Ordered deletions for DNA sequencing and in vitro mutagenesis by polymerase extension and exonuclease III gapping of circular templates.

A simple method is described for generating nested deletions from any fixed point in a cloned inset. Starting with a single-stranded phagemid template, T4 DNA polymerase is used to extend an annealed primer. This leads to a fully double-stranded circular molecule with a nick or small gap just 5' to the primer. Exonuclease III initiates progressive digestion from the resulting 3' end. Removal of timed aliquots and digestion with a single-strand specific endonuclease leads to a series of linear nested fragments having a common end corresponding to the 5' end of the primer. These molecules are circularized and used to transform cells, providing large numbers of deletion clones with targeted breakpoints. The 6-step procedure involves successive additions to tubes, beginning with a single-stranded template and ending with transformation; no extractions, precipitations or centrifugations are needed. Results are comparable to those obtained with standard Exonuclease III-generated deletion protocols, but there is no requirement for restriction endonuclease digestion or for highly purified double-stranded DNA starting material. This procedure provides a strategy for obtaining nested deletions in either direction both for DNA sequencing and for functional analysis.     

Half-site editing: an in vitro mutagenesis procedure for truncating a DNA fragment and introducing a new restriction site

Half-site editing is an in vitro mutagenesis procedure designed for use in making precise plasmid constructions. Like many in vitro mutagenesis techniques, this procedure involves hybridization of a mutagenic oligonucleotide primer to single-stranded template DNA followed by polymerization with DNA polymerase I (Klenow). Half-site editing differs from other techniques in two main ways. First, T4 DNA polymerase treatment truncates the target DNA at a point determined by the primer and repairs any mismatches to the sequence specified by the primer. Second, a blunt-end ligation step is included. This ligation exploits the symmetry inherent in most restriction sites to create a desired restriction site at the truncated end of the target DNA fragment. Half-site editing has been used to place ClaI restriction sites at the 3' end of the yeast pyruvate kinase promoter and at two positions at the 5' end of the yeast acetolactate synthase coding sequence.     

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.     

Restriction site insertion-PCR (RSI-PCR) for rapid discrimination and typing of closely related microbial strains

Taking advantage of point mutations between DNA sequences of closely related microbial strains, PCR primers modified with respect to the target sequence at positions 2-5 near the 3' end were designed to obtain a fragment harbouring an artificial restriction site specific for a given strain. The modified forward primer coupled with a specific reverse primer allows for the amplification of DNA fragments which can be digested with the specific endonuclease only in those strains where the restriction site is inserted by the DNA polymerase. The effectiveness of the method, named restriction site insertion-PCR (RSI-PCR), was tested on isolates of the 'Bacillus cereus group' for the rapid typing and discrimination of these closely related strains.     

Nucleotide sequence of a fragment of SV40 DNA that contains the origin of DNA replication and specifies the 5' ends of "early" and "late" viral RNA. III. Construction of the total sequence of EcoRII-G fragment of SV40 DNA.

Limited T1 RNase digestion of subfragments of the SV40 DNA restriction endonuclease fragment EcoRII-G were prepared and analyzed. The fragments were separately labeled with 32P at their 5' terminus and the terminal sequences analyzed with limited snake venom diesterase digestion. The data permitted us to deduce the nucleotide sequence for EcoRII-G. The sequence contains a stretch of 17 A-T base pairs preceding the DNA complementary to the 5' end of "early" message RNA, a stretch of 27 bases with a perfect 2-fold rotational symmetry near the origin of DNA replication and a perfect tandem repeat of 21 nucleotides.     

Cloning of random-sequence oligodeoxynucleotides

Methods are described for cloning random or highly degenerate nucleotide (nt) sequences. The procedures use synthetically derived mixtures of oligodeoxynucleotides (oligos) whose heterogeneous central portions are bounded at their 5' and 3' ends by sequences recognized by restriction endonucleases. Oligo collections of defined length and nt composition are synthesized by utilizing appropriate concentrations of all four nucleotide precursors during each addition step for the central region. Single-stranded oligos with appropriate 5' and 3' ends can be ligated directly, although inefficiently, into double-stranded (ds) DNA molecules with complementary 5' and 3' extensions produced by restriction endonuclease cleavage. A more general and efficient method is to convert the oligo into a ds form by incubating it with the Klenow (large) fragment of Escherichia coli DNA polymerase I. If the 3' ends are palindromic, two oligo molecules will serve as mutual primers for polymerization. The resulting products are ds molecules containing two oligo units separated by the original 3' restriction site and bounded at each end by the original 5' restriction site. After appropriate restriction endonuclease cleavage, oligo units can be cloned by standard procedures. Analysis of 26 recombinant M13 phages indicates that the nt sequences of the cloned oligos are in good accord with what was expected on a random basis.     

Determination of DNA sequences containing methylcytosine in Bacillus subtilis Marburg.

