A fiber-optic DNA biosensor microarray for the analysis of gene expression.

A fiber-optic biosensor array is described for the simultaneous analysis of multiple DNA sequences. A bundle of optical fibers was assembled with each fiber carrying a different oligonucleotide probe immobilized on its distal end. Hybridization of fluorescently labeled complementary oligonucleotides to the array was monitored by observing the increase in fluorescence that accompanied binding. The approach enables fast (< 10 min) and sensitive (10 nM) detection to multiple DNA sequences simultaneously, with the potential for quantitative hybridization analysis.

Structure of plectonemically supercoiled DNA.

Using electron microscopy and topological methods, we have deduced an average structure for negatively supercoiled circular DNA in solution. Our data suggest that DNA has a branched plectonemic (interwound) form over the range of supercoiling tested. The length of the superhelix axis is constant at 41% of the DNA length, whereas the superhelix radius decreases essentially hyperbolically as supercoiling increases. The number of supercoils is 89% of the linking deficit. Both writhe and twist change with supercoiling, but the ratio of the change in writhe to the change in twist is fixed at 2.6:1. The extent of branching of the superhelix axis is proportional to the length of the plasmid, but is insensitive to superhelix density. The relationship between DNA flexibility constants for twisting and bending calculated using our structural data is similar to that deduced from previous studies. The extended thin form of plectonemically supercoiled DNA offers little compaction for cellular packaging, but promotes interaction between cis-acting sequence elements that may be distant in primary structure. We discuss additional biological implications of our structural data.

Mutation typing using electrophoresis and gel-immobilized Acrydite probes.

A new electrophoresis technology for hybridization-based sequence detection and mutation typing is described. Intrinsic to this approach is copolymerization of specially modified oligonucleotide probes directly into polyacrylamide gels. Electrophoresis of single-stranded samples through gels containing specific immobilized probes results in hybridization-mediated capture of complementary targets. By increasing gel temperature or including denaturants in the buffer, the method can be used to type single-nucleotide polymorphisms. The method can easily be adapted to type mutations in PCR-amplified samples. Acrydite gel technology will also be useful for many other applications, including hybridization-based diagnostics, analysis of gene expression and purification of nucleic acids from biological samples.

Forensic DNA testing on skeletal remains from mass graves: a pilot project in Guatemala.

A reliable method for extracting DNA from teeth was developed and successfully applied to a set of 12 skeletons recovered from two 10-year-old Guatemalan mass graves. Attempts to identify the remains by mitochondrial DNA (mtDNA) testing were hampered by low sequence diversity. Our findings demonstrate the feasibility of using DNA typing to identify victims from mass graves.

High-resolution mapping of carcinogen binding sites on DNA.

We have used a photochemical method to map covalent binding sites of the carcinogen benzo[a]pyrenediol epoxide (BPDE) within DNA from the transcriptional control region of the chicken adult beta-globin gene. Our preliminary low-resolution mapping has demonstrated that this region contains highly preferred BPDE binding sites [Boles, T.C., & Hogan, M.E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5623-5627]. Here, we find that BPDE binding at individual G residues in this region is influenced by nearest-neighbor interactions and also by longer range interactions that may be attributable to sequence-specific variation of DNA secondary structure. Our findings suggest that long poly(dG) sequences should be preferred sites for BPDE action in other genes.

Site-specific carcinogen binding to DNA.

Benzo[alpha]pyrene diol epoxide (BPDE) is a well-studied environmental carcinogen that binds covalently to DNA. Here we describe a photochemical technique that allows us to map BPDE-binding sites within cloned gene sequences. The technique is based upon our observation that, when irradiated with laser light at 355 nm, one single-strand DNA cut is produced at each BPDE binding site. In initial experiments we have studied the distribution of such cuts in cloned DNA from the chicken adult beta-globin gene. We find that BPDE binding in this gene sequence is distinctly nonrandom. While several prominent BPDE-binding sites are evident, a 300-base-pair sequence immediately 5' to the RNA cap site is most strongly attacked by the carcinogen. This region is believed to contain important transcriptional control sequences. We discuss the possibility that sequence-specific binding to such regulatory elements may be an important feature of the mechanism of the carcinogen.

Chi-dependent formation of linear plasmid DNA in exonuclease-deficient recBCD+ strains of Escherichia coli.

Escherichia coli strains carrying mutations in sbcB (exonuclease I) or xthA (exonuclease III) accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids containing the chi sequence, 5'-GCTGGTGG-3'. Chi-dependent formation of high-molecular-weight plasmid DNA is dependent on recA and recF functions. In addition, chi stimulation occurs only in cis. Our data are consistent with models in which RecA and RecF proteins bind to and protect the DNA ends produced by RecBCD-chi interaction.

