EP0690870A1 - Dinucleotide restriction endonuclease preparations and methods of use - Google Patents
Dinucleotide restriction endonuclease preparations and methods of useInfo
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- EP0690870A1 EP0690870A1 EP94912866A EP94912866A EP0690870A1 EP 0690870 A1 EP0690870 A1 EP 0690870A1 EP 94912866 A EP94912866 A EP 94912866A EP 94912866 A EP94912866 A EP 94912866A EP 0690870 A1 EP0690870 A1 EP 0690870A1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
- C12N9/1007—Methyltransferases (general) (2.1.1.)
Definitions
- the present invention relates generally to isolated purified polynucleotides which encode restriction enzymes and to methods of expressing the restriction enzymes from such polynucleotides. More particularly this invention relates to isolated purified polynucleotides which encode Cv/JI and related methods for the production of this enzyme. Other aspects of the invention relate to methods for partially or completely digesting DNA at a dinucleotide sequence. More particularly, this aspect of the invention relates to methods of generating quasi-random fragments of DNA, and methods of cloning, labeling, and sequencing DNA, as well as epitope mapping of proteins.
- the invention also relates to methods for generating sequence-specific oligonucleotides from DNA, without prior knowledge of the nucleic acid sequence of such DNA, and to methods for cloning and labeling DNA after restriction digestion by a two base recognition endonuclease reagent.
- This invention also relates to methods for cloning, labeling, and detecting nucleic acids using two base restriction endonuclease reagents, such as Cv T I, BsuR I, Aci I or CGase I.
- the invention relates to labeling DNA by taking advantage of certain properties of the holo-enzyme of thermostable DNA polymerases.
- Restriction endonucleases are a group of enzymes originally found to be expressed in a wide variety of prokaryotic organisms. More recently they have also been found to be encoded in viral genomes. These enzymes catalyze the selective cleavage of DNA at generally short sequences, often unique to the individual enzyme. This ability to cleave makes restriction endonucleases indispensible tools in recombinant DNA technology. The increased commercial availability of the isolated enzymes has contributed in large part to the enormous expansion in the field of recombinant DNA technology over the last few years.
- Type II enzymes are part of a binary system known as a restriction modification system consisting of a restriction endonuclease that cleaves a specific sequence of nucleotides and a separate DNA modifying enzyme that modifies the same recognition sequence and thereby prevents cleavage by the cognate endonuclease.
- a total of about 2103 restriction enzymes are known, encompassing 179 different type ⁇ specificities (Roberts, et al , Nucl. Acids Res. 20:2167-2180 (1992)).
- type ⁇ restriction enzymes Although there are more than 1200 type ⁇ restriction enzymes, many of them are members of groups which recognize the same sequence. Restriction enzymes that recognize the same sequence are said to be isoschizomers. The vast majority of type ⁇ restriction enzymes recognize specific double-stranded sequences which are four, five, or six nucleotides in length and which display twofold (palindromic) symmetry. A few enzymes recognize longer sequences or degenerate sequences.
- cleavage sites within a palindrome differs from enzyme to enzyme. Some enzymes cleave both strands exactly at the axis of symmetry generating fragments of DNA that carry blunt ends, while others cleave each strand at similar sequences on opposite sides of the axis of symmetry, creating fragments of DNA that carry protruding, single-stranded termini.
- Restriction endonucleases with shorter recognition sequences cut DNA more frequently than those with longer recognition sequences. For example, assuming a 50% G-C content, a restriction endonuclease with a 4-base recognition sequence will cleave, on average, every 4 4 (256) bases compared to every 4" (4096) bases for a restriction endonuclease with a 6-base recognition sequence. Under certain conditions some restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered specificity has been termed "star" (*) activity and is observed only under certain non-standard reaction conditions. The manner in which an enzyme's specificity is altered depends on the particular enzyme and on the conditions employed to induce the star activity.
- Conditions that contribute to star activity include high glycerol concentration, high ratio of enzyme to DNA, low ionic strength, high pH, the presence of organic solvents, and the substitution of Mg + + with other divalent cations.
- the most common types of star activity involve cutting at a recognition sequence having a single base substitution, cutting at sites having truncation of the outer bases of the recognition sequence, and single-strand nicking.
- the following restriction endonucleases show star activity: Ase I, BamH I, BssH II, BsuR I, CviJ I, EcoR I, EcoR V, Hind m, Hinf I, Kpn I, Pst I, Pvu II, Sal I, Sea I, Taq I, and Xmn I.
- Star activity is generally viewed as undesirable, and of little intrinsic value.
- 31 have a 4- base recognition sequence
- 11 have a 5-base recognition sequence
- 127 have a 6- base recognition sequence
- 10 which have recognition sequences of greater than 6 bases.
- a restriction endonuclease has a recognition sequence of less than 4 bases.
- CviJ I may be altered to cleave DNA more frequently. This activity is referred to as CviJ I , for star or altered specificity. However, CviJ I activity is not observed under conditions which favor star activity of other restriction endonucleases.
- the restriction enzyme BsuR I normally recognizes the sequence GGCC and cleaves between the G and C to leave blunt ends. (Heininger, et al. , Gene 1:291-303 (1977)).
- the restriction endonuclease provides a mechanism of defense against foreign DNA molecules (e.g., bacteriophage DNA) by virtue of its ability to distinguish and cleave only exogenous DNA, leaving endogenous bacterial DNA unaffected.
- foreign DNA molecules e.g., bacteriophage DNA
- Viral endonucleases possess the same discerning capabilities, but rather than providing a means for defense, this activity has presumably evolved to cripple the host's ability to replicate its own DNA and allows the virus to assume control of the host's replication machinery.
- Bacteria and viruses which express restriction endonucleases necessarily possess the inherent ability to protect their own genome from cleavage by their endogenous endonuclease.
- the primary mechanism by which this is accomplished is by modifying the organisms own DNA by, for example methylating a base in the recognition sequence which prevents binding and cleavage by the endonuclease. Therefore, to insure viability, the genome of an organism which expresses a restriction endonuclease is almost always heavily modified, usually by methylation of cytosine or adenosine bases.
- the methylase enzyme which modifies the genome acts in tandem with the endonuclease, either as part of an enzyme complex (restriction/modification complex) or as two distinct entities. Therefore, recognizing that an organism expresses an enzyme with endonuclease activity strongly suggests the expression of an associated modifying methylase enzyme (and vice versa) and this association has led to isolation and cloning of a number of commercially available restriction/modification enzymes for use in the laboratory as discussed below.
- restriction endonucleases exists when cleavage of a given sequence is required and no known endonuclease exists which is specific for that particular sequence. Therefore, the continued identification and isolation of unique restriction endonucleases and altered reaction conditions will allow for even more sophisticated manipulation of DNA in vitro.
- Nwanko, D.O. and Wilson, G.G. Gene 64:1-8 (1988), describe the cloning and expression of the Mspl restriction and modification genes isolated from Moraxella sp. This system recognizes the sequence 5 '-CCGG-3 ' and both enzymes are functional in E. coli. Evidence indicates that these genes are transcribed in opposite directions, thus are probably under the control of different promoters. Ashok, K.D. , et al. , Nucleic Acids Research 20: 1579-1585 (1992), describe the purification and characterization of cloned Mspl methyltransferase, over-expressed in E. coli. At low concentrations the enzyme exists as a monomer, but at higher concentrations it exists mainly as a dimer. Polyclonal antibodies to the enzyme cross-react with methyltransferase genes of other modification systems.
- this reference describes the cloning of the Taql and HaeQ. systems from Thermits aquaticus and Haemophilus aegypticus, respectively.
- bacterial DNA was initially purified and digested, and the fragments were then cloned into a vector to produce a bacterial DNA library.
- the library was then transformed into E. coli and the cells were plated. Colonies were then scraped from the plate to form a primary cell library. Plasmid DNA from this cell library was purified and digested with the endonuclease of the two gene system.
- Bacteria which expressed the methylase gene had modified plasmid DNA which was protected from endonuclease activity, while plasmids from bacteria which lacked the intact methylase gene were digested. The resulting, undigested plasmid DNA was then transformed into another bacterial strain and the bacteria were plated. Surviving colonies were again harvested to give a secondary cell library and the entire procedure repeated. Plasmids which code for the complete restriction-modification system presumably survived each round of purification and were enriched. Bacteria which survive several rounds of enrichment were subsequently assayed for both methylase and endonuclease activity.
- Chlorella virus D -3A encodes a unique restriction endonuclease called CviJl (Xia et al. Nucleic Acids Res. 15:6075-6090 (1987)).
- IL-3A is a large, polyhedral, plaque-forming phycodnavirus (Francki,
- the use of a two/three base recognition endonuclease, such as CviJl, to improve numerous conventional molecular biology applications as well as permitting novel applications has been described in co-pending U.S. Patent Application Ser.No. 08/036,481, filed on March 24, 1993.
- the application discloses methods for generating sequence-specific oligonucleotides from DNA without prior knowledge of the nucleic acid sequence of such DNA, and to methods for cloning and labeling DNA after restriction digestion by a two base recognition endonuclease.
- the application also teaches methods for generating quasi-random fragments of DNA, methods for cloning, labeling, and sequencing DNA, as well as epitope mapping of proteins.
- the ability to generate numerous oligonucleotides with perfect sequence specificity or quasi-random distributions of DNA fragments such as is possible with Cvz ' JI has important implications for a number of conventional and novel molecular biology procedures.
- CviJ ⁇ CviJl restriction endonuclease
- the present invention provides purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts thereof) encoding a unique restriction endonuclease, CviJl, as well as polypeptides and variants thereof which display activities characteristic of CviJl. Activities of CVfJI include the recognition of specific DNA sequences, binding to these sequences and cleaving the bound DNA into fragments.
- Preferred DNA sequences of the invention include viral genomic sequences as well as wholly or partially chemically synthesized DNA sequences. Replicas (i.e., copies of the isolated DNA sequences made in vivo or in vitro) of DNA sequences of the invention are also contemplated.
- a preferred DNA sequence is set forth in SEQ ID NO: 2 herein and is contained as an insert in the plasmid pCJH1.4.
- the invention provides purified isolated DNA encoding a JI polypeptide by means of degenerate codons.
- Also provided are autonomously replicating recombinant constructions such as plasmid DNA vectors incorporating vz ' JI sequences and especially vectors wherein DNA encoding CviJl or a Cw ' JI variant is operatively linked to an endogenous or exogenous expression control DNA sequence.
- host cells such as prokaryotic and eukaryotic cells
- host cells are stably transformed with DNA sequences of the invention in a manner allowing the desired polypeptides to be expressed therein.
- Host cells expressing CviJl and CviJl variant products are useful in methods for the large scale production of CviJl and CviJl variants wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the host cells or from the medium in which the cells are grown.
- a preferred host cell is E. coli.
- Still another aspect of the invention is a recombinant CviJl polypeptide.
- the present invention is also directed to a method for restriction endonuclease digestion of DNA comprising the step of digesting DNA with a restriction endonuclease reagent under conditions wherein said DNA is digested at 11 of 16 possible dinucleotide sequences and wherein said dinucleotide sequences are selected from the group consisting of PuCGPu, PuCGPy, and
- the present invention is directed to shotgun cloning of DNA, epitope mapping, and for labeling DNA using the digestion methods of the present invention.
