A novel deletion found during cloning of a synthetic palindromic DNA

A 212-bp palindromic DNA comprising two copies of the left end of bacteriophage Mu was assembled from chemically synthesized oligonucleotides and inserted into plasmid pUC9. When cloned and propagated in Escherichia coli, the palindrome was found to be unstable and was generally lost. However, in a few cases, a precise, asymmetric deletion of one half of the insert was observed. This pattern of deletion suggests that the symmetry axis region of the palindrome was involved as recognition site in the deletion process.     

A rapid method for cloning and sequencing variable-region genes of expressed immunoglobulins

A rapid procedure is described for cloning immunoglobulin V region genes from cells that express them. cDNA is synthesized from mRNA template using primers homologous to the immunoglobulin constant-region genes. Blunt-ended, double-stranded cDNA is obtained by sequential addition of enzymes to a single tube. The cDNA is inserted directly into the M13 vector, which is screened by plaque lifting for the presence of specific inserts. Screening probes can be generated from 32P-labeled single-stranded cDNAs generated from primers different from those used for cloning, or alternatively, from previously cloned V or C gene segments. The ease of cloning a cDNA V region is directly related to the abundance of Ig-specific mRNA within the cell of interest. This method minimizes the number of steps and the time needed to obtain accurate and complete sequences of any expressed Ig V region gene.     

A method for cloning mixtures of long,synthetic oligodeoxynucleotides

A method is described for cloning synthetic oligodeoxynucleotides, which can theoretically be of any length. The method requires only a single oligodeoxynucleotide strand and a vector with two unique restriction sites, one of which is for an enzyme that generates 3′ protruding ends. A mixture of unpurified oligonucleotides containing a wild-type genetic regulatory sequence of the Escherichia coli gnd gene and two mutations of it was cloned into a plasmid carrying a gnd-lacZ protein fusion. Individual cloned oligonucleotides were readily identified by direct DNA sequencing of plasmid templates. The method is rapid, efficient, and has application to gene synthesis and site-directed mutagenesis.     

A vector for sequencing long (40-kb) DNA fragments

An improved vector (pAA113M) has been constructed for sequencing long (40-kb) DNA fragments. The DNA fragment is cloned in the tet gene of the cosmid and subdivided into numerous overlapping segments by IS1-promoted deletions. Plasmids bearing these deletions are fractionated by gel electrophoresis, and shortened further from the opposite end by treatment with specific restriction endonucleases. Thus, a series of short overlapping segments, spread across the entire length of the fragment, become fused to IS1. Each segment can then be sequenced by the dideoxy method using an IS1 primer. The plasmids can replicate either from their normal origin or, in the presence of a filamentous helper phage, from the M13 origin. Hence, each segment can be sequenced as either double-stranded DNA or single-stranded DNA. Sequences of IS1-promoted and restriction enzyme-generated deletions contain overlaps that can be used to assemble the complete 40-kb sequence.     

A new method of sequencing DNA

An entirely new method of sequencing DNA has been devised that does not use electrophoresis, radioactivity, or fluorescence. The method works by measuring pyrophosphate generated by the DNA polymerization reaction. DNA and DNA polymerase are held by a DEAE-Sepharose column and solutions containing different dNTPs are pumped through. The pyrophosphate generated is measured continuously by a device consisting of a series of columns containing enzymes covalently attached to Sepharose. The alternating copolymer poly(dA.dT) is sequenced as an illustration of the method. Future improvements that will facilitate automation are discussed.     

A PCR-after-ligation method for cloning of multiple DNA inserts

Here we present a novel and simple PCR-after-ligation method for efficient assembly of multiple DNA inserts. After initial ligation of multiple inserts and vector, the ligation mixture is used as template for a PCR using a pair of primers flanking the cloning sites on the vector. The fragment with correct size is gel purified and inserted into the vector by conventional two-way ligation. With this method, a recombinant plasmid containing four DNA inserts was correctly constructed. As a control, all of the constructs obtained directly from DNA ligation were found to be self-ligation of the vector.     

A novel type of cloning vectors for ultrarapid chemical degradation sequencing of DNA

The construction and evaluation of a novel type of chemical sequencing vectors (pCSV) is described. These small plasmids bear a unique asymmetric restriction site suitable for direct single-end labeling by filling-in polymerization with a selected radiolabeled nucleoside triphosphate. Thus, a secondary cleavage reaction or segregation step is rendered superfluous. Sequencing gels can be read unambiguously starting from the 3′ penultimate nucleotide. pCSV03 and pCSV27 are prototypes of such sequencing plasmids based on BstEII as the labeling site. In its proximity, restriction sites are present that allow subcloning of the DNA fragments to be sequenced. Approaches to random and progressive sequencing using these vectors are discussed. pCSV31 has two Tth111I sites that allow single-end labeling of inserts on either strand by the use of different labeled nucleoside triphosphates. Thus, bidirectional sequencing of larger inserts (>500 bp) is possible.     

A simple and rapid procedure for sequencing long (40-kb) DNA fragments

A simple procedure has been developed for sequencing long (40-kb) DNA fragments by the dideoxy method. The fragment is cloned in the sequencing cosmid pAA113X by in vitro packaging, and subdivided by a series of overlapping IS1-promoted deletions. The deletions are isolated by positive selection for galactose resistance. Plasmids from several thousand galactose-resistant colonies are fractionated on an agarose gel, and DNA from each fraction is restricted with enzymes (such as SphI and SalI) to shorten each deletion from the opposite end. As a result, a series of short overlapping segments, spread across the entire length of the fragment, are fused to IS1. The plasmids are extracted by a rapid method, arranged according to size, and used for supercoil sequencing with an IS1 primer. Sequences of IS1-promoted deletions contain extensive overlaps that are connected further by restriction enzyme-generated deletions to give the complete 40-kb sequence.     

