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Hybridization between nucleic acid strands immobilized on a solid support with partners in solution is widely practiced in bioanalytical technologies and materials science. An important fundamental aspect of understanding these reactions is the role played by immobilization in the dynamics of duplex formation and disassembly. This report reviews and analyzes literature kinetic data to identify commonly observed trends and to correlate them with probable molecular mechanisms. The analysis reveals that while under certain conditions impacts from immobilization are minimal so that surface and solution hybridization kinetics are comparable, it is more typical to observe pronounced offsets between the two scenarios. In the forward (hybridization) direction, rates at the surface commonly decrease by one to two decades relative to solution, while in the reverse direction rates of strand separation at the surface can exceed those in solution by tens of decades. By recasting the deviations in terms of activation barriers, a consensus of how immobilization impacts nucleation, zipping, and strand separation can be conceived within the classical mechanism in which duplex formation is rate limited by preassembly of a nucleus a few base pairs in length, while dehybridization requires the cumulative breakup of base pairs along the length of a duplex. Evidence is considered for how excess interactions encountered on solid supports impact these processes.

Abbreviations -- Preface -- 1. Introduction to nucleic acid hybridization -- Structure of nucleic acids -- Forces stabilizing DNA structure -- Mismatched base pairs -- Stability of nucleic acids -- Effects of pH -- Effects of temperature -- Renaturation and hybridization -- 2. Types of hybridization and uses of each method -- Solution hybridization -- Filter hybridization -- In situ hybridization -- Polymerase chain reaction -- DNA chips -- Use of more than one hybridization technique -- References -- 3. Solution hybridization: reassociation of DNA -- Reassociation kinetics -- Experimental analysis of a reassociation reaction -- Presentation of data -- Relationship between the rate of reassociation and the sequence complexity of DNA -- C0t curves of eukaryotic DNA show the presence of repeated sequences -- Determining the repetition frequency of an isolated sequence -- Isolation of different components -- Analysis of repetitive DNA -- Examining the relatedness of sequences -- Interspersion of unique and repetitive DNA -- References -- 4. Solution hybridization: RNA:DNA hybridization -- Introduction -- Progress of reaction -- DNA:RNA hybridizations with excess DNA -- DNA:RNA hybridizations in RNA excess -- Estimation of the number of genes being expressed by saturation hybridization -- Estimation of complexity of an RNA population by kinetic analysis of RNA-excess hybridization -- Detection of sequences differentially expressed in two cell types -- References -- 5. Solution hybridization: reaction conditions -- The rate of formation of duplexes -- Temperature -- Salt concentration -- Fragment length -- Other factors -- Preventing thermal breakdown of nucleic acid -- Speeding up the reaction -- Lowering the incubation temperature -- Factors affecting the stability of hybrids -- Mismatches -- References -- 6. Basic types of filter hybridization -- Probing recombinant libraries -- Southern blots -- Northern blots -- Dot blots and slot blots -- References -- 7. Hybridization strategy: long probes -- Introduction -- Factors affecting the rate of hybridization and stability -- of hybrids -- Concentration of nucleic acids -- Length of the probe -- Complexity of the probe -- Base composition -- Salt concentration -- Temperature -- Formamide -- Imperfectly matched sequences -- Hybridization accelerators -- Enzyme-linked probes -- Reaction volume -- Time of hybridization -- References -- 8. Hybridization strategy: oligonucleotide probes -- Introduction -- Factors affecting the hybridization of oligonucleotides and stability of hybrids -- Melting temperature -- Mismatches -- Rate of hybridization -- Salt concentration -- Time period for hybridization -- Hybridization accelerators -- Washing -- Control of stringency -- Optimizing conditions -- Probe design -- Length of the probe -- Single sequence probe -- Mixtures of oligonucleotide probes -- Reducing the complexity of an oligonucleotide pool -- References -- 9. Choice of probe -- Introduction -- Characteristics of probes -- DNA probes -- RNA probes -- Oligonucleotide probes -- Characterizing the probe -- Labeling the probe -- Radioactive probes: general characteristics -- Nonradioactive probes: general characteristics -- Choosing a probe -- Sensitivity required -- DNA or RNA or oligonucleotide? -- End-labeling or uniform labeling? -- References -- 10. Basic techniques: binding of nucleic acid to filters -- Types and properties of support -- Transfer of nucleic acid to filters -- Phage /colony transfer -- Transfer of DNA from gels -- Northern blots -- Dot /slot blots -- Immobilization of nucleic acid to filters -- Baking -- UV fixation -- Alkali fixation -- Hybridization in dried gels -- Experimental procedures -- Preventing degradation of RNA -- Phage /colony transfer -- Southern blots -- Northern blots -- DNA dot /slot blots -- RNA dot /slot blots -- References -- 11. Basic techniques: labeling of probes -- Introduction -- Radioactive probes -- Uniform labeling -- Labeling of DNA by random priming -- Labeling of DNA by nick translation -- Uniform labeling of RNA -- Uniform labeling by chemical methods -- End labeling of nucleic acids -- Labeling at the 5' end -- Labeling at the 3' end -- Removal of nonincorporated nucleotides -- Size exclusion -- Sephadex spin columns -- Ethanol precipitation -- Adsorption to silica particles -- Phenol extraction -- Determining the efficiency of incorporation of label -- Radioactive probes -- Nonradioactive probes -- References -- 12. Basic techniques: prehybridization, hybridization and washing -- Equipment -- Prehybridization -- Hybridization -- Washing -- Protocols for prehybridization, hybridization and washing -- References -- 13. Basic techniques: detection of hybrids -- Radioactive hybrids -- Autoradiography -- Phosphor imaging -- Detection of nonradioactive hybrids -- Chromogenic substrates -- Chemiluminescent substrates -- Enhanced chemiluminescence -- Reuse of filters and probes -- Removal of products of nonradioactive detection systems -- Stripping probe off filters -- Reuse of the probe -- References -- 14. Trouble shooting -- 15. Specific applications of techniques -- Mapping DNA -- Mapping repetitive sequences -- Detection of related sequences -- Discrimination between related sequences -- Zoo /garden blots -- Restriction fragment length polymorphisms -- DNA fingerprinting by hybridization -- Detection of mutations -- Loss of enzyme restriction sites -- Loss of genes -- Expansion of repeated sequences -- Use of allele specific oligonucleotide probes -- Reverse dot blots -- Ligase mediated gene detection -- Repeated sequences in probes -- Suppressing hybridization of repetitive sequences -- Using repetitive sequences as probes -- Semi-quantitative analyses -- Methylation -- Analysis of transcripts -- Preliminary characterization of transcripts -- Refined mapping of transcripts -- Quantitation of RNA transcripts -- Sequences expressed in one cell type and not another -- Hybrid selection of mRNA and hybrid arrest translation -- References -- 16. Current trends -- DNA arrays and chips -- Preparation of DNA arrays -- Preparation of oligonucleotide arrays -- Gene expression analyses -- DNA sequencing by hybridization -- Probes -- Developments in hybridization with oligonucleotide and peptide nucleic acid probes -- Molecular beacons -- References -- Appendices -- Appendix A: Glossary -- Appendix B: Solution hybridization equations -- Appendix C: Useful information -- Appendix D: Composition of stock solutions -- Appendix E: Suppliers -- Index.

