without ligating to the insert fragment. For example, use of phosphatased vectors favors a high insertion frequency because dephosphorylated ends can only ligate to phosphorylated ends supplied by the insert. In utilizing this method, however, the phosphatase is difficult to control. TA vectors, used for cloning PCR-amplified DNA, are designed to be linearized in such a way as to leave single 3′ thymidine overhangs which can ligate only to adenine over-hangs like those left on the 3′ ends of DNA extended by taq (in this method ligation is catalyzed either by DNA ligase or DNA topoisomerase). The TA cloning polymerase method also favors efficient insertion of the PCR product. One limitation of this method is that not all thermostable DNA polymerases used for PCR leave the 3′ adenine. Partial fill-in of most 5′ restriction site overhangs by Klenow polymerase renders them non-circularizable; an insertable fragment is then generated by partial fill-in of an appropriate restriction site (e.g. a vector cleaved with SalI (G!TCGAC), filled in with T and C can be ligated to Sau3Al (!GATC) fragments filled in with G and A).
The fourth widely used technique is referred to as “forced ligation.” This technique makes use of a vector digested with two different restriction enzymes to generate a non-circularizable linear molecule; an insert fragment generated with the same enzymes is ligated in, thereby closing the plasmid. Forced ligation of gel-purifed restriction fragments is the simplest and most efficient method of plasmid construction in situations where segments are transferred from one plasmid to another, or connected together in new combinations, e.g. in mutagenesis, in analysis of gene expression, and in numerous other applications. Double-digested vectors are also convenient for cloning PCR fragments into a construct directly, without the necessity of a TA cloning step, provided that the PCR primers are designed with appropriate restriction sites.
The forced ligation method provides a high percentage of insertion (often greater than 90%), provided that both of the vector sites are fully digested, a criterion which is not always easy to achieve, since many enzymes are of uncertain stability or are inhibited by contaminants present in DNA preparations. Hence demonstration of good digestion is highly desirable before proceeding with the construction; this is also problematic, since complete digestion is impossible to document by agarose electrophoresis when the two sites are in a polylinker; and even when the sites are separated by as much as 10% of the full length of the vector, the desired double cut linear may not be fully resolved from contaminating partially digested molecules. Even a barely detectable residue of partially digested vector leads to an inordinately large number of re-circularized plasmids, requiring a large number of minipreps to be analyzed in search of the desired construct. This is especially troublesome when available insert fragments are present in limited amounts or when lower frequency events, such as triple ligations, are desired.
Because of the uncertainties in obtaining reliable double digests, marker plasmids can be included in mixed test digestions to be sure that both enzymes are active and that neither is inhibited by contaminants in the vector plasmid prep or in a PCR product to be inserted. Suitable marker plasmids can sometimes be found in a plasmid collection for a given pair of enzymes but often none is available, limiting the value of the digest marker maneuver, especially in labs with small plasmid inventories. What is needed is a double digest marker plasmid or set of plasmids which is informative of digestion efficiency for a variety of known restriction endonucleases.
SUMMARY OF THE INVENTION
In one aspect, the invention relates to a plasmid for use in monitoring the efficiency of a restriction endonuclease digestion, comprising at least one spacer segment comprising a nucleic acid sequence that is restriction site-free and at least two polylinker regions containing a plurality of unique restriction sites. The restriction sites are distributed so that digestion of the plasmid with two restriction endonucleases whose sites are represented on the plasmid results in two fragments, each of the fragments being sufficiently different in size from the intact plasmid so as to be readily distinguishable from the plasmid. Generally, fragments that are at least about 15% shorter than the intact plasmid can easily be distinguished from the intact plasmid when visualized on an agarose gel.
In another aspect, the invention relates to a plasmid for monitoring the digestion efficiency of a restriction endonuclease digestion comprising at least one spacer segment comprising a nucleic acid sequence that is restriction site-free and at least two polylinker regions where the polylinker regions contain a plurality of unique restriction sites distributed so that, for any two sites, the two sites are situated within different polylinker regions on at least one plasmid of a set of such plasmids. The polylinker regions are separated by a spacer region of restriction site-free nucleic acid whose length is about 15-85% of the length of the plasmid. Alternatively, the length of the spacer region of restriction site-free nucleic acid may be 20-85%, 30-85%, 40-85% or 50-85% of the length of the intact plasmid. Digestion of the plasmid with two endonucleases whose recognition sites are represented on the plasmid, therefore, results in two fragments, one of the fragments being at least about 15%-85% of the length of the intact plasmid. The plasmid of the present invention further comprises a replication origin and a selectable marker, for example, the amp
R
gene. In one embodiment, the plasmid of the present invention comprises a vector backbone of a plasmid such as pUC, pBR322 or pBS.
In a related aspect, the invention relates to a set of plasmids for use in monitoring the efficiency of a restriction endonuclease digestion, wherein each of the plasmids of the set comprises at least one spacer segment comprising a nucleic acid sequence that is restriction site-free and at least two polylinker regions containing a plurality of unique restriction sites distributed so that, for any two sites, the two sites are situated within different polylinker regions on at least one plasmid of the set and the polylinker regions are separated by a spacer segment whose length is about 15-85% of the length of the plasmid.
In yet another aspect,
Albany Medical College
Dias, Esq. Kathy Smith
Heslin Rothenberg Faley & Mesiti P.C.
Ketter James
Lambertson David
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