A high-throughput method for cloning and sequencing human immunodeficiency virus type 1 integration sites

Integration of retroviral DNA is nonspecific and can occur at many sites throughout chromosomes. However, the process is not uniformly distributed, and both hot and cold spots for integration exist. The mechanism that determines target site specificity is not well understood. Because of the nonspecific and widespread nature of integration, studies analyzing the mechanism and factors that control target site selection require the collection and analysis of a large library of human immunodeficiency virus type 1 (HIV-1) proviral clones. Such analyses are time-consuming and labor-intensive using conventional means. We have developed an efficient and high-throughput method of sequencing and mapping a large number of independent integration sites in the absence of any selection or bias. The new assay involves the use of a modified HIV-1 (NL-Mme) containing a type IIS restriction site, MmeI, at the right end of viral DNA. Digestion of genomic DNA from NL-Mme-infected cells generated viral DNA-containing fragments of a discrete size. Subsequent ligation-mediated PCR yielded short integration site fragments termed Int-tags, which were concatemerized for determining multiple integration sites in a single sequencing reaction. Analysis of chromosomal features and sequence preference associated with integration events confirmed the validity of the new high-throughput assay. The assay will aid the effort in understanding the mechanisms of target site selection during HIV-1 DNA integration, and the described methodology can be adapted easily to integration site studies involving other retroviruses and transposons.     

A method for sequencing single-stranded cloned DNA in both directions

A DNA sequencing method has been developed whereby DNA that has been cloned in a single-stranded bacteriophage vector can be sequenced from both ends. The method involves first making a minus-strand sense template from a single-stranded insert in the vector MJ3mp2 using a flanking primer, and then sequencing the synthesized template using the dideoxynucleotide termination method (Sangeret al., 1977, 1980) with a second primer. Special conditions are described under which the first primer is easily removed after making the templat% and sequencing in the opposite direction can be done in the normal way (Sangeret al., 1980) without separating the double strands. This method renders it possible to read up to twice the amount of sequence data from a long insert and also to check short inserts by producing complementary sequence patterns.     

A versatile primer for DNA sequencing in the M13mp2 cloning system

A primer for DNA sequencing by the chain-termination method in the M13mp2 cloning system was constructed and amplified. The primer was isolated as an EcoRI/AluI restriction fragment. After conversion of the AluI end into an EcoRI end the fragment was cloned in pBR325 from which it can be recovered by cleavage with EcoRI. The primer hybridizes to the single-stranded DNA of the mature M13mp2 phage next to the site of insertion thereby directing DNA synthesis along the inserted DNA.     

A general method for cloning DNA fragments in multiple copies

A general method for the cloning of DNA fragments in tandem arrays is presented. Synthetic directional adapters are attached to the fragment ends to establish complete control over fragment orientation during ligation. Use of these directional adapters thereby ensures the production of direct repeats, an arrangement essential for clone stability. The technique involves a protocol that is both less complex and less time-consuming than previous methods. Using this technique, a 10-min ligation has yielded a plasmid containing 20 copies of a 151-bp fragment.     

A ligation-independent cloning method using nicking DNA endonuclease

Using nicking DNA endonuclease (NiDE), we developed a novel technique to clone DNA fragments into plasmids. We created a NiDE cassette consisting of two inverted NiDE substrate sites sandwiching an asymmetric four-base sequence, and NiDE cleavage resulted in 14-base single-stranded termini at both ends of the vector and insert. This method can therefore be used as a ligation-independent cloning strategy to generate recombinant constructs rapidly. In addition, we designed and constructed a simple and specific vector from an Escherichia coli plasmid back-bone to complement this cloning method. By cloning cDNAs into this modified vector, we confirmed the predicted feasibility and applicability of this cloning method.     

A phage T4 in vitro packaging system for cloning long DNA molecules.

Recombinant plasmid DNAs containing long DNA inserts that can be propagated in Escherichia coli would be useful in the analysis of complex genomes. We tested a bacteriophage T4 in vitro DNA packaging system that has the capacity to package about 170 kb of DNA into its capsid for cloning long DNA fragments. We first asked whether the T4 in vitro system can package foreign DNA such as concatemerized lambda imm434 DNA and phage P1-pBR322 hybrid DNA. The data suggest that the T4 system can package foreign DNA as efficiently as the mature phage T4 DNA. We then tested the system for its ability to clone foreign DNA fragments using the P1-pBR322 hybrid vectors constructed by Sternberg [Proc. Natl. Acad. Sci. USA 87 (1990) 103-107]. E. coli genomic DNA fragments were ligated with the P1 vectors containing two directly oriented loxP sites, and the ligated DNA was packaged by the T4 in vitro system. The packaged DNA was then transduced into E. coli expressing the phage P1 cyclization recombination protein recombinase to circularize the DNA by recombination between the loxP sites situated at the ends of the transduced DNA molecule. Clones with long DNA inserts were obtained by using this approach, and these were maintained as single-copy plasmids under the control of the P1 plasmid replicon. Clones with up to about 122-kb size inserts were recovered using this approach.     

A freeze-squeeze method for recovering long DNA from agarose gels

Long DNA can be recovered from agarose gels after electrophoresis by freezing the gel slices and manually squeezing out liquid containing the DNA. With this method the recoveries of phage T₇ DNA (molecular weight 25 × 10⁶) and the open and closed forms of circular phage PM2 DNA (molecular weight 6 × 10⁶) were about 70%. Sedimentation analysis shows that the extruded DNA has not sustained double- or single-stranded breaks. The extruded DNA can be used without further purification as substrate for the restriction endonuclease Hind_II,III, from Hemophilus influenzae, for DNA·DNA hybridization and for electron microscopy.