- the present invention provides methods for quasi-random fragmenting of DNA using the digestion methods of the present invention under conditions wherein the DNA is only partially cleaved and the site preference of the restriction endonuclease reagent is greatly reduced.
- quasi-random is meant an overlapping population of DNA fragments produced by digesting DNA using the methods of the present inventions without apparent site-preference and which appears as a smear upon electrophoresis in a 1-2 wt. % agarose gel.
- the present invention is also directed to the shotgun cloning and sequencing of quasi-random fragments of DNA produced by the methods of the present invention.
- Quasi- random fragments in the shotgun cloning method of the present invention are produced by partial digestion of DNA with a restriction endonuclease reagent according to the methods of the present invention. More particularly, quasi- random fragments of DNA useful in the cloning method of the present invention are produced by the partial digestion of the DNA to be cloned with Cvi I, BsuR I or with a restriction endonuclease reagent termed CGase I comprising Taq I and Hpa ⁇ . Quasi-random fragments having a length of between about 100 and about
- the present invention is also directed to the generation of quasi-random fragmentation of DNA using the method of the present invention for the purposes of epitope mapping and gene cloning. These quasi-random fragments are expressed either in vitro or in vivo and the smallest fragment containing the desired function is identified by screening assays well known in the art.
- the present invention is also directed to the production of anonymous primers from any DNA without prior knowledge of the nucleotide sequence.
- the present invention provides methods for anonymous primer cloning and sequencing after complete digestion of DNA utilizing CviJ I, BsuR I or CGase I using the methods of the present invention.
- the present invention is directed to methods of labeling and detecting DNA comprising the complete digestion of DNA using the methods of the present invention, followed by a heat denaturation step, to yield sequence specific oligonucleotides.
- an aspect of the present invention involves labeling DNA with sequence specific oligonucleotides of about 20 to about 200 bases in length (with an average size of between 20-60 bases) generated by CviJ I, BsuR I or CGase I digestion of the template DNA.
- the invention is directed to restriction generated oligonucleotide labeling (RGOL) of DNA which comprises the digestion of an aliquot of template DNA with CviJ I followed by a simple heat denaturation step, thereby generating numerous sequence specific oligonucleotides, which can then be utilized for labeling nucleic acids by a number of methods, including primer extension type reactions with a DNA polymerase and various labels, isotopic ornon-isotopic (RGOL-PEL); 5' end labeling with polynucleotide kinase: 3' end labeling using terminal transferase and various labels,isotopic or non-isotopic. Labeling at the 3' end, also referred to as tailing, adds numerous labels per oligonucleotide (1-200), depending on the labeling conditions.
- RGOL restriction generated oligonucleotide labeling
- the invention is also directed to thermal cycle labeling (TCL) which comprises the simultaneous labeling and amplification of probes utilizing CviJ I or CGase I restriction generated oligonucleotides as the starting material.
- TCL thermal cycle labeling
- oligonucleotides are combined with the intact template and subjected to repeated cycles of denaturation, annealing, and extension in the presence of a thermostable DNA polymerase or functional fragment thereof which maintains polymerase activity, deoxynucleotide triphosphates and the appropriate buffer.
- a thermostable DNA polymerase or functional fragment thereof which maintains polymerase activity, deoxynucleotide triphosphates and the appropriate buffer.
- Alpha ⁇ 2 P-dATP or any of the other three deoxynucleotide triphosphates
- biotin-dUTP, fluorescein-dUTP, or digoxigenin-dUTP is incorporated during the extension step for subsequent detection purposes.
- Thermal cycle labeling efficiently labels DNA while simultaneously amplifying large amounts of the labeled probe.
- TCL probes exhibit a 10 fold improvement in detection sensitivity compared to conventional probes.
- the present invention is also directed to TCL in which the thermostable DNA polymerase supplies endogenous primers for enzymatic extension.
- This method is referred to as Universal Thermal Cycle Labeling (UTCL).
- UTCL Universal Thermal Cycle Labeling
- natural DNA of unknown sequence is combined intact with the holo-enzyme of a thermostable DNA polymerase, deoxyribonucleotide triphosphates, and the appropriate buffer.
- the holo-enzyme and its associated endogenous primers are then combined with intact template and subjected to repeated cycles of denaturation annealing and extension.
- Alpha 32 P-dATP, • ⁇ P- dTTP, 2 P-dGTP, 2 P-dCTP, biotin-dUTP, fluorescein-dUTP, or digoxigenin- dUTP is also included in the extension step for subsequent detection purposes.
- Isotopic labels useful in the practice of the present invention include but are not limited to 32 P, 33 P, -"S, ⁇ C and 3 H.
- Non-isotopic labels useful in the present invention include but are not limited to fluorescein biotin, dinitrophenol and digoxigenin.
- the present invention is also directed to an improved method for purifying CviJ I from the algae Chlorella infected with the virus IL-3A.
- the present invention is directed to restriction endonuclease reagents which, under conditions which relax the sequence specificity of one or more restriction endonucleases, cleave DNA at the dinucleotide sequences AT or TA.
- Figure 1 is a map of the plasmid p710 which contains DNA sequences encoding for the IL-3A viral methyltransferase M. wJI;
- Figure 2 is the nucleotide sequence of 5497 bp of cloned IL-3A viral DNA
- Figure 3 is a restriction map of the cloned IL-3A viral DNA, including the identified open reading frames;
- Figure 4 is the DNA sequence of the CviJl gene with its flanking regions. The predicted amino acid sequence is provided below the nucleotide sequences;
- Figure 5A depicts the theoretical frequency and distribution of CviJl restriction generated oligomers of individual lengths
- Figure 5B shows the actual frequency and distribution of CviJl restriction generated oligomers of various lengths
- Figure 6 is a flow chart depicting anonymous primer cloning
- Figure 7 is a photographic reproduction of a gel depicting CviJl restriction digests of pUC19;
- Figure 8 is a photographic reproduction of a gel depicting comparisons of sonicated versus CviJl partially digested DNAs
- Figure 9 A is a photographic reproduction of an agarose gel electrophoresis analysis of size-fractionated DNA by microcolumn chromatography compared to fractionation by agarose gel electroelution;
- Figure 9B-E illustrates additional trials of the same procedures used in Figure 9A
- Figure 10A illustrates the size distribution of DNA fragments produced by partial digestion of DNA by CviJl and fractionated by microcolumn chromatography
- Figure 10B-C illustrates the size distribution of DNA fragments produced by partial digestion of DNA by Cvz ' JI and fractionated by agarose gel electrophoresis;
- Figure 11 is a schematic depiction of the distribution of C JI sites in pUC19.
- Figure 12 is a graph of the rate of sequence accumulation by
- the gene for the restriction endonuclease R. CviJl was cloned into E. coli so as to provide an adequate source of R.CViJI for use as a molecular biological reagent.
- Biologically active CviJl has been purified from E.coli to apparent homogeneity.
- the molecular weight of E.coli derived R.C z ' JI is 32.5 kD by SDS gel electrophoresis.
- N-terminal amino acid sequence analysis of this protein and comparison to the nucleotide sequence of the gene revealed that the translation of this enzyme is probably initiated with a GTG start codon, instead of the usual ATG initiation codon.
- the structural gene is 834 nucleotides in length coding for a protein of 278 amino acids (31.6 kD).
- R.CV ⁇ activity which elutes separately from the 32.5 kD form can be seen in the initial stages of enzyme purification. Trace amounts of a larger molecular weight form have not been observed to date. However, the R.CVz ' JI gene does possess an in-frame upstream ATG codon which if translated would yield a predicted 41.4 kD protein. The structural gene for this potentially larger product is 1074 nucleotides in length coding for a putative protein of 358 amino acids.
- the present invention is also directed to a method for the fragmentation and cloning of DNA using the restriction endonuclease CviJ I under conditions which allow the enzyme to cleave DNA at the dinucleotide sequence GC.
- the present invention is also directed to the cloning of quasi- random fragments of DNA digested using the fragmentation method of the present invention.
- CviJ I Under “relaxed” conditions (in the presence of 1 mM ATP and 20 MM DTT) the specificity of CviJ I can be altered to cleave DNA more frequently and perhaps as frequently as at every GC. This activity is referred to as CvU I . Because of the high frequency of the dinucleotide GC in all DNA (16 bp average fragment size for random DNA), quasi-random libraries may be constructed by partial digestion of DNA with CvU I . A DNA degradation method with low levels of sequence specificity produces a smear of the target DNA when analyzed by agarose gel electrophoresis.
- One aspect of the present invention involves the use of the two/ three base recognition endonuclease CvU I, in conjunction with a simple spin- column method to produce libraries equivalent in final form to those generated by the combination of sonication and agarose gel electroelution.
- the method of the present invention requires fewer steps, a shorter time period, and significantly less substrate (nanogram amounts) when compared to conventional procedures. Both small and large sequencing projects using the methods described herein are within the scope of the present invention.
- Shotgun cloning with CvU I digested DNA is efficient partly because the resulting fragments are blunt ended.
- Other methods currently used to randomly-fragment DNA including sonication, DNAse I treatment, and low pressure shearing, leave ragged ends which must be converted to blunt ends for efficient vector ligation.
- Other than a heat denaturation step to inactivate the endonuclease no additional treatments are required for cloning CvU I restricted DNA.
- the preligation step required to equalize representation of the ends of a DNA molecule prior to sonication or DNAse I treatment is not necessary with CvU I fragmentation.
- CvU I cleaves its cognate recognition site very close to the ends of a linear molecule, as judged by the very small fragments resulting from complete digestion of pUC19 as depicted in Figure 2, lane 1.
- the overall efficiency of shotgun cloning depends not only on the fragmentation process, but also upon the size fractionation procedure used to remove small DNA fragments.
- the efficiency of cloning agarose gel fractionated DNA was found to be unexpectedly variable. Numerous experiments produced an erratic distribution of sized material and the resulting cloned inserts were uniformly small (70% ⁇ 500 bp in one trial, 100% ⁇ 500 bp in another).
- the method of the present invention includes a simple and rapid micro-column fractionation method, which has resulted in three to thirteen times more transformants than agarose gel fractionation. More importantly, the size distribution of the cloned inserts from column-fractionated DNA was skewed toward larger fragments (88% > 500 bp). Micro-column fractionation also eliminates the chemical extraction steps required for agarose fractionated DNA. After the target DNA has been column-fractionated, no further treatments are required for cloning. Combining CvU I partial restriction with micro-column fractionation permits the construction of useful libraries from as little as 200 ng of substrate, an order of magnitude less starting material than recommended for sonication/end-repair and agarose gel fractionation procedures.
- the CvU I reaction represents a unique alternative for controlling the partial digestion of DNA, a technique which is fundamental to the construction of genomic libraries (Maniatis et al. Cell 15:687-701 (1978), and restriction site mapping of recombinant clones (Smith, et al. Nucl. Acids Res. 3:2387-2398 (1976).
- Partial DNA digests are notably variable and are strongly dependent on the concentration and purity of the DNA, the amount of enzyme used, the incubation time, and the batch of enzyme. Partial digestions may also be variable with respect to the rate at which a particular recognition sequence is cleaved throughout the substrate.
- Optimal reaction conditions such as those which render such partial digests independent of one or more of these variables, allows more precise control of the end product.
