Improved procedure for the preparation of DNA restriction fragments suitable for sequencing (original) (raw)
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A simple and rapid procedure for sequencing long (40-kb) DNA fragments
Gene, 1987
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 pAA 13X by in vitro packaging, and subdivided by a series of overlapping ISI-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 ISI. The plasmids are extracted by a rapid method, arranged according to size, and used for supercoil sequencing with an ISI primer. Sequences of ISI-promoted deletions contain extensive overlaps that are connected further by restriction enzyme-generated deletions to give the complete 40-kb sequence. INTRODUCTION Large genomes are generally analyzed by dividing them into many overlapping 40-kb DNA fragments
Using restriction enzymes to improve sequencing by hybridization
2002
The expected number of n-long DNA sequences, which are consistent with a given SBH spectrum, grows exponentially with n. In this work we show that by incorporating data from a small number of restriction enzyme digestion assays (REs), the number of consistent sequences decreases signi cantly. We describe computational techniques that enable the reconstruction of sequences consistent with hybridization plus restriction enzymes (H+RE) spectra. The proposed SB(H+RE) scheme uniquely identi es much longer DNA sequences than SBH alone does.
Rapid DNA purification for restriction fragment length polymorphism analysis
Gene analysis techniques
This paper describes a method for isolation of DNA from blood samples involving a rapid chemical disintegration of proteins with 8 M urea and with a minimum of exposure to phenol. The DNA is further desalted and purified on Sephadex G-25 prepacked disposable columns. DNA isolated in this way was pure enough to be immediately cleaved by restriction enzymes.
Genome Research, 1993
Fast acquisition of unknown nucleotide sequences around a known sequence has important implication in molecular biology, especially in genome mapping. We have developed a method, termed restriction site polymerase chain reaction (RS-PCR), that utilizes specially designed primers that recognize, anneal, and sustain PCR. These primers, termed restriction site oligonucleotides (oligonucleotide primers specific for a given restriction enzyme recognition sequence or RSOs), could be generated corresponding to any restriction enzyme irrespective of the length of the recognition site and used as PCR primers corresponding to the unknown region of a DNA segment. In this method a first round of PCR is carried out in different tubes with a set of RSOs and a primer specific to the known region. A second round of PCR is then performed on the products of the first PCR with the same RSOs and another specific primer internal to the first one. Subsequently, the products of the last round of PCR are t...
BioTechniques, 1998
The determination of unknown DNA sequences around a known locus has important applications in molecular genetics, specifically in genomic walking and genome mapping. Several PCR-based methods have been reported to address this issue, but they often involve multiple, time-consuming steps. We have previously described a technique known as restriction site PCR (RSPCR) that allows sequence acquisition faster than the existing methods. The method involves PCR using four separate universal primers that are representative of given restriction enzyme sites (RS primers), and a specific primer from one end of the known sequence. We have now significantly improved the technique by mixing the four universal primers into one PCR tube with the first specific primer. This is followed by a nested PCR with the mixed RS primers and an internal specific primer, after which the product is sequenced by direct automated sequencing. The technique, called multiplex RS-PCR (mRS-PCR), is reproducible and can...
Acta biochimica Polonica, 2021
DNA indexing is based on a presynthesized library of oligonucleotide adaptors (256 in total), named indexers, and type-IIS restriction endonucleases. It enables amplification and direct analysis of large DNA fragments with low overall redundancy and without subcloning. Here, we describe a detailed protocol for PCR-based amplification of DNA fragments followed by DNA sequencing by indexer walking and provide helpful hints on its practical use. The proposed protocol can be applied to the sequencing of plasmids, cDNA clones, and longer DNA fragments. It can also be used for gap filling at the final stage of genome sequencing projects.
A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP
Nucleic Acids Research, 1997
Partial digestion of DNA fragments is a standard procedure for subcloning analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-methyl-dCTP into the PCR product to protect most of the restriction sites. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reaction done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditional methods. Furthermore, using fluorescein-labeled primers in the reaction, we were able to detect the fluorescein-labeled end fragments resulting from the enzyme digestion using a fluorimager or anti-fluorescein-AP antibody and thus determine the restriction maps.
Comptes Rendus de l'Académie des Sciences - Series III - Sciences de la Vie, 2000
A rapid and simple enzymatic method for the purification of a DNA fragment from a restriction digest was developed. The method is based on the two features of exonuclease III activity: digestion of DNA from a 3'-OH at blunt or recessed ends and failure to initiate digestion at DNA ends with four-base 3' overhangs. Herein, we establish a method for purification of a DNA restriction fragment without any physical separation via gel electrophoresis. The elimination of the ethidium bromide staining and ultraviolet irradiation steps should increase the quality and the safety of the purified DNA, a matter of major concern in the perspective of human gene therapy. In addition, since the method described does not use the visualization of the restriction fragments or their difference in size it can be used to purify a DNA fragment from a pool of DNA fragments with the same size even when microquantities of material are available. © 2000 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS
Two-dimensional agarose gel electrophoresis of restriction-digested genomic DNA
Methods, 1991
Two-dimensional agarose gel electrophoresis of genomic restriction endonuclease digestion products can physically separate fragments that comigrate in conventional agarose gels. To ensure that the results of each gel are reproducible, duplicate gels are prepared. The gels may be analyzed by Southern hybridization, or restriction fragments of known size may be preparatively extracted from agarose plugs. This method can be used to compare the digestion patterns of specific sequences from different organisms in a population, different cell types, or different developmental states.