Mapping and ordering of fragments of BK virus DNA produced by restriction endonucleases.

A total of 51 restriction sites were recognized within the BK virus genome by the combination of 10 different restriction endonucleases. These sites were mapped and oriented relative to one another as well as to the five fragments generated by the digestion of BK virus DNA with HindIII and EcoRI. The result was a comprehensive physical map suitable for in-depth characterization of the functions of BK virus at the molecular level.     

A rapid two-dimensional fractionation of DNA restriction fragments

Genomic DNA that has been digested with a restriction endonuclease and fractionated by electrophoresis on an agarose gel can be recovered on glass-fiber filters by a new blotting scheme. The DNA fragments in each fraction are then digested with a second restriction nuclease and then separated on a slab gel, resulting in a two-dimensional display of the restriction fragments. This rapid fingerprinting technique is useful in the analysis of complex genomes and in the isolation and cloning of particular sequences.     

Mapping the order of DNA restriction fragments

A straightforward method was designed for mapping the order of DNA restriction fragments obtained by a double and two single digestions, without the necessity of using a computer or a radioactive label. All possible solutions compatible with a pre-set level of error in the determination of sequence lengths are obtained. The primary assumptions are given, and the appropriate modifications of the algorithm are presented as a function of any assumptions one is unable (or unwilling) to make. Use of the method in connection with end-labeled fragments is also described.     

A computer program for determining the size of DNA restriction fragments

A computer program has been developed for determining the sizes of DNA restriction fragments from their electrophoretic mobilities. A parabola is fitted to the mobilities of a set of standard fragments of known sizes and the sizes of the unknown fragments are then calculated from the fitted curve. This procedure is shown to yield estimated sizes which are accurate to within a few percentage, as judged by experiments with fragments obtained by digestion of pBR322 with the restriction endonuclease HaeIII. The program, which is written in BASIC, is simple to use and is very much faster than the graphical method that it replaces.     

Repeating restriction fragments of human DNA.

Human DNA digested with Hae III showed multiple repeats of a 170 base pair fragment. The most prominent band was the 340 base pair dimer, estimated to be 0.8% of the entire genome. Eco R1 and Hha I yielded fragments with similar electrophoretic mobility to the Hae III dimer. In each case this band was markedly enriched in DNA reassociating at a 0t of less than or equal to 1. Hybridization of the Hae III dimer to gels eluted on to filters demonstrated that the multiple Hae III fragments and Eco R1 fragments contained compatible sequences. These sequences may comprise a distinct subclass of DNA.     

Multi-priming sequencing: a DNA sequencing method involving restriction enzyme-digested DNA fragments as primers.

An improved strategy for fluorescence-labeled dideoxy chain termination sequencing involving restriction enzyme-digested DNA fragments as primers, which are prepared from the DNA to be sequenced, is described. By using modified nucleoside triphosphates for strand protection in chain termination reactions, newly synthesized chains were detached from a primer at the regenerated recognition site by means of suitable restriction enzyme digestion. The digests could be analyzed with commercial automated DNA sequencers. Thus, by using restriction DNA fragments (double-stranded) as primers, sequence information was obtained from both "minus" and "plus" single-stranded DNA templates without subcloning. Nor is the synthesis of oligonucleotide primers needed. This method, named "Multi-Priming Sequencing," was proven to be time-saving, economical, and effective compared to conventional methods.     

Electrophoresis of DNA restriction fragments in poly-N-acryloyl-tris gels

Poly-N-acryloyl-tris(hydroxymethyl)aminomethane (NAT) gels were evaluated as a matrix for DNA electrophoresis. The resolution of DNA restriction fragments in three poly(NAT)-N,N'-methylenebisacrylamide (Bis) gels (4, 5, and 6%) was compared with the resolution in polyacrylamide (AA)-Bis gels of the same percentage. Poly(NAT) gels were found to give a substantially improved separation of DNA fragments larger than 200 bp. In contrast to poly(AA) gels, DNA fragments of up to 4 kbp were well resolved in the new matrix. By pulse-field electrophoresis the useful separation range of poly(NAT) gels was expanded to at least 23 kbp. For DNA fragments below 10 kbp, the resolution was better than that in a 0.7% agarose gel. Thus poly(NAT) gels are most suitable for the electrophoretic separation of DNA molecules whose size is out of the optimal fractionation range of poly(AA) or agarose gels.     