- Several controlling schemes may be employed, including: the addition of a constant amount of carrier DNA (Kohara et al , Cell 50:495-508 (1987)) , the use of limiting amounts of Mg 2 + (.Albertson et al Nucl Acids Res. 17:808 (1989)), ultraviolet irradiation (Whitaker, et al. Gene 41:129-134), and the combination of a restriction enzyme and a sequence complementary DNA methylase (Hoheisel et al , Nucl. Acids Res. 17:9571-9582 (1989)). Utilizing three different batches of CvU I, and three different DNA templates from five separate preparations, a uniform CvU I partial digestion pattern was obtained that was primarily time-dependent when a constant ratio of 0.3 units of enzyme per ⁇ g of DNA was used.
- reaction conditions for CvU I may be optimized to substantially reduce the site preferences of this enzyme during partial digestion (see Figure 2, lanes 3 and 4). Normally, "star" reaction conditions result in cleavage at new sites.
- star reaction conditions according to the present invention dimethyl sulfoxide [DMSO] and lowered ionic strength
- Another aspect of the present invention involves quasi-random fragmentation of DNA using the method of the present invention for epitope mapping and cloning intact genes.
- the same method as described above for shotgun cloning is utilized, except that an expression vector is used to generate functional proteins from the DNA.
- RGO Restriction-generated oligonucleotides
- CvU I presumably cleaves the recognition sequence GC between the G and C to leave blunt ends (Xia et al , Nucl Acids Res. 15:6075- 6090, (1987)). Because of the high frequency of dinucleotide GC in all DNA (16bp average fragment size for random DNA), a complete CvU I restriction results in numerous fragments which are about 20-200 bp in size.
- restriction fragments are generated from an aliquot of the template itself and are heat-denatured to yield numerous single-stranded oligonucleotides which are of variable length but which are specific for the cognate template.
- Complete CvU I restriction of the small plasmid pUC19 (2689 bp) theoretically yields 314 oligonucleotides after a heat-denaturation step.
- the ability to generate numerous oligonucleotides with perfect sequence specificity is an unusual result of the use of this class of enzyme according to the present invention.
- Such oligonucleotides are uniquely suited for purposes of labeling DNA, as described below.
- CvU I restriction-generated oligonucleotides One application of CvU I restriction-generated oligonucleotides is to directly label them using conventional methods. There are several important advantages in using CvU I restriction-generated oligonucleotides.
- Conventional methods employing synthetic oligonucleotides for detection purposes generally use one oligonucleotide containing one or a few labels.
- a complete CvU I digest generates hundreds of oligonucleotides from a given template, depending on the size of the template, and thus makes hundreds of sites available for labeling, regardless of the labeling scheme utilized. These hundreds of sequence specific restriction-generated oligonucleotides have two important advantages over conventional probes used in nucleic acid detection methods.
- the generation of multiple oligonucleotide probes directed at multiple sites in a given target provides enhanced detection sensitivities compared to synthetic oligonucleotides which are directed at 1 or a few sites in a target.
- the numerous labeled restriction-generated oligonucleotides represent a 10-100 fold amplification of the signal for detection compared to the use of a single oligonucleotide.
- the short length of the restriction-generated oligonucleotides permits more efficient hybridization. This is important for two reasons.
- TCL Thermal Cycle Labeling
- the TCL system employs the two base recognition endonuclease CvU I to generate sequence-specific oligonucleotides from the template DNA itself. These oligonucleotides are combined with the intact template and subjected to repeated cycles of denaturation, annealing, and extension by a thermostable DNA polymerase from, for example, Thermits flavus.
- a radioactive- or non-isotopically-labeled deoxynucleotide triphosphate is incorporated during the extension step for subsequent detection purposes.
- the amplified, labeled probes represent a very heterogeneous mixture of fragments, which appears as a large molecular weight smear when analyzed by agarose gel electrophoresis.
- Primer-primer amplification a side product of this reaction (produced by leaving out the intact template in the TCL reaction), may result in enhanced detection sensitivity, perhaps by forming branched structures.
- Biotin- labeled probes generated by the TCL protocol detect as little as 25 zeptomoles (2.5 x IO "20 moles) of a target sequence.
- a 50 ⁇ l TCL reaction yields as much as 25 ⁇ g of labeled DNA, enough to probe 25 to 50 Southern blots.
- biotin-dUTP-incorporated TCL probes may be routinely detected at a 1:10" dilution, which is 1000 fold more sensitive than RPL, and indicates that a significant degree of net synthesis or amplification of the probe is occurring.
- non-isotopically-labeled TCL probes exhibit a 10-fold improvement in detection sensitivity when compared to RPL-generated probes.
- 32 P-labeled probes generated by the TCL protocol may also detect as little as 50 zeptomoles (2.5 xlO "2 ⁇ moles) of a target sequence.
- oligonucleotides for primer extension labeling of DNA.
- One advantage is the specificity of the primers. All of the oligonucleotides generated by the TCL system are specific for the template utilized, unlike random primer labeling (RPL) which utilizes synthetic oligonucleotides 6-9 bases in length having a random sequence.
- RPL random primer labeling
- the amount of primer required for efficient labeling with the TCL system is only 10 ng, compared to the 10 ⁇ g of random primers utilized for RPL. Due to their short length, random primers anneal very inefficiently above 25- 37°C, thus RPL is limited to DNA polymerases such as Klenow or T7.
- the size of the restriction-generated oligonucleotides are longer than the random primers, which extends the hybridization and extension conditions to include a wide variety of temperatures and polymerases.
- the use of the restriction-generated sequence-specific oligonucleotides results in more efficient hybridization and extension as compared to RPL.
- the TCL system has been optimized for labeling with a thermostable DNA polymerase which allows the option of temperature cycling. After 20 cycles of denaturation and extension, a significant amount of amplified TCL probes can be generated. Most importantly, TCL-labeled probes exhibit a 10 fold improvement in detections sensitivity when compared to RPL- generated probes.
- thermostable DNA polymerases Another aspect of the present invention involves a variation of TCL called Universal Thermal Cycle Labelling (UTCL) in which the extension primers are not supplied by CvUI restriction, but rather, are found endogenously in the enzyme preparations of thermostable DNA polymerases. Random sequence DNA is usually co-purified along with the holo-enzyme preparation of the thermostable UTCL
- DNA polymerases regardless of the source of the enzyme, i.e. native or cloned. However, only the holo-enzyme, and not the exonuclease minus deletion variants, contain the endogenous DNA. Typically, when the holo-enzymes of thermostable polymerases are used in protocols such as the polymerase chain reaction, the presence of such primers can create spurious results. Methods for circumventing the problems of endogenous DNA are described in PCR Protocols: A Guide to Methods and Applications, Eds. M. Innis, et al. , Academic Press, 1990.
- UTCL combines the holo- enzyme of a thermostable polymerase from, for example, Thermus flavus, with the intact DNA template and is subjected to repeated cycles of denaturation, annealing, and extension.
- a radioactive- or non-isotopically-labeled deoxynucleotide triphosphate is incorporated during the extension step for subsequent detection purposes.
- the amplified, labeled probe represents a very heterogenous mixture of fragments, which appears as a large molecular weight smear when analyzed by agarose gel electrophoresis.
- Biotin-labeled probes generated by the UTCL protocol detect as little as 25 zeptomoles (2.5 x IO "20 moles) of a target sequence.
- a 15 ⁇ l UTCL reaction yields as much as 5-10 ⁇ g of labeled DNA, enough to probe 5 to 10 Southern blots.
- biotin-dUTP-incorporated UTCL probes may be routinely detected at a 1:10" dilution, which is 1000 fold more sensitive than RPL, and indicates that a significant degree of net synthesis or amplification of the probe is occurring.
- non-isotopically-labeled UTCL probes exhibit a 10-fold improvement in detection sensitivity when compared to RPL-generated probes.
- 32 P-labeled probes generated by the UTCL protocol may also detect as little as 50 zeptomoles (2.5 xlO moles) of a target sequence.
- the radioactive version of UTCL generates probes having extremely high specific activities, e.g. (about 5 x l ⁇ " cpm/ ⁇ g DNA), which permits 5 to 10-fold lower detection limits than conventional labeling protocols.
- Example 1 provides for the propagation of IL-3A virus and isolation of viral genomic DNA.
- Example 2 addresses the improved expression of a clone for the viral methylase M.CVUI .
- Example 3 describes the strategy for isolating and cloning the viral R. CVUI gene by a forced co-cloning strategy of the M. CviJl gene.
- Example 4 describes the sequencing of cloned IL-3A genomic DNA and identification of the R. CviJl gene.
- Example 5 relates the methods for purification of CVUI to homogeneity from an E.coli strain, DH5 ⁇ .F 'MCR, transformed with a plasmid which encodes the R.CV JI enzyme.
- Example 6 details the amino acid sequence analysis of the purified R. CviJl enzyme.
- Example 7 describes the analysis of CVUI recognition sequences.
- Example 8 relates to a technique for producing restriction generated oligonucleotides using CviJl.
- Example 9 relates the generation of anonymous primers using CviJl.
- Example 10 describes end-labeling of CviJl restriction generated oligonucleotides.
- Example 11 describes primer extension labeling of DNA using restriction generated oligonucleotides.
- Example 12 relates the use of CviJl in thermal cycle labeling of DNA as well as the method of universal thermal cycle labelling.
- Example 13 provides a method for generation of quasi-random DNA fragments using CviJl.
- Example 14 describes fractionation of CviJl digested DNA by size using spin column chromatography.
- Example 15 details the relative cloning efficiency of CviJl digested, size-fractionated DNA by gel elution and chromatographic methods.
- Example 16 describes the comparison of cloning efficiency using lambda DNA fragmented by both sonication and CVUI techniques.
- Example 17 details the use of CviJl fragmentation for shotgun cloning and sequencing.
- Example 18 describes the shotgun cloning of lambda DNA using CVUI.
- Example 19 describes the use of CviJl in epitope mapping techniques.
- Example 20 describes the restriction endonuclease reagent CGase I.
- Cultures were innoculated with 1 X 10" algae cells/ml and grown at 25°C in 250 ml of MBBM in 500 ml Erlenmeyer flasks on a rotary shaker (150 rpm) in continuous light (ca. 30 ⁇ Ei, m ,sec ⁇ * *). Growth was monitored by light scattering measured as ⁇ Qnm and/or by direct cell counts with a hemocytometer. When the cultures reached approximately 1 X 10' algae cells/ml they were innoculated with filter sterilized (0.4 ⁇ m nitrocellulose filter, Nucleopore, Pleasanton, California) IL-3A virus at a multiplicity of infection of 0.01 and incubated for an additional 48 - 72 hours at 25°C.
- filter sterilized 0.4 ⁇ m nitrocellulose filter, Nucleopore, Pleasanton, California
- the crude lysate was then centrifuged at 3000 rpm (2000 xg) for 10 minutes to remove cellular debris.
- Nonidet P-40 was then added to 1 % (v/v) and the virus was pelleted from the supernatant by centrifiiging at 15,000 rpm at 4°C for 75 minutes in a Beckman No. 30 rotor.
- the viral pellet was gently resuspended in 0.05 M Tris-HCl, pH 7.8, and the sample was layered on linear 10 - 40% sucrose gradients equilibrated with 0.05 M Tris-HCl, pH 7.8, and centrifuged for 20 minutes at 20,000 rpm at 4°C in a Beckman SW28 rotor.
