Hydrophobic properties of porcine fibronectin and its functional domains

The relations between surface hydrophobicities and binding properties of the functional domains of porcine plasma fibronectin were investigated. Porcine plasma fibronectin as well as human plasma fibronectin was adsorbed on a hydrophobic column with butyl or phenyl ligands in the presence of 0.5 M ammonium sulfate, and recovered in a single peak by decreasing the concentration of ammonium sulfate to 0 M, indicating that both fibronectins have very high surface hydrophobicities. On digestion with thermolysin, porcine plasma fibronectin yielded five fragments (140-150, 43, 25, 17, and 14 kDa) similar to those reported for human fibronectin, although porcine fibronectin was more resistant to the digestion than human fibronectin. The three heparin-binding fragments were found to have a wide range of surface hydrophobicities, the 140-150 kDa fragment having the lowest, the 25 kDa fragment a higher, and the 14 kDa fragment the highest among all the fragments. The 43 kDa collagen-binding and 17 kDa fragments had surface hydrophobicities as high as that of fibronectin. It is noteworthy that the 43 kDa collagen-binding fragment contributes to the high surface hydrophobicity of intact fibronectin in spite of the high content of carbohydrates.     

A new method for the determination of critical polyethylene glycol concentration for selective precipitation of DNA fragments

Separation strategies based on size-selective precipitation of DNA fragments with polyethylene glycol (PEG) have been used for achieving desired DNA interval in automated sample preparation for next-generation sequencing. By varying PEG concentration, DNA fragments of different sizes can be precipitated onto surfaces of carboxyl-coated paramagnetic particles selectively, and therefore, the desired DNA interval can be obtained. However, one of the crucial points in this approach is to determine the critical PEG concentration for DNA fragment of a certain size. The aim of this work was to develop a convenient and reliable method for accurately determining the critical PEG concentration. In our method, at a fixed concentration of sodium chloride (NaCl), recovered DNA samples obtained with different PEG concentrations were directly quantified, and their concentrations as a function of the PEG concentration were fitted by the logistic function. The critical PEG value was easily and accurately determined from the fitted logistic function. The repeatability and stability of the critical PEG value were assessed, showing an excellent reliability of the method. Based on this method, critical PEG values of different-size DNA fragments were determined at different NaCl concentrations. The effectiveness of the method was also demonstrated by selective precipitation of DNA fragments.     

Pulsed field gradient electrophoresis of DNA digested in agarose allows the sizing of the large duplication unit of a surface antigen gene in trypanosomes

Intact chromosome-sized DNA molecules from eukaryotes may be prepared by performing lysis and enzymic deproteinization on cells embedded in agarose [Schwartz and Cantor, Cell 37 (1984), 67-75]. Here we show that DNA prepared by this method may be cut with restriction enzymes, or modified with site-specific methylases and cut by DpnI. As the DNA remains incorporated in the gel matrix, shear degradation of large fragments is avoided. The fragments can then be sized by conventional or pulsed field gradient gel electrophoresis. Phage lambda genomic oligomers are used as size markers, allowing the estimation of fragment sizes up to about 1200 kb. We apply these techniques to show that activation of the telomeric gene encoding variant surface antigen 1.3 in Trypanosoma brucei strain 427, involves the duplication of a DNA segment that starts between 29 and 42 kb upstream of the gene and to assign a chromosomal fragment into which the duplicated 1.3 gene may have transposed.     

An algorithmic approach to multiple complete digest mapping.

Multiple Complete Digest (MCD) mapping is a method of determining the locations of restriction sites along a target DNA molecule. The resulting restriction map has many potential applications in DNA sequencing and genetics. In this work, we present a heuristic algorithm for fragment identification, a key step in the process of constructing an MCD map. Given measurements of the restriction fragment sizes from one or more complete digestions of each clone in a clone library covering the molecule to be mapped, the algorithm identifies groups of restriction fragments on different clones that correspond to the same region of the target DNA. Once these groups are correctly determined the desired map can be constructed by solving a system of simple linear inequalities. We demonstrate the effectiveness of our algorithm on real data provided by the Genome Center at the University of Washington.     

The influence of fluorescent dye structure on the electrophoretic mobility of end-labeled DNA.

