DNA microarrays with stem-loop DNA probes: preparation and applications.

We have developed DNA microarrays containing stem-loop DNA probes with short single-stranded overhangs immobilized on a Packard HydroGel chip, a 3-dimensional porous gel substrate. Microarrays were fabricated by immobilizing self-complementary single-stranded oligonucleotides, which adopt a partially duplex structure upon denaturing and re-annealing. Hybridization of single-stranded DNA targets to such arrays is enhanced by contiguous stacking interactions with stem-loop probes and is highly sequence specific. Subsequent enzymatic ligation of the targets to the probes followed by stringent washing further enhances the mismatched base discrimination. We demonstrate here that these microarrays provide excellent specificity with signal-to-background ratios of from 10- to 300-fold. In a comparative study, we demonstrated that HydroGel arrays display 10-30 times higher hybridization signals than some solid surface DNA microarrays. Using Sanger sequencing reactions, we have also developed a method for preparing nested 3'-deletion sets from a target and evaluated the use of stem-loop DNA arrays for detecting p53 mutations in the deletion set. The stem-loop DNA array format is simple, robust and flexible in design, thus it is potentially useful in various DNA diagnostic tests.

Multiplex allele-specific target amplification based on PCR suppression.

We have developed a strategy for multiplex PCR based on PCR suppression. PCR suppression allows DNA target amplification with only one sequence-specific primer per target and a second primer that is common for all targets. Therefore, an n-plex PCR would require only n + 1 primers. We have demonstrated uniform, efficient amplification of targeted sequences in 14-plex PCR. The high specificity of suppression PCR also provides multiplexed amplification with allele specificity. Multiplexed PCR was used to develop assays for genotyping DNA samples from cystic fibrosis-affected individuals. The new approach greatly simplifies primer design, significantly increases the PCR multiplexing level, and decreases the overall primer cost. In addition, this assay is more readily amenable to automation and is therefore suitable for high-throughput genetic diagnostics.

An artificial primosome: design, function, and applications.

Double-stranded (ds) DNA is capable of the sequence-specific accommodation of an additional oligodeoxyribonucleotide strand by the peptide nucleic acid(PNA)-assisted formation of a so-called PD-loop. We demonstrate here that the PD-loop may function as an artificial primosome within linear, nonsupercoiled DNA duplexes. DNA polymerase with its strand displacement activity uses this construct to initiate the primer extension reaction at a designated dsDNA site. The primer is extended by several hundred nucleotides. The efficiency of dsDNA priming by the artificial primosome assembly is comparable to the single-stranded DNA priming used in various assays. The ability of the PD-loop structure to perform like an artificial primosome on linear dsDNA may find applications in biochemistry, molecular biology, and molecular biotechnology, as well as for DNA diagnostics. In particular, multiple labels can be incorporated into a chosen dsDNA site resulting in ultrasensitive direct quantification of specific sequences. Furthermore, nondenaturing dsDNA sequencing proceeds from the PD-loop. This approach opens the way to direct isothermal reading of the DNA sequence against a background of unrelated DNA, thereby eliminating the need for purification of the target DNA.

High-level multiplex DNA amplification.

We present data on efficient amplification of large number of DNA targets using a single-tube polymerase chain reaction (PCR). This is a further enhancement of our approach to multiplexed PCR based on PCR suppression, which allows multiple DNA amplification using only one sequence-specific primer per amplicon while the second primer is common for all targets (Broude, N.E., et al., Proc. Natl. Acad. Sci. USA 98, 206-211, 2001). The reaction conditions have been optimized for simultaneous synthesis of 30 DNA targets, mostly consisting of fragments containing single nucleotide polymorphisms (SNP). The size of the amplified fragments, derived from many different human chromosomes, varies from 100 to 600 bp. We conclude that this method has potential for highly multiplexed DNA amplification useful for SNP analyses, DNA diagnostics, and forensics.

PNA openers as a tool for direct quantification of specific targets in duplex DNA.