- the viral band which was present in the center of the gradient as an opaque band, was removed, diluted with 0.05 M Tris-HCl, pH 7.8, and pelleted by centrifugation at 15,000 rpm at 4°C for 120 minutes in a Beckman No. 80 rotor.
- the virus was resuspended in a small volume (10ml) of 0.05 M Tris-HCl, pH 7.8, and stored at 4°C.
- IL-3A viral DNA was purified from the viral particles using a modification of the protocol described by (Miller, S.A., Dykes, D.D., and Polesky, H.I., Nucleic Acids Res. 16:1215 (1988)). Briefly, 100 ⁇ l of IL-3A virus (9.8 X 10 * - * ••* plaque forming units/ml) was diluted with 400 ⁇ l of water and then mixed with 10 ⁇ l TEN (0.5 M Tris-HCl, pH 9.0, 20 mM EDTA, 10 mM NaCl) and 10 ⁇ l of 10% SDS.
- TEN 0.5 M Tris-HCl, pH 9.0, 20 mM EDTA, 10 mM NaCl
- IL-3A was cloned and sequenced by Shields et al, Virology 176:16-24 (1990). Briefly, Sau3A partial digest of Chlorella virus IL-3A was ligated to Bam ⁇ I digested pUC19 and transformed into E. coli strain RR1. This library of plasmids was restricted with Hindlll (AAGCTT) and Sstl (GAGCTC), both of which are inhibited by 5-methylcytidine (5mC) in the AGCT portion of their recognition sequences, and transformed again into RRl cells.
- M.CVUI methylates the internal cytidine in (G/A)GC(T/C/G) sequences. If the M.CVUI gene is cloned and expressed appropriately, the plasmid DNA would be expected to be resistant to HindJU and Sstl restriction.
- the CviJl methyltransferase gene was originally cloned as a 7.2 kb insert, termed pIL-3A.22. Plasmid pIL-3A.22 was only partially resistant to CVUI digestion. Partial digestion is most likely due to the inefficient expression of the M.CVUI gene and the numerous CviJl sites in both the vector (pUC19 has 45 CviJl sites) and in the insert DNA.
- the M. CviJl gene was eventually sublocalized to a region of 3.7 kb by subcloning using methods well known in the art (Ausubel, F.M., Brent, R., guitarist, R.E., Moore, D.D., Seidman, J.G., Smith, J.A.
- CviJl gene by three criteria: (i) it is the only ORF located in the region identified by transposon mutagenesis; (ii) it has amino acid motif s similar to those of other cytosine methyltransferases; and (iii) a 1.6 kb Dr ⁇ l fragment containing the 367 amino acid ORF (1101 bp) produces the methyltransferase. This 1.6 kb M.CVUI encoding fragment was subcloned into the Ec ⁇ RV site of pBluescript KS(-)
- p710 A physical map of the resulting plasmid termed p710 is shown in Figure 1.
- the plasmid p710 was digested with several endonucleases to indirectly test the efficiency of M. CviJl expression. Fully active methylase should render the plasmid DNA completely resistant to digestion by the following enzymes: HaeHl (which recognizes the sequence GGCC), Sacl (which recognizes the sequence GAGCTC), and Hindlll (which recognizes the sequence .AAGCTT).
- the plasmid was partially resistant to HaeUl (90%) and Sacl (90%), and even less resistant to Hindlll (25%) digestion. This lack of complete protection of the plasmid DNA made it impractical to attempt cloning the three/two base restriction endonuclease encoded by the R. CviJl gene. Thus, improvements in the efficiency of M. CviJl expression were required before attempting to clone the R. CVUI gene.
- the translation efficiency of the M.CVUI gene was improved by removing extraneous 5 ' open reading frames, creating a perfect fusion of the lacZ ' Shine-Delgarno sequence with the methyltransferase start codon (see Figure 1). This was achieved by site-specific oligonucleotide mutagenesis, using the oligomer
- mutagenesis reaction was completed using a commercially available kit according to the manufacturer's instruction (Mutagene, Bio-Rad, Hercules, California).
- the oligonucleotide was annealed to the single-stranded plasmid, extended in the presence of T4 DNA polymerase, ligated using T4 DNA ligase, and transformed into competent SURETM cells (Stratagene, La Jolla, California). Transformed cells were then grown overnight as a pool, the DNA isolated and purified.
- Enrichment for the mutagenized plasmids was made possible by virtue of the loss of an Xhol site located in the sequence that was deleted by mutagenesis. Enrichment was accomplished by digesting the isolated, purified plasmid DNA with Xhol, followed by dephosphorylation with calf intestinal alkaline phosphatase (CIAP), and transformed into SURE cells. Plasmid DNA was isolated from 18 individual colonies and the DNA tested for resistance to
- Plasmid DNA from 11 colonies were resistant to Xhol digestion, indicating that they lacked the deleted sequence. Five of these plasmids were restricted with
- the location of the R.CVUI gene on the IL-3A virus genome was inferred as being 3 ' to the M.CVUI gene for two reasons: 1) the cloned DNA sequence 5 ' to the M.CVUI gene did not produce a restriction activity; and 2) several attempts to clone the DNA 3 ' to the M.CVUI gene resulted in deletions/rearrangements of this downstream region. This information permitted a forced co-cloning strategy to obtain the restriction endonuclease gene. This strategy uses a deletion derivative of pBMC5 lacking the 3 ' half of the M.CVUI gene.
- Digestion of the IL-3A genome with the same enzyme used to create the M.CVUI deletion, followed by ligation of the respective DNAs, transformation, and digestion with enzymes incapable of recognizing methylated DNA should force the selection of clones which have a restored M.CVUI gene (and thus active methylase enzyme), as well as downstream DNA.
- enzymes incapable of recognizing methylated DNA e.g., Hae ⁇ l, HindUl, Pvull, CviJl, etc.
- pBMC5 has two Ec ⁇ SI sites, one approximately in the middle of the M. CviJl gene, while the other site lies in the vector DNA, 3 ' to the M.CVUI gene (see Figure 1).
- pBMC5 was restricted with Ec ⁇ SI and ligated at a dilute concentration (10-50 ng/ ⁇ l) to favor circularization without the 3 ' M.CVUI fragment.
- the reaction mixture was then transformed into competent SURE cells and plated on TY agar containing ampicillin. Plasmid DNA from the resulting colonies was tested for the lack of this EcoRl fragment by digestion with Ec ⁇ RI.
- pBMC5RI was used for the subsequent co-cloning work.
- Plasmid pBMC5RI was digested with a& ⁇ RI and dephosphorylated using CLAP. IL-3A genomic DNA was then digested to completion with Eco I. The EcdSl digested pBMC5RI and IL-3A DNAs were combined at a ratio of 1:3 in a ligation reaction using T4 DNA ligase, and the products of the ligation reaction were subsequently used to transform competent SURE cells. The pBMC5RI/IL-3A transformants were not plated, but rather grown overnight in culture as a library or pool of cells. The cells were harvested the next day and DNA was isolated and purified.
- Isolated, purified DNA was digested with HaelU, dephosphorylated with CIAP, and transformed into competent SURE cells. The cells were then plated and grown overnight. Six colonies grew, of which only one containing the plasmid, pCJH1.4, was resistant to Haelll. The plasmid pCJH1.4 was found to encode CVUI restriction activity. Plasmid pCJH1.4 was further characterized to localize the gene for CviJl by deletion analysis, subcloning experiments, and sequencing. The plasmid pCJHl.4 was deposited with the American Type Culture Collection on June 30, 1993 under Accession Number 69341. Example 4
- the Ec ⁇ Rl fragment cloned into pCJH1.4 (as described in Example 3) is 4901 bp in length. Except for the 519 bp corresponding to the 3 ' portion of the M.CVUI gene, the remainder of the 4901 bp EcoR I fragment cloned into pCJHl.4 was sequenced using the SEQUAL DNA Sequencing System (CHIMERx, Madison, WI) by methods well known in the art. Sequencing was accomplished using three approaches: 1) primer walking on pCHJ1.4, 2) cloning various restriction endonuclease digests of pCHJ1.4 into an M13 type sequencing vector; and 3) sequencing various restriction endonuclease deletion derivatives of pCHJ1.4.
- the nucleotide sequence of 5497 bp of IL-3A viral DNA is shown in Figure 2 and set forth in SEQ ID NO.: 2.
- ORF Six open reading frames (ORF) of 1155 bp (ORFl), 468 bp (ORF2), 555 bp (ORF3), 1086 bp (ORF4), 397 bp (ORF5) and 580 bp (ORF6) which could code for polypeptides containing 358 (41.4 kD), 156 (19.4 kD), 185
- ORFs 4-6 do not code for the R.CVUI gene, as the deletion derivative pCdA12, which lacks the DNA between the Aval and BamHI sites (see Figure 3), does produce CviJl restriction endonuclease activity.
- the deletion derivative pCdEB7 lacking the
- ORFl or ORF3 were the most likely candidates for encoding the R.CVUI gene.
- the vertical line in Figure 4 and the associated arrow indicate where the DNA sequence from pJCH1.4 diverges from that of pIL- 3A.22-8 (Shields, S.L., et al, Virology 76:16-24, 1990).
- This open reading (ORFl) frame is believed to represent the CviJl gene because 14 out of 15 N- terminal amino acids from the protein sequence (see Example 6) matched the predicted translation product of the nucleic acid sequence ( Figure 4). Also, the 32.5 kD molecular weight of the homogeneously purified enzyme described in Example 5 matched the predicted translation product of the nucleic acid sequence (31.6 kD) if the encoded protein was translated beginning at the GTG codon located at nucleotides 299 - 301 ( Figure 4), instead of the 5 ' ATG codon located at nucleotides 59 - 61.
- the structural gene was identified to be 834 nucleotides in length, coding for a protein of 278 amino acids (31.6 kD) and is set forth in SEQ ID NO: 4. It is also interesting to note that the CviJl gene was shown to possess an in-frame, upstream ATG codon which if translated could yield a protein with a predicted molecular weight of 41.4 kD ( Figure 4). A larger molecular weight form possessing CVUI restriction activity has not been detected by SDS gel electrophoresis. However, a second peak of CVUI activity which eluted separately from the 32.5 kD form was detected in the initial stages of enzyme purification. The DNA sequence which could theoretically code for a larger form of CviJl would be approximately 1074 nucleotides in length (assuming it starts at the upstream ATG codon) and would code for a protein of 358 amino acids.
- TB medium 12 g Bacto-Tryptone, 24 g Bacto Yeast Extract, 4 ml glycerol, 2.31 g KH2PO4, 12.54 g K 2 HPO , 0.1 g MgSO , 100 ⁇ g/ml ampicillin, and water to 1 liter
- 50 liters of TB medium 12 g Bacto-Tryptone, 24 g Bacto Yeast Extract, 4 ml glycerol, 2.31 g KH2PO4, 12.54 g K 2 HPO , 0.1 g MgSO , 100 ⁇ g/ml ampicillin, and water to 1 liter
- Vigorous aeration was essential for CviJl expression and a typical yield contained 70 g of cell paste after centrifugation.
- the cell pellet was immediately resuspended in lysis buffer A (30 mM Tris-HCl, pH 7.9 at 4°C, 2 mM EDTA, 10 mM beta-mercaptoethanol,
- phenylmethylsulfonyl fluoride (PMSF), 20 ⁇ g/ml benzamidine, 2 ⁇ g/ml 0-phenantroline, 0.7 ⁇ g/ml pepstatin) at a volume of 3 ml of buffer A per 1 g of cells.
