Specific deletion of DNA sequences between preselected bases.

Blunt-end ligation of a "filled-in" HindIII, Sal I, Ava I or Bcl I restriction site with a DNA fragment having A, G, C, or T as the terminal 3' nucleotide regenerates the corresponding restriction site. A combination of this property with the action of BAL 31 nuclease which progressively removes base-pairs from the ends of linear DNA, can generate deletions extending to desired pre-selected nucleotides, and introduces unique restriction sites at those positions. Similarly other restriction sites can be used to select for the deletion of sequences between specific di-, tri-, tetra- and penta-nucleotides. Using this method, 10 base pairs were deleted from the end of a restriction fragment carrying the late promoter for bacteriophage T7 gene 1.1, to create a molecule with a unique restriction site at the initiation codon for translation.     

Adriamycin-mediated introduction of a limited number of single-strand breaks into supercoiled DNA

It was reported previously that Adriamycin converts form I covalently closed circular, supercoiled bacteriophage PM2 DNA to the relaxed circular form II DNA; no form III linear DNA was produced as a result of the extracellular action of Adriamycin in the presence of NADH-dehydrogenase. When form II DNA, produced by the action of Adriamycin, was treated with the BAL 31 nuclease, a single sharp DNA band after agarose gel electrophoresis indicated the presence of only full-length linear form III DNA. As one of its activities, the BAL 31 nuclease introduces a single-strand break in the complementary strand opposite a preexisting single-strand break. When form II DNA, produced by the action of gamma irradiation, was reacted with the BAL enzyme, the resulting linear DNA molecules exhibited a broad range of molecular weights, indicating the presence of many single-strand breaks in the substrate form II DNA. When the Adriamycin-produced form II DNA was treated with restriction endonucleases that cleave PM2 DNA at a single site, either with or without pretreatment with the BAL enzyme, the formation of only full-length linear DNA was observed. Thus, the drug is capable of introducing one or only a very limited number of single-strand breaks into supercoiled DNA; furthermore, these breaks are introduced at random sites along the DNA molecules.     

Construction of plasmid vectors with unique PstI cloning sites in a signal sequence coding region

We have constructed a series of plasmids with unique PstI restriction sites within or near the pre-penicillinase signal sequence for protein secretion. To do this, we devised a rapid, simple method to eliminate undesirable unique restriction sites within plasmids while maintaining antibiotic resistance. We thus obtained a plasmid with a conveniently located, unique HincII site in the penicillinase gene of plasmid pBR322 which was used to generate, with BAL 31 exonuclease, deletions extending into the region encoding the signal sequence. DNA inserted into these plasmids can be translated in all three reading frames both including signal sequence, or starting immediately beyond it.     

A new strategy to create ordered deletions for rapid nucleotide sequencing

A method is described for generating ordered deletions using previously published techniques but a new strategy. This method is simpler than the published ones and has many advantages. Target DNA is cloned in both orientations into one of the unique restriction enzyme sites adjacent to the complementary region of the commercially available primers in bacteriophage M13. Ordered unidirectional deletions are created using BAL 31 nuclease and religating into M13 vector DNA without the need of purifying BAL 31-digested DNA from a gel.     

Specific cleavage of DNA molecules at RecA-mediated triple-strand structure

A novel procedure to cleave DNA molecules at any desired base sequence is presented. This procedure is based upon our finding that double-stranded DNA molecules at a site where RecA-mediated triple-stranded DNA structure with a complimentary deoxyoligonucleotide is located can be cleaved by a single-strand specific nuclease, such as nuclease S1 or BAL31, between the first base at the 5′ termini of the deoxyoligonucleotides and the nearest base proximal to the 5′ termini. Accordingly, the sequence as well as the number of the cleavage sites to be cleaved can be custom designed by selecting deoxyoligonucleotides with specific base sequences for triple-stranded DNA formation. The basic characteristics of the cleavage reaction and typical applications of the procedure are presented with actual results, including those which involve cleavage of complex genomic DNA at the very sites one desires.     

Developmentally coordinated en masse excision of a highly repetitive element in E. crassus

The E. crassus Tec1 element is present in greater than 10(4) copies in the micronuclear genome but is absent from the macronuclear genome. During formation of a macronucleus from a micronucleus, a majority of the Tec1 elements appear as extrachromosomal circles. The circular and integrated forms of Tec1 have been characterized by restriction mapping to produce consensus maps and by sequence analysis of the element's termini. The circular forms are resistant to BAL31 and have the restriction map expected if the element excises at the end of its inverted repeats. DNA sequence analysis of a circular form confirms that the inverted repeats are in a head-to-head configuration. Excision of Tec1 occurs very early during macronuclear development as the DNA begins to replicate to form polytene chromosomes.     

