Ligation-independent cloning of PCR products (LIC-PCR)
TL;DR: A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones, and the procedure is applied for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.
Abstract: A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5'-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3'-ends. The 3'-terminal sequence can be removed by the action of the (3'----5') exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5'-extending single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.
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TL;DR: An isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase is described.
Abstract: We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.
8,117 citations
TL;DR: Specific aspects of sample collection, cell lysis, nucleic acid extraction, PCR amplification, separation of amplified DNA, application of nucleic probes and data analysis are covered.
Abstract: After nearly 10 years of PCR-based analysis of prokaryotic small-subunit ribosomal RNAs for ecological studies it seems necessary to summarize reported pitfalls of this approach which will most likely lead to an erroneous description on the microbial diversity of a given habitat. The following article will cover specific aspects of sample collection, cell lysis, nucleic acid extraction, PCR amplification, separation of amplified DNA, application of nucleic probes and data analysis.
2,171 citations
TL;DR: Cl cloning of polymerase chain reaction products as blunt-ended fragments requires enzymatic processing to remove of the 3' overhang using an enzyme with 3' to 5' exonuclease activity.
Abstract: Although the polymerase chain reaction (1,2) (PCR) can be used to produce a large amount of a specific DNA from a complex source, cloning the PCR products has not proven to be straightforward. Restriction endonuclease sites are often incorporated into the oligonucleotide primers used for amplification, so that cleavage of the product will create sticky ends that can theoretically be ligated to an equivalently cut vector (3). Unfortunately, many restriction endonucleases fail to cleave when their recognition sequences are located within a few base pairs of the end of a DNA fragment (4, 5). Ligation-independent PCR cloning schemes (6, 7, 8) involve the addition of at least 12 bases to the 5' end of the primer, which can increase the cost of synthesis substantially when done routinely. Attempts to clone PCR products as blunt-ended fragments have been very inefficient, due to the template-independent terminal transferase activity of Taq polymerase, which results in the addition of a single nucleotide at the 3' end of the fragment (9, 10). This nucleotide is almost exclusively an adenosine, due to the strong preference of the polymerase for dATP (9). Thus, cloning the products as blunt-ended fragments requires enzymatic processing to remove of the 3' overhang using an enzyme with 3' to 5' exonuclease activity (11).
1,196 citations
TL;DR: Progresses ranging from the identification of novel genes and pathogens to the quantitation of characterized nucleotide sequences and some recent developments in instrumentation, methodology, and applications of the PCR are presented.
Abstract: The polymerase chain reaction (PCR) has dramatically altered how molecular studies are conducted as well as what questions can be asked. In addition to simplifying molecular tasks typically carried out with the use of recombinant DNA technology, PCR has allowed a spectrum of advances ranging from the identification of novel genes and pathogens to the quantitation of characterized nucleotide sequences. PCR can provide insights into the intricacies of single cells as well as the evolution of species. Some recent developments in instrumentation, methodology, and applications of the PCR are presented in this review.
1,178 citations
TL;DR: A new cloning method, sequence and ligation–independent cloning (SLIC), which allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing, which allows much greater versatility in the generation of recombinant DNA for the purposes of synthetic biology.
Abstract: We describe a new cloning method, sequence and ligation–independent cloning (SLIC), which allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing. SLIC mimics in vivo homologous recombination by relying on exonuclease-generated ssDNA overhangs in insert and vector fragments, and the assembly of these fragments by recombination in vitro. SLIC inserts can also be prepared by incomplete PCR (iPCR) or mixed PCR. SLIC allows efficient and reproducible assembly of recombinant DNA with as many as 5 and 10 fragments simultaneously. SLIC circumvents the sequence requirements of traditional methods and functions much more efficiently at very low DNA concentrations when combined with RecA to catalyze homologous recombination. This flexibility allows much greater versatility in the generation of recombinant DNA for the purposes of synthetic biology.
992 citations
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...The SLIC reactions described here that do not use RecA bear a resemblance to ligation-independent clonin...
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TL;DR: A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction, which significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Abstract: A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.
17,663 citations
TL;DR: Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmid molecules.
11,144 citations
TL;DR: In the chapter, the IG, which has the potential to form five hairpin structures, is represented schematically and important regions designated and the origin of replication of the (+) strand is stated most important to the functioning of M 13KO7.
Abstract: Publisher Summary This chapter discusses the production of single-stranded plasmid DNA. The chapter focuses on M13KO7 and its uses. The chapter reviews M13 biology; M13 mutants play a vital role in the functioning of M13KO7. M13 is a phage that contains a circular single-stranded DNA (ssDNA) molecule of 6407 bases packaged in a filamentous virion, which is extruded from the cell without lysis. It can infect only cells having F-pili to which it binds for entering the cell. The phage genome consists of nine genes encoding 10 proteins and contains an intergenic region of 508 bases. Phage replication consists of three phases: (1) ss-double strand (ds), (2) ds-ds, and (3) ds-ss. The intergenic region (IG) structure contains regions important for four phage processes: (1) the sequences necessary for the recognition of an ssDNA by phage proteins for its efficient packaging into viral particles; (2) the site of synthesis of an RNA primer that is used to initiate strand synthesis; (3) the initiation; and (4) the termination of (+) strand synthesis. In the chapter, the IG, which has the potential to form five hairpin structures, is represented schematically and important regions designated. The origin of replication of the (+) strand is stated most important to the functioning of M 13KO7.
2,491 citations
TL;DR: Results demonstrate that template instruction is not an absolute requirement for the catalysis of nucleotidyl transfer reactions by DNA polymerases.
Abstract: DNA polymerases catalyze the addition of deoxyribonucleotides onto DNA primers in a template-directed manner. The requirement for template instruction distinguishes these enzymes from other nucleotidyl transferases, such as terminal deoxynucleotidyl transferase, that do not utilize a template. An oligonucleotide substrate was used to characterize a novel, non-templated nucleotide addition reaction carried out by DNA polymerases from a variety of procaryotic and eucaryotic sources. The products of the reaction, in which a deoxyribonucleotide was added to the 3' hydroxyl terminus of a blunt-ended DNA substrate, were analyzed by electrophoresis on high resolution, denaturing polyacrylamide gels. DNA polymerase from Thermus aquaticus, polymerase alpha from chick embryo, rat polymerase beta, reverse transcriptase from avian myeloblastosis virus, and DNA polymerase I from Saccharomyces cerevisiae all carried out the blunt-end addition reaction. The reaction required a duplex DNA substrate but did not require coding information from the template strand. These results demonstrate that template instruction is not an absolute requirement for the catalysis of nucleotidyl transfer reactions by DNA polymerases.
877 citations
TL;DR: A method is described for directly cloning enzymatically amplified segments of genomic DNA into an M13 vector for sequence analysis and promises to be a rapid method for obtaining reliable genomic sequences from nanogram amounts of DNA.
Abstract: A method is described for directly cloning enzymatically amplified segments of genomic DNA into an M13 vector for sequence analysis. A 110-base pair fragment of the human beta-globin gene and a 242-base pair fragment of the human leukocyte antigen DQ alpha locus were amplified by the polymerase chain reaction method, a procedure based on repeated cycles of denaturation, primer annealing, and extension by DNA polymerase I. Oligonucleotide primers with restriction endonuclease sites added to their 5' ends were used to facilitate the cloning of the amplified DNA. The analysis of cloned products allowed the quantitative evaluation of the amplification method's specificity and fidelity. Given the low frequency of sequence errors observed, this approach promises to be a rapid method for obtaining reliable genomic sequences from nanogram amounts of DNA.
783 citations