- the cell suspension was then passed through a Manton-Gaulin cell disrupter (Gaulin Corporation, Everett, MA) twice and centrifuged for 1 hr (8000 RPM, Sorvall GS3 Rotor) at 4°C.
- buffer B (10 mM K/PO 4 , pH 7.2, 0.5 mM EDTA, 10 mM beta- mercaptoethanol, 50 mM NaCl, 10% glycerol, 0.05% Triton X-100, 50 ⁇ g/ml PMFS, 20 ⁇ g/ml benzamidine, 2 ⁇ g/ml o-phenanthroline, 0.7 ⁇ g/ml pepstatin).
- the dissolved protein solution was then dialysed (14kD cut-off) for 12 hours against three 1 liter changes of buffer B.
- the dialyzed solution was then diluted to 600 ml with buffer B and applied to a 5 x 20 cm phosphocellulose Pll (Whatman) column (flow rate 100 ml/hr). The column was then washed with 1.5 liter of buffer B followed by a 0 - 1.5 M NaCl gradient in buffer B (5 liters). R.CviJl eluted at approximately 600 mM NaCl. The active fractions were then pooled and concentrated to 50 ml with a 76 mm Amicon YM10 membrane.
- the resulting solution was then diluted to 300 ml with buffer C (20 mM Tris-acetate, pH 7.4 at 4°C, 2 mM EDTA, 10 mM beta-mercaptoethanol, 50 mM NaCl, 10% glycerol, 0.01 % Triton X-100, 50 ⁇ g/ml PMFS, 20 ⁇ g/ml benzamidine, 2 ⁇ g/ml o-phenanthroline, 0.7 ⁇ g/ml pepstatin) and applied to 2.5 x 7 cm Heparin- Sepharose column at a flow rate of 25 ml/hr. After a 400 ml wash with buffer B, R.CviJl was eluted with a
- the molecular weight of the purified CviJl was determined by comparison to known protein standards on a denaturing 10% SDS polyacrylamide gel and a single band migrating with an apparent molecular weight of 32.5 kilodaltons was seen indicating that by these criteria, CviJl was purified to homogeneity.
- LYS glutamic acid
- GLU glutamic acid
- LEU leucine
- ALA alanine
- TLE isoleucine
- GNN glutamine
- the dinucleotide GC is found at 205 sites in pUC19. These GC sites (shown in Table 2) can be divided into four classes based on their flanking
- Pu/Py structure the normal recognition sequence (N) and three potential classes of relaxed sites (R2 and R3). As seen in Table 2, the fraction of such NGCN sites which belong to each classification is roughly equal (22.0%-27.8 %). A total of 200 CviJl restricted junctions were analyzed by sequencing 100 cloned inserts.
- CviJl cleaves all NGCN sites except for PyGCPu. As CviJl cleaves 12 out of 16 possible NGCN sites, it may be referred to as a 2.25-base recognition endonuclease.
- oligonucleotide Due to the high frequency of CvUI or CviJl restriction, it is possible to generate useful oligonucleotides by digestion and a heat denaturation step as described above. The size and number of the resulting oligonucleotides are important for subsequent applications such as those described above. If for example, an oligonucleotide is to be used with a large genome, it has to be long enough so that the sequence detected has a probability of occuring only once in the genome. This minimum length has been calculated to be 17 nucleotides for the human genome (Thomas, C.A., Jr. Prog. Nucl Acid Res. Mol. Biol, 5:315
- Oligonucleotides used for sequencing or PCR amplification are generally 17-24 bases in length. Oligomers of shorter length will often bind at multiple positions, even with small genomes, and thus will generate spurious extension products. Thus, an enzymatic method for generating oligomers should ideally result in polymers greater than 18 bases in length.
- the theoretical number of pUC19 CVfJI restriction-generated oligomers is 314 (157 CviJl restriction fragments x 2 oligomers/fragment), the size distribution of which is shown in panel A of Figure 5. Most of the expected CviJl restriction-generated oligomers (about 75%) are smaller than 20 bp . This assumes that CviJl is capable of restricting DNA to very small fragments, the shortest of which would be 2 bp. However, in practice, about 93 % of the cloned CviJl fragments were 20-56 bp in size, and 3% of the fragments generated by
- CviJl were smaller than 20 bp (panel B of Figure 5). This suggests that CviJI is not able to bind or restrict those fragments below a certain threshold length. Since the smallest observed fragment is 18 bp, it may be assumed that this length is the minimal size which can be generated from a given larger fragment. Whatever the reason for this phenomenon, CviJl treatment of DNA produces a relatively small range of oligomers (mostly 20-60 bases in length), most of which are a perfect size class for molecular biology applications.
- Primers are critical tools in many molecular biology applications such as PCR, sequencing, and as probes. Anonymous primers are useful as sequencing primers for genomic sequencing projects, as probes for mapping chromosomes, or to generate oligonucleotides for PCR amplification.
- the Anonymous Primer Cloning (APC) method is a variation of shotgun cloning in that unknown sequences of DNA are being randomly cloned.
- anonymous primer cloning utilizes a complete CviJl digest to restrict large DNAs into small fragments 20-200 bp in size. These small fragments are cloned into a unique vector designed for excising the anonymous DNA as labeled primers.
- the strategy for this method is illustrated in Figure 6.
- Plasmid pFEM has a unique arrangement of the restriction sites for MboU and Fokl, which permits DNA cloned into the Ec ⁇ RV site to be excised without associated vector DNA. This is possible because Fokl cleaves 9/13 bases to the left of the recognition site shown in pF ⁇ M and MboU cleaves 8/7 bases to the right of the recognition site shown in pF ⁇ M, which is well into the cloned anonymous sequence.
- flanking primer After MboU or Fokl restriction, a known flanking primer is annealed (primer 1 or 2) and extended using a DNA polymerase and dNTPs.
- the m-im p r is previously end-labeled, or alternatively, one or more radioactive.
- the labeled anonymous primer After denaturation of the newly synthesized DNA and separation from its cognate template, the labeled anonymous primer is ready for use in sequencing the original template from which it was subcloned. The presence of the pFEM vector sequence fused to the anonymous sequence does not influence the enzymatic extension of this primer from its unique binding site, as the vector
- DNA is at the 5' end and the unique sequence is located at the 3' end (all polymerases extend 5' to 3')-
- Both the top and bottom strand primers may be excised from pFEM due to the symmetrical placement of restriction sites and flanking primer binding sites. Thus, two primers may be derived from each cloning event.
- APC is particularly well suited to the genomic sequencing strategy of Church and Gilbert Proc Natl. Acad Sci. USA 81:1991-1995 (1984), although its utility is not limited thereto.
- CviJl provides the ability to generate sequence-specific oligonucleotides ranging in size from 20-200 bases in length with an average length of 20-60 bases.
- Sequence specific oligonucleotides generated by CV/JI digestion may be labeled directly at the 5 '-end or at the 3 '-end using techniques well known in that art. For example, 5 '-end labeling may be accomplished by either a forward reaction or an exchange reaction using the enzyme T4 polynucleotide kinase.
- Homopolymeric tailing is another standard labeling technique useful in the labeling of CviU -generated sequence specific oligonucleotides.
- This reaction involves the addition of - ⁇ P-labeled nucleotides to the 3 '-end of the sequence specific oligonucleotides using a terminal deoxynucleotide transferase.
- Commonly used labeling techniques typically employ a single oligonucleotide directed to a single site on the target DNA and containing one or a few labels.
- Oligonucleotides generated by the method of the present invention are directed to many sites of a target DNA by virtue of the fact that they are generated from a sample of the target sequence.
- the hybridization of multiple oligonucleotides allows a significantly enhanced sensitivity in the detection of target sequences.
- the short length of the labeled oligonucleotides used in the methods of the present invention allows a reduction in hybridization time from overnight (as is used in conventional methods) to 60 mins.
- Another aspect of the present invention includes methods for labeling DNA which include the generation of oligonucleotide primers by complete digestion with C JI , followed by heat denaturation.
- PEL-RGO requires three steps: 1) generating the sequence-specific oligonucleotides by CviU restriction of the template DNA; 2) denaturation of the template and primer; and 3) primer extension in the presence of labeled nucleotide triphosphates.
- Plasmid DNA may be prepared by methods known in the art such as the alkaline lysis or rapid boiling methods (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)).
- the vector should be linearized to ensure effective denaturation.
- a restriction fragment may be labeled after separation on low melting point agarose gels by methods well known in the art.
- template DNA to be labeled is divided into two aliquots; one is used to generate the sequence specific oligonucleotide primers and the other aliquot is saved for the primer annealing and extension reaction.
- a typical reaction mix for generating sequence-specific oligonucleotides is assembled in a microcentrifuge tube and includes: 100 ng DNA; 2 ⁇ l 5x CviJI buffer; 0.5 ⁇ l CviU (Ixxl ⁇ l); sterile distilled water to 10 ⁇ l final volume.
- Cv ⁇ U 5X restriction buffer includes: 100 mM glycylglycine (Sigma, St. Louis, Missouri, Cat. No.
- CviJI is obtained from CHIMERx (Madison, Wisconsin). The reaction mix is incubated at 37°C for 30 min, followed by the inactivation of CviJI by heating at 65°C for 10 min.
- the CviJI -restricted DNA may be used directly without further purification, or it may be stored at -20 C for several months for subsequent labeling reactions.
- CviJI restriction fragments appear as a low molecular weight smear in the 20-200 bp range.
- a template-primer cocktail was prepared by mixing 10 ng of linearized pUC19 DNA template with 20 ng pUC19 sequence- specific oligonucleotides (prepared as described above) and the mixture is brought to a final volume of 17 ⁇ l with sterile distilled water. The template-primer mixture is denatured in a boiling water bath for 2 minutes and immediately placed on ice.
- the following labeling mixture is then added to the template-primer mix:2.5 ⁇ l 10X labeling buffer (500 mM Tris HCl at pH 9.0, 30 mM MgCl 2 , 200 mM (NH 4 ) 2 SO 4 , 20 ⁇ M dATP, 20 ⁇ M dTTP, 20 / ⁇ M dGTP, 0.4% NP-40); 5.0 ⁇ l [ ⁇ - 32 P] dCTP (3000Ci/mmol, lO ⁇ Cil ⁇ l New England Nuclear, Catalog No. NEG013H); 0.5 ⁇ l Thermits flavus DNA polymerase (5u/ ⁇ l) (Molecular Biology Resources, Milwaukee, Wisconsin); up to 25 ⁇ l final volume with distilled water. The reaction was incubated at 70°C for 30 min and then stopped by adding 2 ⁇ l of 0.5M EDTA at pH 8.0 to the reaction mix.
- 10X labeling buffer 500 mM Tris HCl at pH 9.0, 30 mM MgCl 2 ,
- the efficiency of the labeling reaction is gauged by the percentage of radioisotope incorporated into labeled DNA.
- One microliter of the labeling reaction is added to 99 ⁇ l of lOmM EDTA in a microcentrifuge tube. This serves as the source of diluted probe for total and trichloroacetic acid (TCA)-precipitable counts.