We report a new approach for target quantification directly within DNA duplex. Our assay is based on the formation of a new biomolecular structure, the PD-loop. The approach takes advantage of a selective hybridization of a probe to double-stranded DNA (dsDNA), which is locally opened by a pair of bis-PNA oligomers. To optimize the technique, several experimental formats are tested with the use of PNA and oligonucleotide probes. The highest sensitivity is achieved when the hybridized probe is extended and multiply labeled with 125I-dCTP by DNA polymerase via strand displacement in the presence of single-strand binding (SSB) protein. In this case, the PNA-assisted probe hybridization combined with the method of multiphoton detection (MPD) allows to monitor sub-attomolar amounts of the HIV-1 target on the background of unrelated DNA at sub-nCi level of radioactivity. The developed robust methodology is highly discriminative to single mutations, thus being of practical use for DNA analysis.

PCR based targeted genomic and cDNA differential display.

We previously described a targeted genomic differential display method (TGDD: Broude NE, Chandra A, Smith CL. Differential display of genomic subsets containing specific interspersed repeats. Proc. Natl. Acad. Sci. USA 1997;94:4548-53). In that method, presently characterized as method I, targeting was accomplished by capturing DNA fragments containing specific a sequence by hybridization with complementary single-stranded DNA. The captured fragments were amplified by PCR. Here, we describe method II where targeting is accomplished by PCR using primers specific to the target sequence. Method II takes advantage of PCR suppression to eliminate fragments not containing the target sequence (Siebert PDA, Chenchik A, Kellogg DE, Lukyanov KA and Lukyanov SA. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 1995;23:1087-1088). Targeting focuses analysis on and around interesting areas and additionally serves to reduce the complexity of the amplified subset. These approaches are useful to amplify genome subsets containing a variety of targets including various conserved sequences coding for cis-acting elements or protein motifs.

High polymorphism level of genomic sequences flanking insertion sites of human endogenous retroviral long terminal repeats.

The polymorphism at the multitude of loci adjacent to human endogenous retrovirus long terminal repeats (LTRs) was analyzed by a technique for whole genome differential display based on the PCR suppression effect that provides selective amplification and display of genomic sequences flanking interspersed repeated elements. This strategy is simple, target-specific, requires a small amount of DNA and provides reproducible and highly informative data. The average frequency of polymorphism observed in the vicinity of the LTR insertion sites was found to be about 12%. The high incidence of polymorphism within the LTR flanks together with the frequent location of LTRs near genes makes the LTR loci a useful source of polymorphic markers for gene mapping.

Probing the genetic population structure of Trypanosoma cruzi with polymorphic microsatellites.

We describe here the identification of eight polymorphic microsatellite loci with (CA)n repeats in the Trypanosoma cruzi genome based on the affinity capture of fragments using biotinylated (CA)12 attached to streptavidin-coated magnetic beads. The presence of two peaks in PCR amplification products from individual clones confirmed that T. cruzi is diploid. Hardy-Weinberg and linkage disequilibrium analyses suggested that sexual reproduction is rare or absent and that the population structure is clonal. Several strains, especially those isolated from nonhuman sources, showed more than two alleles in many loci demonstrating that they were multiclonal. The phylogenetic analysis of T. cruzi based on microsatellites revealed a great genetic distance among strains, although the strain dispersion profile in the Wagner network was in general agreement with the species dimorphism found by PCR amplification of the divergent region of the rRNA 24Salpha gene.

Differential display of genome subsets containing specific interspersed repeats.