Over the past 10 years, fluorescent end-labeling of DNA fragments has evolved into the preferred method of DNA detection for a wide variety of applications, including DNA sequencing and PCR fragment analysis. One of the advantages inherent in fluorescent detection methods is the ability to perform multi-color analyses. Unfortunately, labeling DNA fragments with different fluorescent tags generally induces disparate relative electrophoretic mobilities for the fragments. Mobility-shift corrections must therefore be applied to the electrophoretic data to compensate for these effects. These corrections may lead to increased errors in the estimation of DNA fragment sizes and reduced confidence in DNA sequence information. Here, we present a systematic study of the relationship between dye structure and the resultant electrophoretic mobility of end-labeled DNA fragments. We have used a cyanine dye family as a paradigm and high-resolution capillary array electrophoresis (CAE) as the instrumentation platform. Our goals are to develop a general understanding of the effects of dyes on DNA electrophoretic mobility and to synthesize a family of DNA end-labels that impart identically matched mobility influences on DNA fragments. Such matched sets could be used in DNA sequencing and fragment sizing applications on capillary electrophoresis instrumentation.     

A procedure for the construction of linear restriction fragment maps of a 50- to 100-kb DNA.

A simple and effective procedure for the construction of linear restriction fragment maps was developed. Using a two-enzyme digestion, two-dimensional (2-D) electrophoresis procedure, all the restriction fragments in a 50- to 100-kb DNA can be individually resolved and displayed on a 2-D plane. This 2-D gel pattern, with appropriate markers, provides a fixed set of x, y coordinates for each fragment obtained from the single and double digestion as well as the relationship between the two steps. A matrix is constructed from the 2-D pattern. The vertical column shows all the singly digested individual fragments and their sizes obtained from each restriction enzyme treatment, and the dividing horizontal row shows all the doubly digested DNA fragments and their sizes after treatment with two enzymes. The order of arrangement is always from the smallest to the largest fragments. Using this matrix, two linear DNA restriction maps for these two enzymes can be simultaneously constructed in a self-reconfirming manner. As examples for this procedure, we describe the construction of two linear restriction fragment maps, a combination of EcoRI and BamHI digestion as well as a combination of EcoRI and HindIII digestion of lambda-phage DNA.     

A method for separating DNA fragments by electrophoresis in polyacrylamide concentration gradient slab gels

Electrophoresis on slab gels containing a linear gradient of polyacrylamide concentration has been used to separate DNA fragments obtained by restriction of viral DNAs. A simple method of preparing gradient gels using a sucrose density-gradient mixer and preexisting slab gel apparatus is described. DNA fragments of molecular weights 7 × 10⁴–14 × 10⁶ have been fractionated on gels of 3.5–7.5% and 2.5–7.5% acrylamide concentration. In addition to the wide range of fragment sizes which may be run on a single gel, a further advantage of the system is that much sharper bands are obtained compared to conventional constant concentration gels, thus improving resolution.In the molecular-weight range below 5 × 10⁶, for bands whose terminal velocities in the polyacrylamide concentration gradient approach zero, an approximately linear relationship holds between the logarithms of the molecular weights of the fragments and the logarithms of the distances they have migrated in the gel. Thus, by choosing a suitable upper limit to the concentration gradient, the gel system provides a method for estimating approximate molecular weights of unknown DNA fragments, by comparing their mobilities to known standards.     

A quantile method for sizing optical maps.

Optical mapping is an integrated system for the analysis of single DNA molecules. It constructs restriction maps (noted as "optical map" ) from individual DNA molecules presented on surfaces after they are imaged by fluorescence microscopy. Because restriction digestion and fluorochrome staining are performed after molecules are mounted, resulting restriction fragments retain their order. Maps of fragment sizes and order are constructed by image processing techniques employing integrated fluorescence intensity measurements. Such analysis, in place of molecular length measurements, obviates need for uniformly elongated molecules, but requires samples containing small fluorescent reference molecules for accurate sizing. Although robust in practice, elimination of internal reference molecules would reduce errors and extend single molecule analysis to other platforms. In this paper, we introduce a new approach that does not use reference molecules for direct estimation of restriction fragment sizes, by the exploitation of the quantiles associated with their expected distribution. We show that this approach is comparable to the current reference-based method as evaluated by map alignment techniques in terms of the rate of placement of optical maps to published sequence.     