The methylcytosine-containing sequences in the DNA of Bacillus subtilis 168 Marburg (restriction-modification type BsuM) were determined by three different methods: (i) examination of in vivo-methylated DNA by restriction enzyme digestion and, whenever possible, analysis for methylcytosine at the 5' end; (ii) methylation in vitro of unmethylated DNA with B. subtilis DNA methyltransferase and determination of the methylated sites; and (iii) the methylatability of unmethylated DNA by B. subtilis methyltransferase after potential sites have been destroyed by digestion with restriction endonucleases. The results obtained by these methods, taken together, show that methylcytosine was present only within the sequence 5'-TCGA-3'. The presence of methylcytosine at the 5' end of the DNA fragments generated by restriction endonuclease AsuII digestion and the fact that in vivo-methylated DNA could not be digested by the enzyme XhoI showed that the recognition sequences of these two enzymes contained methylcytosine. As these two enzymes recognized a similar sequence containing a 5' pyrimidine (Py) and a 3' purine (Pu), 5'-PyTCGAPu-3', the possibility that methylcytosine is present in the complementary sequences 5'-TTCGAG-3' and 5'-CTCGAA-3' was postulated. This was verified by the methylation in vitro, with B. subtilis enzyme, of a 2.6-kilobase fragment of lambda DNA containing two such sites and devoid of AsuII or XhoI recognition sequences. By analyzing the methylatable sites, it was found that in one of the two PyTCGAPu sequences, cytosine was methylated in vitro in both DNA strands. It is concluded that the sequence 5'-PyTCGAPu-3' is methylated by the DNA methyltransferase (of cytosine) of B. subtilis Marburg.     

DNA microarrays with unimolecular hairpin double-stranded DNA probes: fabrication and exploration of sequence-specific DNA/protein interactions

We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on.     

A primer-based approach to genome walking

A genome walking strategy based on annealing and ligation of single-stranded DNA primers to 3′ overhangs following restriction endonuclease digestion was developed. A set of primers contains 4 nucleotides at the 3′ end that are complementary to overhangs formed by restriction endonucleasesApaI;PstI;SacI andSphI. Following ligation, 5′ end overhangs are formed on the DNA, which serves as sites for the adaptor primers and nested primers for PCR amplification in combination with the gene-specific primers. This strategy was verified by the amplification of up to 4 kb of a potato leafroll virus full-length infectious clone. The procedure could be adopted to target any upstream and downstream regions flanking known sequences within the plant genome.     

Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA

Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA.     

Restriction maps and restriction fragment length polymorphisms of the human 21-hydroxylase genes

Restriction maps were constructed for the two human 21-hydroxylase genes (21-OHA and 21-OHB) by using DNA from subjects homozygous for a deletion of each gene. Comparing the patterns of these two genes, a KpnI restriction site occurred in the 21-OHA gene in place of a TaqI site in the 21-OHB gene about 1-kb from the 5' end of the gene, and an extra EcoRI site was located 500 bp 5' to the common EcoRI site. The DNA of fourteen unrelated normal subjects was digested with nine restriction endonucleases (AccI, BamHI, BgIII, EcoRI, HindIII, KpnI, MspI, SacI and TaqI). Restriction fragment length polymorphisms were found with EcoRI, HindIII and AccI that resulted from polymorphic endonuclease sites outside the genes.     

Mechanism of replication of human mitochondrial DNA. Localization of the 5' ends of nascent daughter strands

Human mitochondrial DNA contains two physically separate and distinct origins of DNA replication. The initiation of each strand (heavy and light) occurs at a unique site and elongation proceeds unidirectionally. Animal mitochondrial DNA is novel in that short nascent strands are maintained at one origin (D-loop) in a significant percentage of the molecules. In the case of human mitochondrial DNA, there are three distinct D-loop heavy strands differing in length at the 5' end. We report here the localization of the 5' ends of nascent daughter heavy strands originating from the D-loop region. Analyses of the map positions of 5' ends relative to known restriction endonuclease cleavage sites and 5' end nucleotides indicate that the points of initiation of D-loop synthesis and actual daughter strands are the same. In contrast, the second origin is located two-thirds of the way around the genome where light strand synthesis is presumably initiated on a single-stranded template. Mapping of 5' ends of daughter light strands at this origin relative to known restriction endonuclease cleavage sites reveals two distinct points of initiation separated by 37 nucleotides. This origin is in the same relative genomic position and shows a high degree of DNA sequence homology to that of mouse mitochondrial DNA. In both cases, the DNA region within and immediately flanking the origin of DNA replication contains five tightly clustered tRNA genes. A major portion of the pronounced DNA template secondary structure at this origin includes the known tDNA sequences.     

Organization of delta-crystallin genes in the chicken.

Double-stranded DNA was synthesized from delta-crystallin mRNA prepared from lens fibers of 15-day-old chick embryos and cloned at the Pst I site of the plasmid pBR322. Using the cloned cDNA and single-stranded cDNA as hybridization probes, a number of genomic DNA fragments containing delta-crystallin gene sequences have been cloned from the partial and complete EcoRI digests of chick brain DNA. One of the clones from the partial digests contains a DNA fragment that consists of four EcoRI fragments of 7.6 kb, 4.0 kb, 2.6 kb, and 0.8 kb. The gene sequences reside in the (5')7.6 kb - 0.8 kb - 4.0 kb (3') fragments. Electron microscopy has provided evidence that the cloned DNA fragment includes the entire gene sequences complementary to delta-crystallin mRNA except for the 3' terminal poly(A) tail, and that the delta-crystallin gene is interrupted by at least 13 intervening sequences. Another clone contains a genomic fragment that consists of two EcoRI fragments of 3.0 kb and 11 kb. The DNA fragment in the latter clone represents a different delta-crystallin gene, as judged by restriction endonuclease mapping and by electron microscopy.     