A genomic differential display method was developed that analyzes many restriction fragment length polymorphisms simultaneously. Interspersed repeat sequences were used to reduce DNA sample complexity and to target genomic subsets of interest. This work focused on trinucleotide repeats because of their importance in human inherited diseases. Immobilized repeat-containing oligonucleotides were used to capture genomic DNA fragments containing sequences complementary to the oligonucleotide. Captured fragments were amplified by PCR and fluorescently labeled using primers complementary to the repeat sequence and/or to the known sequences ligated to the ends of the restriction fragments. The labeled PCR fragments were displayed by size on a high-resolution automated fluorescent DNA sequencing instrument. Although there was a conservation in the overall pattern of displayed genome subsets, many clear and reproducible differences were detected when genomes from different individuals were compared. Fewer differences were detected within, than between, monozygotic twin pair genomes. In control experiments, the method distinguished between Huntington disease alleles with normal and expanded CAG repeat lengths.

Parallel Processing in Genome Mapping and Sequencing

Conventional genome mapping and sequencing involves the analysis and processing of individual samples and pieces of experimental data. Although these methods work, it is quite clear that more efficient and less expensive methods are needed. Our top down physical mapping experiments have focused on the parallel processing of information from multiple samples at one time. This approach has aided the construction of genomic restriction maps and allowed us to assess the degree of large-scale conservation across wide regions of the human genome. The principles of parallel processing were applied in top down experiments that ordered an overlapping cosmid library from the 14-Mb Schizosaccharomyces pombe genome. This approach produced an eight-fold increase in efficiency in clone ordering over similar efforts. Recently, we have developed an enhanced sequencing by hybridization protocol that allows DNA sequence information to be collected on a large number of samples at once. Our current research focuses on applying parallel processing principles to make genome-wide comparisons between pairs of samples for analyzing disease states.

Efficient preparation of short DNA sequence ladders potentially suitable for MALDI-TOF DNA sequencing.

Duplex probes with five-base single-stranded overhangs were developed for positional sequencing by hybridization [Broude et al., Proc Natl Acad Sci USA 91:3072-3076, 1994]. The partially duplex probes can be employed to capture single-stranded oligonucleotide targets and form primer-template complexes. Recently we showed that partially duplex probes can prime Sanger sequencing reactions on immobilized, but non-ligated long single-stranded targets (approximately 500 nucleotide) [Fu et al., Proc Natl Acad Sci, in press]. Here immobilized, non-ligated partially duplex probes were used to capture and sequence short single-stranded targets. This strategy is capable of rapidly preparing large numbers of samples for future mass spectrometric DNA sequencing.

A DNA sequencing strategy that requires only five bases of known terminal sequence for priming.

We have previously reported an enhanced version of sequencing by hybridization (SBH), termed positional SBH (PSBH). PSBH uses partially duplex probes containing single-stranded 3' overhangs, instead of simple single-stranded probes. Stacking interactions between the duplex probe and a single-stranded target allow us to reduce the probe sizes required to 5-base single-stranded overhangs. Here we demonstrate the use of PSBH to capture relatively long single-stranded DNA targets and perform standard solid-state Sanger sequencing on these primer-template complexes without ligation. Our results indicate that only 5 bases of known terminal sequence are required for priming. In addition, the partially duplex probes have the ability to capture their specific target from a mixture of five single-stranded targets with different 3'-terminal sequences. This indicates the potential utility of the PSBH approach to sequence mixtures of DNA targets without prior purification.

Calcium is necessary in the cell response to EM fields.

Previous research showed that exposure of human HL-60 cells to extremely low frequency electromagnetic fields increases the steady-state levels of some mRNAs. Modifications in calcium flux have been suggested as a means of amplifying electromagnetic signals, and induced changes in calcium influx could hypothetically lead to gene activation. The present experiments tested the role of calcium in the response of cells to electromagnetic fields. Steady state transcript levels for c-fos and c-myc were determined under conditions of low extracellular calcium. The present study confirms that calcium plays a role in the response of cells to electromagnetic fields.

Enhanced DNA sequencing by hybridization.