Control of partial digestion combining the enzymes dam methylase and MboI.

A method is described which allows the preparation of reproducible partial digests without previous establishment of the incubation conditions. It is based on a combined application of dam methylase and the restriction endonuclease MboI, both recognizing the sequence 5'-GATC-3' but MboI unable to cut the methylated site. Due to their competition for the same substrate the DNA is partially digested, with the size of the resulting fragments strongly dependent on the ratio of enzymes. The Km of the dam methylase was determined to be 115 ng DNA/microliters indicating a variance in fragment sizes generated at low DNA-concentrations. This effect is minimized above 150 ng/microliters. Any influence of digestion time is avoided, because the reaction runs until complete modification of all sites. The dependence on enzyme concentration and presence of agarose was checked. Knowledge of these parameters allows an accurate prediction of fragment sizes generated at different conditions. The technique was successfully used to construct libraries from different sources, in particular chromosome-specific libraries from small amounts of flow-sorted material.     

Map of restriction sites on bacteriophage T4 cytosine-containing DNA for endonucleases bamHI, BglII, KpnI, PvuI, SalI, and XbaI.

A complete map of the cleavage sites of restriction endonucleases BamHI, BglII, KpnI, PvuI, SalI, and XbaI was determined for the cytosine-containing DNA of a bacteriophage T4 alc mutant. The 56 sequence-specific sites were assigned map coordinates based on a least-squares analysis of measured fragment lengths. Altogether, the lengths of 118 fragments from single and double enzyme digestions were measured by electrophoresis of the fragments in agarose gels. DNA fragments of known sequence or DNA fragments calibrated with fragments of known sequence were used as standards. The greatest deviation between an experimentally measured fragment length and its computed map coordinates was 3.0%; the average deviation was 0.8%. The total length of the wild-type T4 genome was calculated to be 166,200 base pairs.     

Using molecular sizes of simple sequence repeats vs. discrete binned data in assessing probability of ancestry: application to maize hybrids

Most inferential methods for profiling genotypes based upon the use of DNA fragments use molecular-size data transcribed into discrete bins, which are intervals of DNA fragment sizes. Categorizing into bins is labor intensive with inevitable arbitrariness that may vary between laboratories. We describe and evaluate an algorithm for determining probabilities of parentage based on raw molecular-size data without establishing bins. We determine the standard deviation of DNA fragment size and assess the association of standard deviation with fragment size. We consider a pool of potential ancestors for an index line that is a hybrid with unknown pedigree. We evaluate the identification of inbred parents of maize hybrids with simple sequence repeat data in the form of actual molecular sizes received from two laboratories. We find the standard deviation to be essentially constant over the molecular weight. We compare these results with those of parallel analyses based on these same data that had been transcribed into discrete bins by the respective laboratories. The conclusions were quite similar in the two cases, with excellent performance using either binned or molecular-size data. We demonstrate the algorithm's utility and robustness through simulations of levels of missing and misscored molecular-size data.     

Rapid DNA fingerprinting of pathogens by flow cytometry

BACKGROUND: A new method for rapid discrimination among bacterial strains based on DNA fragment sizing by flow cytometry is presented. This revolutionary approach combines the reproducibility and reliability of restriction fragment length polymorphism (RFLP) analysis with the speed and sensitivity of flow cytometry. METHODS: Bacterial genomic DNA was isolated and digested with a rare-cutting restriction endonuclease. The resulting fragments were stained stoichiometrically with PicoGreen dye and introduced into an ultrasensitive flow cytometer. A histogram of burst sizes from the restriction fragments (linearly related to fragment length in base pairs) resulted in a DNA fingerprint that was used to distinguish among different bacterial strains. RESULTS: Five different strains of gram-negative Escherichia coli and six different strains of gram-positive Staphylococcus aureus were distinguished by analyzing their restriction fragments with DNA fragment sizing by flow cytometry. Fragment distribution analyses of extracted DNA were approximately 100 times faster and approximately 200,000 times more sensitive than pulsed-field gel electrophoresis (PFGE). When sample preparation time is included, the total DNA fragment analysis time was approximately 8 h by flow cytometry and approximately 24 h by PFGE. CONCLUSIONS: DNA fragment sizing by flow cytometry is a fast and reliable technique that can be applied to the discrimination among species and strains of human pathogens. Unlike some polymerase chain reaction (PCR)-based methods, sequence information about the bacterial strains is not required, allowing the detection of unknown, newly emerged, or unanticipated strains.     