Growth plate chondrocytes undergo sequential differentiation to form the resting zone, the proliferative zone (PZ), and the hypertrophic zone (HZ). The important role of microRNAs (miRNAs) in the growth plate was previously revealed by cartilage-specific ablation of Dicer, an enzyme essential for biogenesis of many miRNAs. To identify specific miRNAs that regulate differentiation of PZ chondrocytes to HZ chondrocytes, we microdissected individual growth plate zones from juvenile rats and performed miRNA profiling using a solution hybridization method and miRNA sequencing. Thirty-four miRNAs were differentially expressed between the PZ and the HZ, and we hypothesized that some of the miRNAs that are preferentially expressed in the PZ may promote proliferation and inhibit hypertrophic differentiation. Consistent with this hypothesis, transfection of inhibitors for four of these miRNAs (mir-369-3p, mir-374-5p, mir-379-5p, and mir-503-5p) decreased proliferation in primary epiphyseal chondrocytes. The inhibitors for three of these miRNAs (mir-374-5p, mir-379-5p, and mir-503-5p) also increased expression of multiple genes that are associated with chondrocyte hypertrophic differentiation. We next hypothesized that preferential expression of these miRNAs in the PZ is driven by the parathyroid hormone–related protein (PTHrP) concentration gradient across the growth plate. Consistent with this hypothesis, treatment of primary chondrocytes with a parathyroid hormone (PTH)/PTHrP receptor agonist, PTH1-34, increased expression of mir-374-5p, mir-379-5p, and mir-503-5p. Taken together, our findings suggest that the PTHrP concentration gradient across the growth plate induces differential expression of mir-374-5p, mir-379-5p, and mir-503-5p between the PZ and the HZ. In the PZ, the higher expression levels of these miRNAs promote proliferation and inhibit hypertrophic differentiation. In the HZ, downregulation of these miRNAs inhibits proliferation and promotes hypertrophic differentiation.