Physical analysis and mapping of the Mycoplasma pneumoniae chromosome.

Field inversion gel electrophoresis was used for analysis of the chromosome of Mycoplasma pneumoniae. The restriction endonuclease SfiI (5'-GGCCNNNNNGGCC-3') generated 2 M. pneumoniae DNA fragments of approximately 437 and 357.5 kilobase pairs (kbp), whereas 13 restriction fragments ranging in size from 2.4 to 252.0 kbp resulted from digestion with ApaI (5'-GGGCCC-3'). Totaling the sizes of the individual restriction fragments from digestion with SfiI or ApaI yielded a genome size of 794.5 or 775.4 kbp, respectively. A physical map of the M. pneumoniae chromosome was constructed by using a combination of techniques that included analysis by sequential or partial restriction endonuclease digestions and use as hybridization probes of cloned M. pneumoniae DNA containing ApaI sites and hence overlapping adjacent ApaI fragments. Genetic loci for deoC, rrn, hmw3, and the P1 gene were identified by using cloned DNA to probe ApaI restriction fragment profiles.     

Replication of phage G4. A novel and simple system for the initiation of deoxyribonucleic acid synthesis.

Conversion in vitro of single-stranded circular DNA of phage G4 (related to phage phiX174) to the double-stranded replicative form (RF-II) depends on a novel and relatively simple group of three proteins: a priming protein of approximately 65,000 daltons, the DNA unwinding protein, and the DNA polymerase III holoenzyme. Stimulation by ATP and GTP suggests an RNA synthetic step in the priming of DNA synthesis. The synthetic strand in the RF-II contains a small gap at a unique position relative to the template strand; the 5' end of the gap is about 250 nucleotide residues (5% of the genome length) away from the single site of cleavage by a restriction endonuclease (Eco RI).     

A novel multistep method for generating precise unidirectional deletions using BspMI, a class-IIS restriction enzyme

A novel approach is described that permits the introduction of unidirectional deletions into a cloned DNA fragment, in a precisely controlled manner. The method is based on the use of a special vector and a class-IIS restriction endonuclease, BspMI, which produces staggered cuts 4 and 8 nucleotides (nt) to the 3' from its recognition site 5'-ACCTGC-3'. The DNA fragment is inserted into the pUC19-based plasmid, which contains a unique BspMI recognition site, and the appropriate number of cleavage-and-deletion cycles is performed, each cycle removing 4 bp. Since the recognition site is not affected by the BspMI cleavage, no recloning of the DNA fragment is necessary. Each cycle consists of BspMI cleavage, removal of the 4-nt single-stranded cohesive ends with mung bean nuclease (MB), and blunt-end ligation to recircularize the plasmid. The shortened plasmid is reintroduced into the host, after one or after several such 4-bp deletion cycles. When DNA is inserted into the multiple cloning site in the lacZ alpha gene, the progress of 4-bp removal can be followed by determining the Lac phenotype, since removal of multiples of 3 bp retains the reading frame while other kinds of deletions distort (or restore) the reading frame. Loss of pre-existing restriction sites or creation of new ones also permits monitoring the progress of the deletion process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effective method of oligonucleotide-controlled mutagenesis of DNA fragments

A procedure has been designed for changing specific nucleotides in a DNA sequence with efficiency. The method involves the use of the specially constructed cloning vectors pBRS1, pHS1, and pHS2. These plasmids are derivatives of pBR322 in which the EcoRI-HindIII region has been replaced by synthetic duplexes carrying SmaI, HpaI and XhoI sites, in addition to EcoRI and HindIII sites. The DNA fragment to be mutagenized is cloned in pHS1 (or pBRS1, or pHS2) using restriction sites close to the SmaI and HpaI sites. The recombinant plasmid obtained is digested with one of these enzymes to produce double-stranded DNA with blunt ends. This linear DNA is a substrate for exonuclease III (or T4 DNA polymerase). The digestion under controlled conditions produces duplex with protruding single-stranded 5'-regions which include the site of the desired mutation. The synthesis of DNA by DNA-polymerase I (Klenow's fragment), primed in part by the synthetic oligonucleotide containing the desired mutation, leads to the linear heteroduplex. The closed circular heteroduplex is formed by ligation. After transformation into E. coli, DNA replication generates homoduplexes, some of which contain the mutation. Colony hybridization with the same 32P-labeled oligonucleotide is used to select mutants. The yield of the mutants is 10-20%. This technique can be extended to replicative form of M13 vectors. It can be also applied to any DNA sequence which has a unique site of restriction endonuclease generating blunt ends.