An enhanced version of DNA sequencing by hybridization (SBH), termed positional SBH (PSBH), has been developed. PSBH uses duplex probes containing single-stranded 3' overhangs, instead of simple single-stranded probes. Stacking interactions between the duplex probe and a single-stranded target should provide enhanced stringency in distinguishing perfectly matched 3' sequences. A second enhancement is the use of enzyme-catalyzed steps, instead of pure physical hybridization. The feasibility of this scheme has been investigated using biotinylated duplex probes containing single-stranded 5-base 3' overhangs, immobilized on streptavidin-coated magnetic beads. Ligation of a single-stranded target, hybridized to the single-stranded region of the duplex probes, provided enhanced discrimination of perfectly matched targets from those containing mismatches. In distinction to the serious complications caused by base composition effects in ordinary SBH, there was little effect of base composition in PSBH. The hardest mismatch to discriminate was the one furthest from the phosphodiester bond formed by ligation. However, mismatches in this position were efficiently discriminated by 3' extension of the duplex probe using a template-dependent DNA polymerase. These results demonstrate that PSBH offers considerable promise to facilitate actual implementations of SBH.

Tissue-specific expression of Na,K-ATPase beta-subunit. Does beta 2 expression correlate with tumorigenesis?

We have found a substantial decrease in the level of Na,K-ATPase beta 2-subunit mRNA in xenografts of human renal, lung hepatocellular carcinomas in nude mice as compared with corresponding normal tissues, as well as in the neuroblastoma cell line as compared with the neuron primary cell culture. The level of beta 1 mRNA is decreased in kidney and lung tumor cells, but is unchanged in hepatocellular carcinoma. In the neuroblastoma cell line the level of beta 1 subunit mRNA was found to be higher then in neuron primary cell culture. The level of alpha 1 mRNA in investigated tumors was the same as in normal tissues. These results may give evidence of the involvement of beta 2-subunit in the process of tumorigenesis as was shown for some other adhesion molecules.

[The hormonal regulation of mRNA Na,K-ATPase expression in rat kidneys in postnatal ontogeny]. / Gormonal'naia reguliatsiia ékspressii mRNK Na,K-ATFazy v pochkakh v postnatal'nom ontogeneze.

A study was made of effects of aldosterone, aldosterone+dexamethasone, and aldosterone+spironolactone on Na,K-ATPase mRNA expression in renal cortex of adult and 10 day old rats, when kidney is not sensitive to the hormone injection. It is shown that hormonal induction of synthesis of Na,K-pump mRNA occurs in the early postnatal period apart from mineral corticoid receptors. It seems probable that aldosterone exerts its action in 10 day old rats by interaction with glucocorticoid receptors inducing synthesis of different amounts of alpha- and beta-subunits of Na,K-ATPase.

A comparative analysis of the putative regulatory regions in human genes for the alpha-subunit family of Na(+)-K+ ATPase.

The sequences of a 1.5 kb long stretch of the 5' flanking region of the gene for the alpha 3 isoform of the catalytic subunit of human Na(+)-K+ ATPase (located on chromosome 19) and of more than a 2 kb stretch of the 5' flanking region of the gene for the alpha 2 isoform (located on chromosome 1) have been determined. Transcription start sites for the gene for the alpha 3 isoform have been mapped at positions -152 and -155 relative to the translation initiation codon by primer extension analysis and S1-nuclease mapping of mRNA from human brain. The 5' flanking region of the gene for isoform alpha 3 contains a CCAAT box on the noncoding chain and six putative Sp1 binding sites. Absence of a conventional TATA box and a high GC content are other features of the region. The 5' upstream region of the gene for the alpha 2 isoform contains potential TATA and CCAAT boxes and one potential Sp1 binding site. Upstream of the putative TATA box there is an octanucleotide repeat, GGGGGAGA, which is also found in several eukaryotic genes in analogous positions. Pairwise comparison of the putative 5' regulatory regions of the genes coding for the different isoforms of the Na(+)-K(+)-ATPase catalytic subunit shows the existence of conserved elements, as well as of oligonucleotide blocks with very different structures. It is suggested that the differences in the primary structure of the 5' upstream regions may provide the basis for tissue-specific expression of the Na(+)-K(+)-ATPase isoforms.