95 Orientational ordering of short DNA fragments in the polymer matrix

As a vital part of modern nanotechnology, nanofabrication aims to develop nanoscale components and nanomaterials in large quantities at relatively low cost. The promising strategy is the bottom-up self-assembly techniques of chemical assembly and molecular recognition to bring together individual atoms, molecules, or supramolecular building blocks to form useful constructs. The DNA-DNA self-assembly seems to be the key point regulating the polymer composites formation. We address the mixture of a flexible polymer with short double-strand DNA fragments, where the persistence length is in comparable with the contour length of the molecule. We investigate the conditions affecting the orientational order formation of short double-strand DNA fragments, immersed in the flexible polymer. It is shown that short double-strand DNA fragments exhibit the formation of a liquid crystalline ordered phase, in dependence on the value of the Flory–Huggins parameter, aspect ratio , and the attraction energy (Mamasakhlisov et al., 2009; Todd et al., 2008) of the double strand DNA molecules and volume fraction of polymer.     

A method for sequencing restriction fragments with dideoxynucleoside triphosphates.

A rapid enzymatic approach is described for the sequence analysis of a 5' terminally labelled restriction fragment. It involves limited nicking of the strands of the molecule throughout the sequence by pancreatic DNAase I. The 3' hydroxyl groups exposed by each nick are then used to prime chain extension by DNA polymerase I in four separate reactions. Each reaction uses one of the four chain terminating dideoxynucleoside triphosphates (ddNT-PSs), together with the four deoxynucleoside triphosphates (dNTPs). In a single reaction all the 3' ends are terminated in positions of the same base, which is different for each of the four reactions. When the products of these reactions are resolved by gel electrophoresis according to size, a sequence can be deduced from the pattern of radioactive bands. Sequences can be determined onwards from 10-20 residues from the 5' labelled end. The length of sequence which can be determined is only limited by the resolution of the gel.     

A method for generating sticky-end PCR products which facilitates unidirectional cloning and the one-step assembly of complex DNA constructs

We have developed and tested a method for the restriction enzyme-independent generation of sticky-end PCR products. The method is suitable for use with a proof-reading polymerase such as pfu, or any other heat-stable polymerase which produces a blunt-end product. The technique can be used to achieve unidirectional cloning of PCR products with an efficiency greater than 90%. Because the sequences of the sticky ends are defined by the user and potentially can be of any length, the method can also be exploited for the one-step construction of recombinant plasmids from multiple functional cassettes, without the use of restriction enzymes.     

A novel method for producing partial restriction digestion of DNA fragments by PCR with 5-methyl-CTP.

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.     

Interaction of the MvaI restriction enzyme with synthetic DNA fragments

The cleavage of synthetic DNA duplexes by the restriction endonuclease MvaI has been studied. The main result of the cleavage experiments is that MvaI cleaves unmodified duplexes in two single strand scissions in separate events and that the two strands are cleaved at significantly different rates. One strand nicks within the recognition site do not affect the cleavage. Furthermore, neither a pyrophosphate internucleotide bond modification in one strand nor the absence of one phosphate group at the central dA-residue of the recognition site do inhibit the cleavage of the second strand.     

Two-dimensional electrophoretic display of restriction fragments from genomic DNA

We have developed a procedure for the resolution of restriction enzyme digests of mammalian genomic DNA in two dimensions. Fragments from a first digestion are separated on a column of purified agarose containing a second restriction enzyme in the absence of the divalent cation required for enzyme activity. After enzyme activation and digestion, the fragments are resolved on an agarose slab gel. We have digested rat genomic DNA and found in the ethidium-stained pattern a variety of features which have not been described previously.     

Quantitation of specific fragments in DNA restriction digests: application to the cohesive fragments in lambda DNA digests

A procedure for the quantitation of reactions between specific members of a set of DNA restriction fragments is presented. Quantitation of the cohesive fragments in NruI nuclease digests of lambda DNA is used as an example. Restriction fragments are resolved on agarose gels and their amounts are estimated from densitometer scans of photographic negatives of ethidium bromide-stained gels. A linear relationship is found between the peak height of given fragment on the scan and the logarithm of the molecular weight of the fragment, arising in part from the stoichiometry of the digest; this relationship allows simple interpolation between the peak heights of the nonreacting fragments in each gel lane to determine the theoretical maximal amount of each reactive fragment in that gel lane. Similar procedures should be applicable to enzymatic ligation or to site-specific cleavage of specific restriction fragments or to autoradiographic detection of the fragments. Since each lane of the gel is analyzed independently, the method is largely self-correcting for variations in amounts applied to the gel.     

A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity

A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.