Sulfur shows totally opposite faces in environmental and energy issues. Sulfur containing gases, H2S and SO2, are dominant air pollutants, while sulfur plays as the clean energy carrier for Li-Sulfur (Li-S) batteries. Herein, through confining the gaseous comproportionation reaction in water, between H2S and SO2 that normally produces uncontrollable aggregates, we develop a completely clean approach to convert the air pollutants into water-dispersed sulfur nanoparticle (WDS). The WDS is contaminant-free, size-controllable, and more promisingly solution processable, and is thus ideal for achieving solution hybridization with other materials for various applications. As a typical example, an aqueously fabricated WDS/carbon nanotube composite delivers the theoretical capacity of sulfur at 0.5 A g−1, and a capacity of ~750 mAh g−1 even at a high current density of 5.0 A g−1. In addition to the normal use of sulfur as cathode, a new-concept WDS/carbon interlayer with a low sulfur content has been designed to inhibit the shuttling of polysulfides, greatly improving the cycling performance of the Li-S battery. The proposed approach gives a good example to turning pollutants into clean energy carriers by green chemistry.

63 INTRODUCTION 65 MATERIALS AND METHODS 67 Animals 67 Hormone Treatment 67 Blood Sampling 67 Biopsies 68 Radioimmunoassay 70 Nucleic Acid Isolation 70 Solution Hybridization 71 Statistical Analysis 71 RESULTS 73 Acute Response 73 Chronic Response 75 Correlation of Measurements 77 DISCUSSION 80 LITERATURE CITED 85 SECTION III. THE RELATIONSHIP OF THYROID STATUS 88 TO GH AND IGF-1 IN PLASMA AND IGF-1 mRNA IN LIVER AND SKELETAL MUSCLE OF CATTLE ABSTRACT 90 INTRODUCTION 91 MATERIALS AND METHODS 92 Animals 92

Here, we present a set of RNA-based probes for whole mitochondrial genome in-solution enrichment, targeting a diversity of mammalian mitogenomes. This probes set was designed from seven mammalian orders and tested to determine the utility for enriching degraded DNA. We generated 63 mitogenomes representing five orders and 22 genera of mammals that yielded varying coverage ranging from 0 to >5400X. Based on a threshold of 70% mitogenome recovery and at least 10× average coverage, 32 individuals or 51% of samples were considered successful. The estimated sequence divergence of samples from the probe sequences used to construct the array ranged up to nearly 20%. Sample type was more predictive of mitogenome recovery than sample age. The proportion of reads from each individual in multiplexed enrichments was highly skewed, with each pool having one sample that yielded a majority of the reads. Recovery across each mitochondrial gene varied with most samples exhibiting regions with gaps or ambiguous sites. We estimated the ability of the probes to capture mitogenomes from a diversity of mammalian taxa not included here by performing a clustering analysis of published sequences for 100 taxa representing most mammalian orders. Our study demonstrates that a general array can be cost and time effective when there is a need to screen a modest number of individuals from a variety of taxa. We also address the practical concerns for using such a tool, with regard to pooling samples, generating high quality mitogenomes and detail a pipeline to remove chimeric molecules.

We were motivated by three novel technologies, which exemplify a new design paradigm in high throughput genomics: nanostring TM, DNA-mediated Annealing, Selection, extension, and Ligation DASL TM, and multiplex real-time quantitative polymerase chain reaction (QPCR). All three are solution hybridization based, and all three employ on 10–1000 DNA sequence probes in a small volume, each probe specific for a particular sequence in a different human gene. nanostring TM uses 50-mer, DASL and multiplex QPCR use ∼20-mer probes. Assuming a 1-nM probe concentration in a 1 μL volume, there are 10− 9 × 10− 9 × 6.23 × 1023 or 6.23 × 105 molecules of each probe present in the reaction compared to 10–1000 target molecules. Excess probe drives the sensitivity of the reaction. We are interested in the limits of multiplexing, i.e., the probability that in such a design a particular probe would bind to any other, sequence-related probe rather than the intended, specific target. If this were to happen with appreciable frequency, this would result in much reduced sensitivity and potential failure of this design. We established upper and lower bounds for the probability that in a multiplex assay at least one probe would bind to another sequence-related probe rather than its cognate target. These bounds are reassuring, because for reasonable degrees of multiplexing (103 probes) the probability for such an event is practically negligible. As the degree of multiplexing increases to ∼106 probes, our theoretical boundaries gain practical importance and establish a principal upper limit for the use of highly multiplexed solution-based assays vis--a-vis solid-support anchored designs. WIREs Comput Stat 2015, 7:394–399. doi: 10.1002/wics.1364 For further resources related to this article, please visit the WIREs website.