- 2 ⁇ l of diluted probe is spotted onto the center of a glass fiber filter disc (Whatman number 934- AH). The disc is then allowed to dry and is then placed in a vial containing scintillation cocktail for counting total radioactivity in a liquid scintillation counter.
- the factor 50 is derived from using 2 ⁇ l of a 1:100 dilution for TCA precipitation.
- the number 27 converts this back to the total reaction volume (which is the reaction volume plus 2 ⁇ l of stop solution).
- Theoretical yield ⁇ Ci of dNTPs x 4 x 330
- Unincorporated radioactive label may be removed using standard methods well known in the art. Comparisons were made between PEL-RGO vs RPL under similar conditions, and it was observed that a detection limit of 100 fg was seen using PEL-RGO labeled DNA compared to a detection limit of 500 fg with RPL, using a radiolabeled probe.
- TCL Thermal Cycle Labeling
- TCL of DNA requires two general steps: 1) generation of the sequence-specific oligonucleotides by CviU restriction of the template DNA; and 2) repeated cycles of denaturation, annealing, and extension in the presence of a thermostable DNA polymerase or a functional fragment thereof which maintains polymerase activity. Optimal results are obtained after 20 such cycles, which is best performed in an automated thermal cycling instrument such as a Perkin-Elmer Model 480 thermocycler. In conjunction with such an instrument, about 1.5 hr. is required to complete this protocol.
- Non-radioactive labeling of DNA using TCL is accomplished by mixing: 10 pg - 100 ng linearized template, 50 ng CviJI -digested primers (prepared as described above), 1.5 ⁇ l 10X labeling buffer, 0.5 ⁇ l Thermits flavus
- DNA polymerase (5u/ ⁇ l) (Molecular Biology Resources, Inc., Milwaukee, Wisconsin), 1 ⁇ l of ImM Biotin-11-dUTP (Enzo Diagnostics, New York, New York), 1.5 ⁇ l each of dATP, dCTP, and dGTP (2 mM), and 1.0 ⁇ l 2mM dTTP.
- Radioactive labeling of DNA using TCL was accomplished by mixing 10 pg - 100 ng of CviJI generated primers, 10 pg-25 ng of linearized template, 1.5 ⁇ l of 10X labeling buffer, 5 ⁇ l of 32 P-dCTP (3000 Ci/mmole, 10 ⁇ Ci/ ⁇ l or 40 ⁇ Ci/ ⁇ l), 0.5 ⁇ l of Thermits flavus DNA polymerase (5u/ ⁇ l), and 0.5 ⁇ l each of dATP, dGTP, and dTTP (1 mM) was added. The reaction mix was brought to a volume of 15 ⁇ l with deionized H2O, overlaid with mineral oil and cycled through 20 rounds of denaturation, annealing and extension.
- a typical cycling regimen employed 20 cycles of denaturation at 91°C for 5 sec, annealing at 50°C for 5 sec and extension at 72°C for 30 sec. The reaction is then terminated by adding 1 ⁇ l of 0.5M EDTA, pH 8.0.
- the amplified, labeled probe is a very heterogeneous mixture of fragments, which appears as a smear when analyzed by agarose gel electrophoresis.
- Universal thermal cycle labeling (UTCL) is a method according to the present invention for efficiently labeling double-stranded DNA while simultaneously amplifying large amounts of labeled probe. UTCL is unique in that no sequence information is required regarding the template.
- the extension primers are suppled endogenously via the holo-enzyme of the thermostable DNA polymerase and any anonymous DNA template can be labeled by repeated cycles of denaturation, annealing, and extension in the presence of a labeled deoxynucleotide triphosphate.
- Optimal results are obtained after 20 such cycles, which is best performed in an automated thermal cycling instrument such as a Peririn-Elmer Model 480 thermocycler. In conjunction with such an instrument, about 1.5 hr are required to complete this protocol. If a thermal cycler is not available these reactions may be performed using heat blocks. As a few as 5 cycles may yield probes with acceptable detection sensitivies.
- Non-radioactive labeling of DNA using UTCL is accomplished by mixing: 10 ng linearized template, 1.5 ⁇ l 10X labeling buffer, 0.5 ⁇ l Thermits flavus DNA polymerase (5u/ ⁇ l) (Molecular Biology Resources, Inc., Milwaukee,
- Radioactive labeling of DNA using UTCL was accomplished by mixing: 10 pg-100 ng of linearized template, 1.5 ⁇ l of 10X labeling buffer, 5 ⁇ l of 32 P-dCTP (3000 Ci/mmole, 10 ⁇ Ci/ ⁇ l or 40 ⁇ Ci/ ⁇ l), 0.5 ⁇ l of Thermits flavus DNA polymerase (5u/ ⁇ l), and 0.5 ⁇ l each of dATP, dGTP, and dTTP (1 mM) was added. The reaction mix was brought to a volume of 15 ⁇ l with deionized H2O, overlaid with mineral oil and cycled through 20 rounds of denaturation, annealing and extension.
- a typical cycling regimen employed 20 cycles of denaturation at 91°C for 5 sec, annealing at 50°C for 5 sec and extension at 72°C for 30 sec. The reaction is then terminated by adding 1 ⁇ l of 0.5M EDTA, pH 8.0.
- the amplified, labeled probe is a very heterogeneous mixture of fragments, which appears as a smear when analyzed by agarose gel electrophoresis.
- the unbound sites on the membrane are blocked using a blocking buffer for 15 min at 25°C.
- Streptavidin-alkaline phosphatase (Gibco-BRL Gaithersburg, Maryland, Cat. No. 9545A) is added to the blocking buffer (0.058 M Na 2 HPO 4 , 0.017 M NaH 2 PO 4 , 0.068 M NaCl, 0.02% sodium azide, 0.5% casein hydrolysate, 0.1 % Tween-20) at a 1:5000 dilution and incubated for a 30 min. , and the membrane is rinsed 3 times for 10 min.
- wash buffer lx PBS [0.058 M Na 2 HPO 4 , 0.017 M NaH 2 PO 4 , 0.068 M NaCl], 0.3% Tween, 0.2% sodium azide
- AP buffer 100 mM NaCl, 5 mM MgCl 2 , 100 mM Tris-Cl pH 9.5
- AP buffer 100 mM NaCl, 5 mM MgCl 2 , 100 mM Tris-Cl pH 9.5
- AP buffer 100 mM NaCl, 5 mM MgCl 2 , 100 mM Tris-Cl pH 9.5
- AP buffer 100 mM NaCl, 5 mM MgCl 2 , 100 mM Tris-Cl pH 9.5
- AP buffer containing 4.0 ⁇ l/ml nitro blue tetrazolium (NBT) Sigma Cat. No. N6639
- Sigma Cat. No. B6777 5-bromo-4-chloro-3-indoly
- Detection Sensitivities 2 P-labeled probes generated by the protocol above described labelling detect as little as 25 zeptomoles (2.5 x IO "2 " moles) of a target sequence. As little as 10 pg of template DNA is enough to synthesize 5-10 ng of radiolabeled probe, which is sufficient for screening 5 Southern blots.
- the radioactive versions of TCL and UTCL facilitate extremely high specific activities of labeled probe (about 5 x IO 9 cpm/ ⁇ g DNA), which permits 5-10 fold lower detection limits than conventional labeling protocols.
- the thermal cycling permits probe amplification.
- Biotin-labeled probes generated by the TCL and UTCL protocols detect as little as 25 zeptomoles (2.5 x IO "2 " moles) of a target sequence.
- a 15 ⁇ l TCL or UTCL reaction yields as much as 5-10 ⁇ g of labeled DNA, enough to probe 5 to 10 Southern blots.
- Biotin-labeled TCL and UTCL probes provide a 10 fold greater detection sensitivity when compared to RPL biotin probes.
- the thermal cycling permits probe amplification.
- Non-radioactive, biotinylated probes labeled by the TCL and UTCL methods were shown to have detection limits that are identical to the radioactive probes. These methods have the advantage of eliminating the need to work with hazardous radioactive materials without sacrificing sensitivity. In addition, results are obtained from non-isotopic probes in 3-4 hours compared to 3-4 days for radiolabeled probes. The ability to substitute non-radioactive probes for radioactive probes may be very useful to clinical laboratories, which do not use radioisotopes but do need greater detection sensitivities. Research laboratories favor the use of non-isotopic systems if detection sensitivity is not an issue. The non-isotopic labeling version of the TCL and UTCL systems represent a major improvement in labeling DNA probes.
- Non-radioactive probes generated by the methods of the present invention are also useful in the detection of RNA in situ.
- An advantage of this system is that labeling protocols of the present invention yield highly sensitive non-radioactive probes, and the size of the probes are predominantly in the small molecular weight range and can therefore penetrate the tissue easily, unlike RPL. Because non-radioactive probes labeled using the labeling protocols of the present invention have the same detection limits as do radioactive probes similarly labeled, it is within the scope of this invention to use either radioactive or non-radioactive probes for probing, for example, Southern blots, Northern blots, for in situ hybridization for the detection of mRNA or DNA in cells or tissue directly, and for colony or plaque lifts.
- Shotgun cloning and sequencing requires the generation of an overlapping population of DNA fragments. Therefore, conditions were established for the partial digestion of DNA with Cv ⁇ U to produce an apparently random pattern, or smear, of fragments in the appropriate size range.
- Conventional methods for obtaining partially restricted DNA include limiting the incubation time or limiting the amount of enzyme used in the digestion. Initially, agarose gel electrophoresis and ethidium bromide staining of the treated DNA were utilized to assess the randomness and size distribution of the fragments.
- Cvi. ⁇ was obtained from CHIMERx (Madison, Wisconsin). Digestion of pUC19 DNA for limited time periods, or with limiting amounts of CviJI under normal or relaxed conditions, did not produce a quasi-random restriction pattern, or smear. Instead, a number of discrete bands were observed, as shown in Figure 7, lane 3 for the Cv ⁇ U partial digestion of pUC19. Complete digests of pUC19 under normal and CviJI buffer conditions are shown in lanes 1 and 2 respectively. These results show that, under these relaxed conditions, Cvi. ⁇ has a strong restriction site preference.
- Lanes 1-4 pUC19 DNA (1.0 ⁇ g) was run after digestion at 37°C in a 20 ⁇ l volume for the following times and conditions: Lane 1, complete CviJI digest (1 unit of enzyme for 90 min in 50 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 , 50 mM NaCl); Lane 2, complete Cv ⁇ U digest (1 unit of enzyme for 90 min in 50 mM Tris-HCl, pH 8.0,10 mM MgCl 2 , 50 mM NaCl, 1 mM ATP, 20
- Lane 4 partial CviJI digest (0.5 units of eirzyme for 60 min in 10 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 , 10 mM NaCl, 1 mM ATP, 20 mM DTT, 20% v/v DMSO); and Lane 5, uncut pUC19 (1.0 ⁇ g).
- DNA was mechanically sheared by sonication utilizing a Heat Systems Ultrasonics (Farmingdale, New York) W-375 cup horn sonicator as specified by Bankier et al, Methods in Enzymology 155:51-93 (1987). DNA fragmented by this method has random single-stranded overhanging ends (ragged ends).
- CviJI digested, and sonicated samples were size fractionated by agarose gel electrophoresis and electroelution, or by spin columns packed with the size exclusion gel matrix, Sephacryl S-500 (Pharmacia LKB, Piscataway N.J.) to eliminate small DNA fragments.