AFLP: a new technique for DNA fingerprinting.

A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.     

Characterization of human chromosomal DNA sequences which replicate autonomously in Saccharomyces cerevisiae.

We have characterised two restriction fragments, isolated from a "shotgun" collection of human DNA, which function as autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Functional domains of these fragments have been defined by subcloning and exonuclease (BAL 31) deletion analysis. Both fragments contain two spatially distinct domains. One is essential for high frequency transformation and is termed the Replication Sequence (RS) domain, the other, termed the Replication Enhancer (RE) domain, has no inherent replication competence but is essential for ensuring maximum function of the RS domain. The nucleotide sequence of these domains reveals several conserved sequences one of which is strikingly similar to the yeast ARS consensus sequence.     

Vectors containing infrequently cleaved restriction sites for use in BAL 31 nuclease-assisted and end-label-mediated analysis of cloned DNA fragments

Cloning vectors derived from plasmids pUC8 and pUC18 and phage M13mp10 were constructed so as to have multiple cloning sites (MCS) flanked by the recognition/cleavage sites for the Sfi I and Not I restriction nucleases. Cleavage of vectors containing cloned DNA fragments with either of the infrequently cleaving Sfi I or Not I endonucleases will usually yield linear DNAs cleaved only at the corresponding site in the MCS, so that the cloned insert can be degraded unidirectionally by the duplex exonuclease activity of the BAL 31 nucleases until an amount equal to the length of the vector has been degraded. The ends of the above constructs resulting from cleavage with Not I or Sfi I can readily be labeled, with labeling at only the terminus of the cloned DNA available for the Sfi I site. The BAL 31 nuclease-mediated procedures enhance a previous technique for mapping of restriction enzyme fragments, allow for localization of sequences in cloned segments for which a probe is available, and improve a method for sequencing cloned inserts through the production of sets of nested unidirectional deletions from either end of the parent cloned fragment. The advantages of end-label-mediated restriction site mapping using the above vectors over existing such procedures are also demonstrated.     

Structure and physical map of Rhodopseudomonas sphaeroides bacteriophage RS1 DNA.

We analyzed, by restriction endonuclease mapping and electron microscopy, the genome of the lytic Rhodopseudomonas sphaeroides-specific bacteriophage RS1 and characterized it as a linear molecule of approximately 60 to 65 kilobases. When the DNA from purified phage particles was examined by several independent methods, considerable size heterogeneity was apparent in the RS1 DNA. This size heterogeneity was concluded to be of biological origin, was independent of the specific host strain used to propagate virus, and was not due to the presence of host DNA within or nonspecifically associated with purified virions. In addition, treatment of RS1 DNA with either BAL 31 nuclease or DNA polymerase I Klenow fragment revealed that several distinct regions exist within the viral chromosome which contain free 3' hydroxyl groups. A restriction endonuclease map of the RS1 genome was constructed by using the restriction endonucleases EcoRI, ClaI, KpnI, BamHI, MluI, SmaI, and BclI; thereby allowing the positioning of some 40 restriction sites within the viral genome. The results are discussed in terms of the significance and the possible biological origin of the unique features discovered within the phage RS1 DNA.     

Site specificity of bleomycin-mediated single-strand scissions and alkali-labile damage in duplex DNA.

Form II PM2 DNA, which contained bleomycin-mediated single-strand breaks, was purified and treated with the extracellular endonuclease from Alteromonas BAL 31. This enzyme cleaves the phosphodiester backbone opposite a single-strand break to yield a double-strand break. The locations of these double-strand breaks were determined relative to the cleavage sites produced by the restriction enzyme HindIII. The experimental procedure was as follows. Form I PM2 DNA was treated with bleomycin to produce alkali-labile bonds. These were hydrolyzed by alkali treatment and the DNA, now containing single-strand breaks, was purified and treated with the BAL 31 enzyme and the HindIII enzyme to determine the positions of the original alkali-labile bonds. It was found that the single-strand breaks and alkali-labile bonds were introduced at preferred sites on the PM2 genome, since electrophoretic analyses of the DNA after the HindIII digestion revealed DNA bands of discrete sizes. The molecular weights of the DNA fragments produced by these treatments indicate that single-strand breaks and alkali-labile bonds occur at the same sites as those previously determined for direct double-strand scissions introduced by bleomycin at neutral pH. Some of the specific sites of double-strand scissions mediated by bleomycin at neutral pH (Lloyd et al., 1978b) are also shown here to be relatively more reactive than other sites when the DNA contains superhelical turns.     