Plasmid recombination by the RecBCD pathway of Escherichia coli.

Previously, we demonstrated that exonuclease I-deficient strains of Escherichia coli accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids carrying the chi sequence (5'- GCTGGTGG-3') (M. M. Zaman and T. C. Boles, J. Bacteriol. 176:5093-5100, 1994). Since high-molecular weight linear DNA is believed to be the natural substrate for RecBCD-mediated recombination during conjugation (A. J. Clark and K. B. Low, p. 155-215, in K. B. Low, ed., The Recombination of Genetic Material, 1988), we analyzed the recombination frequencies of chi+ and chi0 plasmids in sbcB strains. Here, we report that chi sites stimulate plasmid recombination frequency by 16-fold in sbcB strains. Chi-stimulated plasmid recombination is dependent on RecBCD but is independent of RecF pathway genes. The distribution of recombination products suggests that high-molecular-weight linear plasmid DNA is a substrate for RecBCD-mediated recombination. Surprisingly, our data also suggest that chi+ plasmids also recombine by the RecBCD pathway in rec+ sbcB+ cells.

DNA structure equilibria in the human c-myc gene.

We have employed analytical S1 nuclease analysis to identify sites with altered DNA secondary structure in the human c-myc gene. We have mapped several sites of that kind in vitro at one-base resolution but have focused our attention on one particularly stable conformational isomer which occurs approximately 270 base pairs upstream from the preferred transcription origin. We have analyzed the kinetics of that conformational equilibrium as a function of supercoil density and enzyme concentration and find that DNA structure in this region is adequately modeled as a two-state equilibrium between an undistorted (S1 nuclease insensitive) and a distorted (S1-sensitive) state. We find that at fixed supercoil density, S1 nuclease cleavage at this DNA segment can be altered in vitro by a DNA sequence change as far away as 1500 bases. We also find that the S1 nuclease cleavage at this site can be dramatically enhanced by the binding of small RNA molecules. On the basis of an analysis of S1 cutting kinetics and an analysis of DNA sequence at the S1 cleavage site, we conclude that RNA may bind directly to DNA, thereby shifting the underlying conformational equilibrium. Together, these data suggest that as a class, short RNA molecules could serve as site-specific regulatory elements in the myc gene and elsewhere.

The helical repeat of underwound DNA in solution.

Closed circular DNA was relaxed with a topoisomerase in the presence of varying concentrations of the intercalating dye, ethidium bromide, to create underwound, planar DNA rings. We directly determined the helical repeat of these DNA molecules by the Gaussian center method and found that it varied as a simple predicted function of the degree of underwinding and the helical repeat of relaxed, dye-free DNA. We discuss these results in light of a recent mathematical treatment of DNA structure which predicts that the helical repeat of supercoiled DNA molecules in solution obeys the same function.

Analysis of the structure of dimeric DNA catenanes by electron microscopy.

We analyzed the structure of open-circular and supercoiled dimeric DNA catenanes generated by site-specific recombination in vitro. Electron microscopy of open-circular catenanes shows that the number of duplex crossings in a plane is a linear function of the number of catenane interlinks (Ca/2), and that the length of the catenane axis is constant, independent of Ca. These relationships are similar to those observed with supercoiled DNA. Statistical analyses reveal, however, that the conformations of the individual rings of the catenanes are similar to those of unlinked circles. The distribution of distances between randomly chosen points on separate rings depends strongly on Ca and is consistent with a sharp decrease in the center-of-mass separation between rings with increasing Ca. Singly linked supercoiled catenanes are seen by microscopy to be linked predominantly through terminal loops in the respective superhelices. The observations suggest that chain entropy is a major factor determining the conformation of DNA catenanes.

Immobilization of acrylamide-modified oligonucleotides by co-polymerization.

A flexible chemistry for solid phase attachment of oligonucleotides is described. Oligonucleotides bearing 5'-terminal acrylamide modifications efficiently co-polymerize with acrylamide monomers to form thermally stable DNA-containing polyacrylamide co-polymers. Co-polymerization attachment is specific for the terminal acrylamide group. Stable probe-containing layers are easily fabricated on supports bearing exposed acrylic groups, including plastic microtiter plates and silanized glass. Attachment can be accomplished using standard polyacrylamide gel recipes and polymerization techniques. Supports having a high surface density of hybridizable oligonucleotide (approximately 200 fmol/mm2) can be produced.