- Spin columns (0.4 cm in diameter) were packed to a height of 1.3 cm by adding 1 ml of Sephacryl S-500 slurry and centrifuging at 2000 RPM for 5 minutes in a Beckman CPR centrifuge. The columns were rinsed 3 times with 1 ml aliquots of 100 mM Tris-HCl (pH 8.0) by centrifugation at 2000 RPM for 2 min.
- 0.2-2.0 ⁇ g of fragmented DNA in a total volume of 30 ⁇ l was applied to the column.
- DNA fragments larger than 500 bp was recovered in the column eluant after spinning at 2000 RPM for 5 minutes.
- the capacity of this micro-column procedure is 2 ⁇ g of DNA.
- Agarose gel electrophoresis and electroelution are described in detail by Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor N. Y.
- sonicated DNA was treated according to the procedure outlined by Bankier et al Methods in Enzymology 155:51-93 (1987), where 2.0 ⁇ g of sonicated lambda DNA is combined with 10 units of the Klenow fragment of DNA polymerase I,
- Figure 8 shows comparisons of the size distributions of sonicated DNA versus DNA that was partially digested with Cv ⁇ U .
- Lanes M a 1 kb DNA ladder was run.
- Lanes 1-3 untreated ⁇ DNA (0.25 ⁇ g), sonicated ⁇ DNA (1.0 ⁇ g), and CviJI partially-digested ⁇ DNA (1.0 ⁇ g) were run, respectively.
- Lanes 4-6 untreated pUC19 (0.25 ⁇ g), sonicated pUC19 (1.0 ⁇ ⁇ g), and CviJI partially-digested pUC19 (1.0 ⁇ g) were run, respectively.
- Fragmentation of a large substrate such as lambda DNA (45 kb) revealed essentially no banding differences between the Cv ⁇ U method and sonication, as demonstrated in Figure 8, lanes 2 and 3.
- pUC19 DNA that was partially digested with CviJI gave a size distribution or "smear" that closely resembled that achieved with sonication ( Figure 8, lanes 5 and 6).
- the minor bias evident with a small molecule such as pUC19 was not detectable with a larger substrate such as lambda DNA.
- the intensity and duration of sonic treatment affects the size distribution of the resulting DNA fragments.
- the amount of data obtained by the shotgun sequencing approach is substantially increased if fragments of less than 500 bp are eliminated prior to the cloning step.
- Small fragments yield only a portion of the sequence data which may be collected from polyacrylamide gel based separations and, thus, such small fragments lower the efficiency of this strategy.
- Agarose gel electrophoresis followed by electroelution is commonly used to size fractionate DNA prior to shotgun cloning (Bankier et al, Methods in Enzymol. 155:51-93 (1987)). Approximately three hours are required to prepare the agarose gel, electrophorese the sample, electroelute fragments larger than 500 bp, perform phenol-chloroform extractions, and precipitate the resulting material.
- Figures 9A-E CviJI -fragmented lambda DNA by agarose gel electrophoresis are shown in Figures 9A-E.
- Figures 9A-D illustrate the following.
- Lane M Lane M
- Small DNA fragments may also be removed by passing the sample through a short column of Sephacryl S-500. Approximately 15 min. are needed to prepare the column and 5 min. to fractionate the DNA by this method.
- large quantities of the cloning vector and ligation cocktail were prepared, ligation reactions and transformation of competent E. coli were performed on the same day, numerous redundant controls were performed, and all cloning experiments were repeated twice. Ligation reactions were carried out overnight at 12°C in 20 ⁇ l mixtures using the following conditions: 25 mM Tris-HCl (pH 7.8), 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, DNA, and 2000 units of T4 DNA ligase.
- T agar (Messing, Methods in Enzymol 101:20-78 (1983)) containing 20 ⁇ g/ml ampicillin, 25 ⁇ l of a 2% solution of isopropylthiogalactoside (IPTG) and 25 ⁇ l of a 2% solution of 5-dibromo-4-chloro-3-indolylgalactoside (X-GAL).
- IPTG isopropylthiogalactoside
- X-GAL 5-dibromo-4-chloro-3-indolylgalactoside
- This cloning vector was chosen because it permits a simple blue to white visual assay to indicate whether a DNA fragment was cloned (white) or not (blue) (Messing, Methods in Enzymol 101:20-78 (1983)).
- Cloning efficiencies reflect the number of ampicillin-resistant colonies/ng pUC19 DNA.
- CIAP represents treatment with calf intestinal alkaline phosphatase used to dephosphorylate HincII-digested pUC19 to minimize self- ligation.
- CF refers to DNA that was fractionated on Sephacryl S-500 columns as described above.
- GFE1 and GFE2 refer to two runs wherein DNA was fractionated by agarose gel electrophoresis and electroeluted.
- ⁇ refers to bacteriophage ⁇ DNA. These trials represent repeated experiments in which ⁇ DNA fragments generated by CviJI partial digestion were ligated to HincII-linearized, dephosphorylated pUC19 and transformed into DH5 ⁇ F' competent cells described above.
- the first three rows in Table 2 show controls performed to establish a baseline to better evaluate the various treatments.
- Supercoiled pUC19 transforms E. coli 10 times more efficiently than the Hi/icII-digested plasmid and 150-260 times more efficiently than the HincII-digested and dephosphorylated plasmid.
- the number of blue and white colonies which resulted from transforming HincU- cut and dephosphorylated pUC19 was determined both before and after treatment with T4 DNA ligase in order to differentiate these background events from cloning inserts.
- the background of blue colonies (which represent the uncut and/or non-dephosphorylated population of molecules) averaged 0.4 % , compared to supercoiled plasmid.
- the background of white colonies (which presumably results from contaminating nucleases in the enzyme treatments or genomic DNA in the plasmid preparations) after HincII-digestion, dephosphorylation, and ligation of pUC19 averaged 0.014% as compared to the supercoiled plasmid.
- the void volume containing partially digested DNA was directly ligated to linear, dephosphorylated pUC19 and 43 resulting clones were analyzed for insert size.
- the DNA for this experiment is the same as that shown in Figure 9A, lane 2.
- Figure 10B a CvJI partial digest of 5 ⁇ g of ⁇ DNA was size fractionated by agarose gel electroelution. The eluted DNA was phenol-extracted and ligated to linear, dephosphorylated pUC19, and the resulting 40 clones were analyzed for insert size.
- the DNA for this experiment is the same as that shown in Figure 9A, lane 3.
- Figure IOC the procedure is the same as in Figure 9B, except the DNA for this experiment came from Figure 9B, lane 3.
- ⁇ DNA was fragmented by each of these methods and ligated to pUC19 which was linearized with HincU and dephosphorylated to minimize self-ligation.
- DNA/treatment Blue White Blue White Supercoiled pUC19 30000 ⁇ 10 16000 ⁇ 10 pUC19/#i «clI/CIAP 150 ⁇ 1 31 1 pUC19/ffi « I/CIAP/ 100 ⁇ 1 15 1 T4 DNA ligase ⁇ //4M+pUC19 200 400 73 250 ⁇ /CwJI Partial + pUC19 100 160 97 340 ⁇ /Sonicated + pUC19 - 11 29 ⁇ /Sonicated/ER 1 17 10 10 44
- Cloning efficiencies represent the number of ampicillin-resistant colonies/ng pUC19 DNA.
- CIAP indicates treatment with calf intestinal alkaline phosphatase.
- ER 1 and ER 2 are end repair methods described in Example 13.
- ⁇ refers to bacteriophage lambda.
- the indicated trials represent repeated experiments in which two identical sets of lambda DNA fragments generated by Alul complete digestion, CviJl partial digestion, or sonication were each ligated to HmdI-linearized, dephosphorylated pUC19 and transformed into DH5 ⁇ .F' competent cells.
- the cloning efficiencies reported are the average of triplicate platings of each ligation reaction.
- the number of white colonies obtained when unfractionated CVzTf treated ⁇ DNA was cloned into pUC19 ranged from 160-340/ng vector, versus 68- 90 white colonies/ng vector if the same material was fractionated.
- Sonicated ⁇ DNA is a poor substrate for ligation, compared to CviJI treatment, as indicated by the roughly ten-fold reduced cloning efficiencies.
- Enzymatic repair of the ragged ends produced by sonication results in an increased cloning efficiency.
- ER 1 conditions may not be optimal, as an alternate end repair reaction (ER 2) (as described in Example 13) resulted in greater numbers of white colonies (63 and 100/ng vector for fractionated and unfractionated DNA, respectively).
- ER 2 an alternate end repair reaction
- a ten-fold excess of reagents and enzymes were utilized to repair the sonicated DNA, which apparently improved the efficiency of cloning such molecules by two to three fold.
- Table 3 sections A and B show that Cv ⁇ U partial digestion results in three to sixteen times the number of white colonies than sonicated ER 1 -treated DNA.
- pUC19 DNA was digested under Cv ⁇ U conditions and size fractionated as described above.
- the fractionated DNA was cloned into the EcoRV site of M13SPSI, a lacZ minus vector constructed by adding an Ec ⁇ KV restriction site to wild type M13 at position 5605.
- M13SPSI lacks a genetic cloning selection trait, therefore after ligation of the pUC19 fragments into the vector the sample was restricted with EcoRV to reduce the background of nonrecombinant plaques.
- Bacteriophage M13 plaques were picked at random and grown for 5-7 hours in 2 ml of 2XTY broth containing 20 ⁇ l of a DH5 ⁇ F' overnight culture. After centrifugation to remove the cells, single-stranded phage DNA was purified using SephaglassTM as specified by the manufacturer (Pharmacia LKB, Piscataway New Jersey). The single-stranded DNA was sequenced by the dideoxy chain termination method using a radiolabeled M13-specific primer and Bst DNA polymerase (Mead et al, Biotechniques 11:76-87 (1991)).
- the first 100 bases of 76 randomly chosen clones were sequenced to determine which CviU recognition site was utilized, the orientation of each insert and how effectively the cloned fragments covered the entire molecule, as shown in Figure 11.
- the positions of the 45 normal CviU sites (PuGCPy) in pUC19 are indicated beneath the line labeled "NORMAL" in the Figure 11.
- the 160 CviU* sites (GC) are indicated beneath the line labeled "R ⁇ L.AX ⁇ D” in Figure 11.
- the marks above these lines indicate the CviU pUC19 sites which were found in the set of 76 sequenced random clones.
- the frequency of cloning a particular site is indicated by the height of the line, and the left or right orientation of each clone is also indicated at the top of each mark.
- the data presented in Figure 11 demonstrate that, under CviU partial conditions, normal CviU sites are preferentially restricted over relaxed (CviU ) sites. Of the 76 clones that were analyzed, only 13%, or 1 in 7, had sequence junctions corresponding to a relaxed CviU site. Thirty-five of the forty-five possible normal restriction sites were cloned, as compared to eight of the possible one hundred sixty relaxed sites.
- the GC sites in pUC19 may be divided into four classes based on their flanking Pu/Py structure.
- the fraction of GC sites observed in pUC19 which belong to each classification is roughly equal (22.0-27.8%).
- a striking difference was found between the observed distribution in pUC19 of normal and relaxed (Rl, R2, R3) CviU recognition sites and the distribution revealed by shotgun cloning and sequence analysis of CviU -treated DNA.
- Figure 11 depicts a linear map of pUC19 showing the relative position of the lacZ' gene ( peptide of /3-galactosidase gene) and ampicillin resistance gene (Amp).