General method of preparation of uniformly 13C, 15N-labeled DNA fragments for NMR analysis of DNA structures

Summary ¹³C, ¹⁵N labeling of biomolecules allows easier assignments of NMR resonances and provides a larger number of NMR parameters, which greatly improves the quality of DNA structures. However, there is no general DNA-labeling procedure, like those employed for proteins and RNAs. Here, we describe a general and widely applicable approach designed for preparation of isotopically labeled DNA fragments that can be used for NMR studies. The procedure is based on the PCR amplification of oligonucleotides in the presence of labeled deoxynucleotides triphosphates. It allows great flexibility thanks to insertion of a short DNA sequence (linker) between two repeats of DNA sequence to study. Size and sequence of the linker are designed as to create restriction sites at the junctions with DNA of interest. DNA duplex with desired sequence and size is released upon enzymatic digestion of the PCR product. The suitability of the procedure is validated through the preparation of two biological relevant DNA fragments.The first two authors contributed equally to this work.     

A universal platform for sensitive and selective colorimetric DNA detection based on Exo III assisted signal amplification

Rapid growth of available sequence data has made the detection of nucleic acids critical to the development of modern life sciences. Many amplification methods based on gold nanoparticles and endonuclease for sensitive DNA detection have been developed. However, these approaches require specific target sequence for endonuclease recognition, which cannot be fulfilled in all systems. Replacing the restriction enzyme with a nuclease that does not require any specific recognition sequence may offer a universally adaptable system. Here we have developed a novel homogeneous, colorimetric DNA detection method, which consists of Exo III, a linker DNA, and two DNA-modified gold nanoparticles. This system is simple, low-cost, sensitive and selective. By coupling cyclic enzymatic cleavage and gold nanoparticle for signal amplification, our system provides a colorimetric detection limit of 15 pM, which is 3 orders of magnitude more sensitive than that of a general three-component sandwich assay format. Due to the intrinsic property of Exo III, our method shows excellent detection selectivity for single-base discrimination. More importantly, superior to other methods based on nicking and FokI endonuclease, our target sequence-independent platform is generally applicable for DNA sensing. This new approach could be widely applied to sensitive nucleic acids detection.     

An improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned in a plasmid.

A strategy for kilo-base sequencing of a target DNA cloned in plasmid pWR34 is described. A long target DNA is progressively shortened from one end, by digestion with BAL31 nuclease or exonuclease III and nuclease S1, followed by cleaving off the shortened vector DNA. The family of the shortened target DNA molecule is next cloned in between the StuI site on one end, and a cohesive-ended restriction site on the other end, within the polylinker region of pWR34. DNA fragments cloned into this plasmid are sequenced directly by using a synthetic oligonucleotide primer, which binds to one side of the polylinker region using the dideoxynucleotide chain-termination method. The plasmid DNA, easily obtained by adoption of a rapid mini-preparation, is usually pure enough for direct DNA sequencing. Thus, both strands of any DNA several thousand base pairs in length can be completely sequenced (using two different primers) with ease within a short time, without the need for constructing a physical map.     

New cloning vectors and techniques for easy and rapid restriction mapping

We have modified plasmid, phage lambda and cosmid cloning vectors to be of general use for easily and unambiguously determining restriction maps of recombinant DNA molecules. Each vector is constructed so that it contains the rarely found NotI restriction site joined to a short synthetic linker sequence that is followed by a multiple cloning site. DNA cloned into these vectors may be restriction-mapped by either of two methods. In one technique, the cloned DNA is completely digested with NotI, followed by partial digestion with any other restriction enzyme. After electrophoresis and transfer to a nylon membrane, the fragments are hybridized to a labeled probe complementary to the NotI linker. In the second technique, referred to as recession hybridization detection, cloned DNA is digested with NotI and then briefly treated with exonuclease III to recess the 3' ends. After hybridizing a labeled complementary oligodeoxynucleotide to the single-stranded 5' end containing the linker sequence, the DNA is partially digested with another restriction enzyme, electrophoresed and the gel is exposed to x-ray film. With either method the size of each labeled fragment corresponds directly to the distance that a restriction site is located from the NotI linker terminus. Methods for obtaining partial restriction enzyme digests have been devised so that as many as 20 different enzymes may be conveniently mapped on a single gel in little more than a day. The vectors and techniques described may also be adapted to automated or semi-automated devices that read fragment lengths and calculate the resulting restriction map.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonrandom DNA sequencing of exonuclease III-deleted complementary DNA

The nonrandom DNA sequence analysis procedure of Poncz et al. [Proc. Natl. Acad. Sci. USA 79, 4298-4302 (1982)] was extensively modified to permit the determination of complementary DNA (cDNA) sequences containing G-C homopolymer regions. The recombinant cDNA plasmid was cleaved at a unique restriction enzyme site close to the cDNA and treated with Exonuclease III under controlled conditions to generate a set of overlapping fragments having deletions 50-1500 bases in length at the free 3' termini. After removal of single-stranded DNA regions by Bal31 and DNA polymerase I large fragment, the unique restriction enzyme site was recreated by blunt end ligation of synthetic oligonucleotides to the deleted DNA fragments and restriction enzyme digestion. The cDNA fragment was excised from the cloning vector using a second different restriction enzyme having a unique site that flanks the cDNA fragment and subsequently force-cloned into either M13 mp10 or mp11. This method should also be particularly useful for the sequencing of other types of DNA molecules with lengths 1500 bp or smaller.     