Nuclear genes coding for four subunits of the yeast ubiquinol-cytochrome c reductase complex are present in single copies in the haploid genome and at least two of these are located on different chromosomes

Summary Genes coding for the 40 kilodaltons (kDa), 17-kDa, 14-kDa and 11-kDa subunits of the ubiquinol-cytochrome c reductase in yeast are present in single copies in the haploid genome. We have mapped each gene to a unique genomic environment and demonstrate that integration of cloned segments into nuclear DNA by homologous crossing-over with the endogenous gene results in the replacement of the corresponding chromosomal restriction fragment by fragments of predicted sizes. Chromosomal mapping, carried out by the procedure of Falco and Botstein 1983, indicates that the gene for the 17-kDa subunit lies on chromosome VI and that for the 11-kDa subunit on chromosome XII.     

Fractionation and molecular weight determination of deoxyribonucleic acid fragments using agarose column chromatography

DNA fragments of several sizes have been produced by shearing E. coli DNA under different pressures. These fragments have been used to demonstrate that column chromatography on agarose Bio-Gel A-15M can provide a rapid, inexpensive fractionation and sizing method for single-stranded nucleic acids having masses between 10⁵ and 10⁶ daltons. Both chromatographic and electrophoretic analysis of the sheared DNA indicated that discrete fragment populations were produced at each shearing pressure and that these fragments were distributed essentially symmetrically around a mean piece size. The average molecular weight of the several DNA fragment distributions was determined electrophoretically by comparison with standard DNA fragments obtained from restriction endonuclease cleavage of SV40 viral DNA. The molecular weights of the denatured, sheared fragments (single-stranded) ranged from 1.25 × 10⁵ to 7.4 × 10⁵. The single-stranded DNA fragments were chromatographed over agarose Bio-Gel A-15M and a linear relationship was found to exist between the mobilities and logarithms of the molecular weights. Readily available tRNA, 5s RNA, and φX174 single-stranded circular DNA chromatographed at the extremes of the linear relationship and could be used to calibrate the column chromatography.     

Bends in SV40 DNA: use of mutagenesis to identify the critical bases involved.

Five fragments of DNA exhibiting sequence directed bends were isolated from the Simian Virus 40 genome using a two-dimensional polyacrylamide gel fractionation. The bend sites were mapped for each fragment using the circular permutation test. All five sites have multiple, short runs of A residues with helical spacing typical of other bent fragments. Base pairs important for the bends were determined for one fragment by utilizing a random, single base pair mutagenesis. Of 28 mutants with decreased or increased bends, 14 had alterations that could be interpreted to affect the spaced runs of A residues, supporting their role in bends as predicted by the ApA wedge model. One major mutation was not explainable by existing models. The remaining minor mutations may only be due to small, local DNA conformational changes in the surrounding B-DNA.     

Alterations in DNA helix stability due to base modifications can be evaluated using denaturing gradient gel electrophoresis

DNA molecules that differ by a single base-pair can be separated by denaturing gradient gel electrophoresis due to the sequence-specific melting properties of DNA. Base modifications such as methylation are also known to affect the melting temperature of DNA. We examined the final position of DNA fragments containing either 5-methyl-cytosine or 6-methyl-adenine in denaturing gradient gels. The presence of a single methylated base within an early melting domain resulted in a well-resolved shift in fragment position relative to the unmethylated sequence. In addition, fragments containing hemimethylated and fully methylated sites could be distinguished, and a proportionally larger shift was observed with an increasing number of methylated bases. Denaturing gradient gel electrophoresis thus provides a sensitive method for analyzing the methylation state of DNA, which is not dependent on the presence of restriction enzyme cleavage sites. We also demonstrate that denaturing gradient gel electrophoresis can be used to obtain a quantitative estimate of the change in helix stability caused by modification of one or two bases in a complex DNA sequence. Such estimates should allow more accurate modeling of melting of natural DNA sequences.     