- the marks extending beneath the top line show the relative position of the 45 normal CviU sites (PuGCPy) present in pUC19.
- the marks above the line are the cleavage sites found from sequencing the CviU partial library.
- the height of the line indicates the number of clones obtained from cleavage at that site, and the orientation of the flag designates the right or left orientation of the respective clone.
- the marks extending beneath the second line show the relative positions of the 160 CVfJI sites (GC) present in pUC19. Those marks above the line were found from sequencing the CviU partial library.
- the bottom portion of Figure 11 shows the relative position and orientation of the first 20 clones sequenced, assuming a 350 bp read per clone. CviJI cleavage at relaxed sites appears to be important in "filling gaps" left by normal restriction. The primary goal of this effort was to determine the efficacy of these methods for rapid shotgun cloning and sequencing. For these purposes, only 100 bases of sequence data were acquired per clone. However, if 350 bases of sequence had been determined from each clone, then the entire sequence of pUC19 would have been assembled from the overlap of the first 20 clones ( Figure
- the horizontal dashed line demarcates the 2686 bp length of pUC19.
- the theoretical accumulation curve expected for a process in which sequence information is acquired in a totally random fashion is also shown.
- the smooth curve is a continuous plot of the discrete function S(N) where
- N is the number of clones sequenced
- L is the length of clone insert in bp
- c is the redundancy of coverage or LN/G (where G is length of fragment being sequenced in bp)
- ⁇ 1- ⁇ , where ⁇ is the fraction of length that two clones must share.
- ⁇ -- ⁇ t amounts of substrate was tested utilizing 200 ng of CvUI -digested lambda DNA.
- CviU recognizes the sequence GC (except for PyGCPu) in the target DNA. Under partial restriction conditions the length of fragment may be controlled by incubation time.
- Epitope mapping using CviU partial digests involves generating DNA fragments of 100-300 bp from a cDNA coding for the protein of interest, by methods described in Example 13, inserting them into an M13 expression vector, plating out on solid media, lifting plaques onto a membrane, screening for binding to the ligand of interest, and picking the positive plaques for isolation of the DNA, which is then sequenced to identify the epitope.
- the same epitope may be expressed as a small fragment or a larger fragment.
- This approach allows one to determine the smallest fragment containing the epitope of interest using functional assays such as binding to an antibody or other ligand, or using a direct assay for activity.
- linkers may be added to the fragments or the insert may be dephosphorylated to ensure that each fragment is cloned alone without ligation of multiple inserts.
- the expression vectors recommended for subcloning of the CviU fragments are Lambda Zap (Stratagene, LaJolla, California) or bacteriophage M13-epitope display vectors.
- An advantage of using an M13-based vector is that the peptide or protein of interest may be displayed along with the M13 coat protein and does not require host cell lysis in order to analyze the protein of interest.
- the lambda-based vectors yield plaques and hence the protein can be directly bound to a membrane filter.
- CGase I refers to a restriction endonuclease reagent which cleaves DNA at the dinucleotide CG.
- CGase I activity is based on the combined star activities of the restriction endonucleases Hpa II and Taq I. Under normal reaction conditions (10 mM Bis Tris Propane-HCl pH 7.0, 10 mM MgCl 2 , 1 mM DTT; 1 unit of enzyme/ ⁇ g DNA, 37°C for 1 hr), Hpa ⁇ recognizes CCGG and cleaves after the first C to leave a 2-base 5' overhang.
- the restriction endonuclease Taq I recognizes TCGA and cleaves after the T to leave a 2-base 5' overhang.
- Taq I activity which decrease the cleavage specificity of Taq I (10 mM Tris-HCl pH 9.0, 5 mM MgCl 2 , 6 mM 2-mercaptoethanol, 20% DMSO; 2000 units of enzyme/ ⁇ g DNA, 65°C for 1 hr)
- Hpa ⁇ Hpa It activity which are also compatible with Taq I activity: 5 mM KC1, 10 mM Tris-HCl pH 8.5, 10 mM MgCl 2 , 1 mM DTT, 15% DMSO, 100 ug/ml BSA (CGase buffer); 50 units of enzyme/ ⁇ g DNA 50° C for 1 hr.
- the Hpa II recognition sites were determined by cloning and sequencing Hpa II restricted fragments. The characterized Hpa ⁇ recognition sequences are as follows:
- Taq I 400 units/ ⁇ g DNA
- Hpa II 50 units/ ⁇ g DNA
- CGase I restriction of natural DNA results in fragments ranging from 20-200 bp in length (average 20-60 bp). Heat denaturation of these fragments generates numerous oligonucleotides of variable length but precise specificity for the cognate template as was the case with CviJ I digestion.
- CGase I restriction of natural DNA results in fragments ranging from 20-200 bp in length (average 20-60 bp). Heat denaturation of these fragments generates numerous oligonucleotides of variable length but precise specificity for the cognate template as was the case with CviJ I digestion.
- DNA without regard to sequence composition, genetic origin, or prior sequence knowledge is one of the properties that CGase I shares with CviJ I .
- the generation of numerous oligonucleotides by CviJ I or CGase I results in a form of probe or primer amplification not practical using conventional means of organic synthesis.
- the present invention contemplates the interchangeability of CGase I with CviJ I in all of the applications described herein.
- CviJ I was prepared by a modification of the method described by Xia et al, Nucl. Acids Res. 15:6025-6090 (1987). Chlorella NC64A cells
- IL-3A infected Chlorella NC64A cells Five grams of IL-3A infected Chlorella NC64A cells were suspended in a glass homogenization flask with 15 g of 0.3 mm glass beads in buffer A (10 mM Tris-HCl pH 7.9, 10 mM 2-mercaptoethanol, 50 ⁇ g/ml phenylmethylsulfonyl fluoride (PMSF), 20 ug/ml benzamidine, 2 ⁇ g/ml o- phenanthroline). Cell lysis was carried out at 4000 rpm for 90 sec in a Braun MSK mechanical homogenizer (Allentown, PA) with cooling from a CO 2 tank.
- buffer A 10 mM Tris-HCl pH 7.9, 10 mM 2-mercaptoethanol, 50 ⁇ g/ml phenylmethylsulfonyl fluoride (PMSF), 20 ug/ml benzamidine, 2 ⁇ g/ml o- phenanthroline.
- PEI polyethyleneimine
- the resulting pellet was dissolved in 20 ml of buffer B (20 mM Tris-acetate pH 7.5, 0.5 mM EDTA, 10 mM 2-mercaptoethanol, 10% glycerol, 30 mM KCl, 50 ug/ml PMSF, 20 ⁇ g/ml benzamidine [Sigma, St. Louis, Missouri], 2 ⁇ g/ml o-phenanthroline [Sigma]) and dialysed against 500 ml of buffer B with 3 changes. The dialysed solution was then applied to 1 x 6 cm Heparin-Sepharose (Pharmacia LKB, Piscataway, New Jersey) column. After a 50 ml wash with buffer B, a 100 ml gradient of 0 to 0.7
- CvU I was eluted by a 100 ml gradient of 0 to 0.7 M NaCl in buffer C.
- CvU I activity separated from non-specific nucleases.
- CviJ I containing fractions were pooled and diluted in 4 volumes of buffer C and applied to a 1 x 4 cm hydroxyapatite HTP column (BioRad,
- CviJ I was eluted by a 0 to 0.7 M potasium phosphate (pH 7.4) gradient in buffer C. Active fractions containing CviJ I activity and lacking non-specific nuclease activity were pooled and were dialysed overnight against storage buffer (50 mM potassium phosphate 200 mM KCl, 0.5 mM EDTA, 50% glycerol, 20 ug/ml PMSF were pooled) and stored at -20°C.
- storage buffer 50 mM potassium phosphate 200 mM KCl, 0.5 mM EDTA, 50% glycerol, 20 ug/ml PMSF were pooled
- MOLECULE TYPE DNA (genomic)
- GGTATATCTA CACCAGTTGC ATTCGTAGAA ATTAATGAAG ACGCACAAAA ATTCTTGAAA 120
- AAAAGAATTG CGGAAGAGAA AAAAAGAATT GCACTTATAG AAAAACAACG AATTGCGGAA 1260
- GGTTCCATAT TATATCTTAC ATTCACTGGT TTTAGCGCAT TAAATGGGCA CTTGGAGAAT 2040
- ATCTGTATAC ATATCACTTG GTTCGAAATG AAAATCGTAG TCCCAATTAG GTACGTTCCA 4620
- MOLECULE TYPE DNA (genomic)
- GAG AAA AAA AGA ATT GCA CTT ATA GAA AAA CAA CGA ATT GCG GAA GAG 195 Glu Lys Lys Arg He Ala Leu He Glu Lys Gin Arg He Ala Glu Glu 45 50 55
- MOLECULE TYPE DNA (genomic)
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US5075232A (en) * | 1988-07-28 | 1991-12-24 | New England Biolabs, Inc. | Method for producing the nlavi restriction endonuclease and methylase |
US5053330A (en) * | 1989-03-13 | 1991-10-01 | New England Biolabs, Inc. | Method for producing the mwoi restriction endonuclease and methylase |
-
1994
- 1994-03-24 AU AU65245/94A patent/AU681650B2/en not_active Ceased
- 1994-03-24 EP EP94912866A patent/EP0690870A4/en not_active Withdrawn
- 1994-03-24 WO PCT/US1994/003246 patent/WO1994021663A1/en not_active Application Discontinuation
- 1994-03-24 CA CA002159081A patent/CA2159081C/en not_active Expired - Fee Related
Non-Patent Citations (6)
Title |
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C. POLISSON ET AL.: "AciI, a unique restriction endonuclease from Arthrobacter citreus which recognizes 5' CCGC 3'" NUCLEIC ACIDS RESEARCH, vol. 18, no. 19, 1990, page 5911 XP002056258 * |
FITZGERALD M C ET AL: "RAPID SHOTGUN CLONING UTILIZING THE TWO BASE RECOGNITION ENDONUCLEASE CVIJI." NUCLEIC ACIDS RES 20 (14). 1992. 3753-3762. CODEN: NARHAD ISSN: 0305-1048, XP000674737 * |
G. SHEARER : "Multienzyme nested deletion {MEND} technique for creation of unidirectional deletions in cloned DNA" ANALYTICAL BIOCHEMISTRY, vol. 223, 1994, pages 105-110, XP002056259 * |
See also references of WO9421663A1 * |
SWAMINATHAN N ET AL: "Restriction generated oligonucleotides utilizing the two base recognition endonuclease CviJI." NUCLEIC ACIDS RESEARCH 22 (8). 1994. 1470-1475. ISSN: 0305-1048, XP000674735 * |
Y. XIA ET AL.: "IL-3A virus infection of a Chlorella-like gene alga induces a DNA restriction endonuclease with novel sequence specificity" NUCLEIC ACIDS RESEARCH, vol. 15, no. 15, 1987, pages 6075-6090, XP000674738 * |
Also Published As
Publication number | Publication date |
---|---|
AU681650B2 (en) | 1997-09-04 |
CA2159081A1 (en) | 1994-09-29 |
WO1994021663A1 (en) | 1994-09-29 |
CA2159081C (en) | 2000-11-21 |
EP0690870A4 (en) | 1998-05-20 |
AU6524594A (en) | 1994-10-11 |
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