A general method for inserting specific DNA sequences into cloning vehicles

A general method has been developed to introduce any double-stranded DNA molecule into cloning vehicles at different restriction endonuclease sites. In this method a chemically synthesized decadeoxyribonucleotide duplex, containing a specific restriction endonuclease sequence, is joinlex DNA is cut by the same restriction endonuclease to generate the cohesive ends. It is then inserted into the restriction endonuclease cleavage site of the cloning vehicle. To demonstrate the feasibility of this new method, we have inserted separately the synthetic lac operator DNA at the Bam I and HindIII cleavage sites of the plasmid pMB9 DNA.     

Mycoplasma restriction: identification of a new type of restriction specificity for DNA containing 5-methylcytosine.

Mycoplasma bacteriophage L51 single-stranded DNA and L2 double-stranded DNA are host cell modified and restricted when they transfect Acholeplasma laidlawii JA1 and K2 cells. The L51 genome has a single restriction endonuclease MboI site (recognition sequence GATC), which contains 5-methylcytosine when the DNA is isolated from L51 phage grown in K2 cells but is unmethylated when the DNA is from phage grown in JA1 cells. This GATC sequence is nonessential, since an L51 mutant in which the MboI site was deleted was still viable. DNA from this deletion mutant phage was not restricted during transfection of either strain K2 or JA1. Therefore, strain K2 restricts DNA containing the sequence GATC, and strain JA1 restricts DNA containing the sequence GAT 5-methylcytosine. We conclude that K2 cells have a restriction system specific for DNA containing the sequence GATC and protect their DNA by methylating cytosine in this sequence. In contrast, JA1 cells (which contain no methylated DNA bases) have a newly discovered type of restriction-modification system. From results of studies of the restriction of specifically methylated DNAs, we conclude that JA1 cells restrict DNA containing 5-methylcytosine, regardless of the nucleotide sequence containing 5-methylcytosine. This is the first report of a DNA restriction activity specific for a single (methylated) base. Modification in this system is the absence of cytosine methylating activity. A restriction-deficient variant of strain JA1, which retains the JA1 modification phenotype, was isolated, indicating that JA1 cells have a gene product with restriction specificity for DNA containing 5-methylcytosine.     

BAL 31 nuclease as a probe in concentrated salt for the B-Z DNA junction

The BAL 31 nuclease, an extracellular nuclease from A. espejiana, specifically recognizes and cleaves the salt induced conformational junction between B and Z-DNA. Short segments of (dC-dG) left-handed Z-helix, comprising approximately 1% of the total DNA, are specifically detected within two different recombinant plasmids. The BAL 31 enzyme is highly resistant to inactivation by the presence of high concentrations of a variety of electrolytes that stabilize left-handed helices, is active at physiological pH, and can be used to probe both linear and circular DNAs. Additionally, the nuclease cleaves left-handed (dC-dG)n only very poorly, if at all. Thus, the BAL 31 nuclease can be utilized as a probe for helical junctions and consequently for segments of left-handed DNA that might exist within predominantly right-handed naturally occurring genomes.     

The MURFI linker for multiple reading frame insertion of a sense or nonsense codon into DNA.

Blunt-end palindromic DNA linkers with a central restriction site have been designed for the multiple reading frame insertion (abbreviated MURFI) of a sense or nonsense codon into DNA. We have utilized an amber MURFI linker, 5'CTAG TCTAGA CTAG3' to disrupt the lacZ gene, yielding truncated beta-galactosidase proteins. Conditional disruption of the tetr gene in E. coli has also been demonstrated. Nonsense codon MURFI linkers permit conditional fusion of multiple gene products while sense codon linkers can add structural elements (e.g. beta-turn, cationic segment, hydrophobic segment) or a desired amino acid to a protein (e.g. methionine, cysteine). Shotgun or alternatively site-directed insertion of the symmetric linkers is possible. The over-all length of the linker may be adjusted to retain the original reading frame, matching nucleotide additions or subtractions at recipient DNA sites. If a linker restriction site occurs elsewhere in the target DNA, single linker copies may still be inserted using non-phosphorylated linkers.