A high-resolution, fluorescence-based, semiautomated method for DNA fingerprinting

We describe a fluorescence-based method for the automated analysis of DNA fragments on polyacrylamide gels. A single-stranded oligonucleotide primer (18-mer) with a fluorochrome covalently bound to its 5'-end is annealed to a synthetic oligonucleotide to create a double-stranded oligonucleotide linker with a 5'-overhang complementary to a restriction enzyme site. Cosmid or plasmid DNA is digested with the appropriate restriction enzyme and then ligated to the fluorochrome-labeled linker. The labeled restriction fragments are loaded on a denaturing polyacrylamide gel in a commercially available DNA sequencer. As the restriction fragments migrate through the gel, they intersect a laser beam which excites the fluorochrome-labeled fragment. Fluorescence emission data are captured on a computer in real time and analyzed after the completion of electrophoresis. Fragment length is nearly linearly related to migration time. This method offers very near single-base resolution up to 400 bases and the ability to quantitate fragment size up to 2000 bases. The fluorochrome-labeling chemistry relies on straightforward enzymatic reactions and can be performed in a single reaction tube. Because four different fluorochromes can be used, each of 16 lanes on the gel can be used to analyze four different digest reactions, one in each color. One of the fluorochromes can be used to label size standards in each lane, eliminating interlane variability and allowing more precise estimates of fragment size. We apply the method to the analysis of overlapping cosmids.     

Interaction of Hoechst 33258 and echinomycin with nucleosomal DNA fragments containing isolated ligand binding sites

We have examined the interaction of Hoechst 33258 and echinomycin with nucleosomal DNA fragments which contain isolated ligand binding sites. A 145 base pair fragment was prepared on the basis of the sequence of tyrT DNA, which contained no CpG or (A/T)(4) binding sites for these ligands. Isolated binding sites were introduced into this fragment at discrete locations where the minor groove is known to face toward or away from the protein core when reconstituted onto nucleosome core particles. The interaction of ligands with target sites on these nucleosomal DNA fragments was assessed by DNase I footprinting. We find that Hoechst 33258 can bind to single nucleosomal sites which face both toward and away from the protein core, without affecting the nucleosome structure. Hoechst binding is also observed on nucleosomal fragments which contain two or more drug binding sites, though in these cases the footprints are accompanied by the presence of new cleavage products in positions which suggest that the ligand has caused a proportion of the DNA molecules to adopt a new rotational positioning on the protein surface. Hoechst 33258 does not affect nucleosome reconstitution with any of these fragments. In contrast, the bifunctional intercalating antibiotic echinomycin is not able to bind to single nucleosomal CpG sites. Echinomycin footprints are observed on nucleosomal fragments containing two or more CpG sites, but there are no changes in the cleavage patterns in the remainder of the fragment. Echinomycin abolishes nucleosome reconstitution when included in the reconstitution mixture.     

一种新的DNA分子克隆方法

DNA分子克隆是基本的分子生物学实验技术,传统的分子克隆方法大多需经过酶切链接过程,但在某些情况下,没有合适的酶切位点往往会成为阻碍克隆进行的障碍.本文描述了一种新的分子克隆方法,称为不依赖酶切和链接的分子克隆（RLIC）.利用RLIC,将3种不同大小的DNA片段克隆到3种不同载体,证明了这种方法的有效性和可靠性.由于该方法不受限制性酶切序列限制,省去了酶切连接步骤,因此具有很大的灵活性和简便性,在分子生物学研究方面有广泛应用前景.     

PFGE MAPPER: a tool to aid in the analysis of pulse field gel electrophoresis maps

Pulse field gel electrophoresis mapping is an important techniquefor characterizing large segments of DNA and constructing long-rangerestriction maps. We have developed a tool, PFGE MAPPER, toaid in the construction of pulse field electrophoresis gel maps.This tool helps construct pulse field gel maps from single anddouble digest experiments visualized by hybridization with singlecopy probes. The program is written in Think C and runs on Macintoshcomputers. An intuitive interface allows the user to interactivelymodify fragment sizes or errors, select fragments for analysisand recalculate the maps. Maps can be printed or saved for laterviewing. After constructing and saving several maps in a region,PFGE MAPPER can be used to refine and extend the overall mapby merging individual maps. This tool should be useful for constructinglong-range restriction maps of genomic DNA and yeast artificialchromosomes.