PREMISE OF THE STUDY Both incomplete lineage sorting and reticulation have been proposed as causes of phylogenetic incongruence. Disentangling these factors may be most difficult in long-lived, wind-pollinated plants with large population sizes and weak reproductive barriers. METHODS We used solution hybridization for targeted enrichment and massive parallel sequencing to characterize low-copy-number nuclear genes and high-copy-number plastomes (Hyb-Seq) in 74 individuals of Pinus subsection Australes, a group of ~30 New World pine species of exceptional ecological and economic importance. We inferred relationships using methods that account for both incomplete lineage sorting and reticulation. KEY RESULTS Concatenation- and coalescent-based trees inferred from nuclear genes mainly agreed with one another, but they contradicted the plastid DNA tree in recovering the Attenuatae (the California closed-cone pines) and Oocarpae (the egg-cone pines of Mexico and Central America) as monophyletic and the Australes sensu stricto (the southern yellow pines) as paraphyletic to the Oocarpae. The plastid tree featured some relationships that were discordant with morphological and geographic evidence and species limits. Incorporating gene flow into the coalescent analyses better fit the data, but evidence supporting the hypothesis that hybridization explains the non-monophyly of the Attenuatae in the plastid tree was equivocal. CONCLUSIONS Our analyses document cytonuclear discordance in Pinus subsection Australes. We attribute this discordance to ancient and recent introgression and present a phylogenetic hypothesis in which mostly hierarchical relationships are overlain by gene flow.

Background Targeted sequencing is a powerful tool with broad application in both basic and translational sciences. Relatively low on-target rates for most current targeted sequencing studies influence the required coverage and data quality for subsequent applications. Results We present an improved targeted sequencing method that uses two rounds of in solution hybridization with probes synthesized from genomic clone templates, termed dCATCH-Seq. Independent captures of two large continuous genomic regions across three cell types within the human major histocompatibility complex (MHC) that spans ~3.5 Mb and a ~250 kb region on chromosome 11 demonstrated that dCATCH-Seq was highly reproducible with ~95% capture specificity. Comprehensive analyses of sequencing data generated using the dCATCH-Seq approach also showed high accuracy in the detection of genetic variants and HLA typing. The double hybridization capture approach can also be coupled with bisulfite sequencing for DNA methylation profiling of both CpG and non-CpG sites. Conclusions Altogether, dCATCH-Seq is a powerful and scalable targeted sequencing approach to investigate both genetic and epigenetic features. Electronic supplementary material The online version of this article (10.1186/s12864-017-4159-7) contains supplementary material, which is available to authorized users.

Whole genome sequencing of buffalo is yet to be completed, and in the near future it may not be possible to identify an exome (coding region of genome) through bioinformatics for designing probes to capture it. In the present study, we employed in solution hybridization to sequence tissue specific temporal exomes (TST exome) in buffalo. We utilized cDNA prepared from buffalo muscle tissue as a probe to capture TST exomes from the buffalo genome. This resulted in a prominent reduction of repeat sequences (up to 40%) and an enrichment of coding sequences (up to 60%). Enriched targets were sequenced on a 454 pyro-sequencing platform, generating 101,244 reads containing 24,127,779 high quality bases. The data revealed 40,100 variations, of which 403 were indels and 39,218 SNPs containing 195 nonsynonymous candidate SNPs in protein-coding regions. The study has indicated that 80% of the total genes identified from capture data were expressed in muscle tissue. The present study is the first of its kind to sequence TST exomes captured by use of cDNA molecules for SNPs found in the coding region without any prior sequence information of targeted molecules.

The aim of the present study was to investigate differences in the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus at different stages of the estrous cycle as well as in inseminated sows at estrus and during early pregnancy by use of solution hybridization and in relation to plasma levels of estradiol and progesterone. Uterine samples were collected at different stages of the estrous cycle and after insemination/early pregnancy. In the endometrium, the expression of ERalpha mRNA and PR mRNA was similar for cyclic and early pregnant groups. Both were highest at early diestrus/70 h after ovulation and ERalpha mRNA was lowest at late diestrus/d 19 while PR mRNA was lowest at diestrus and late diestrus/d 11 and d 19. The expression of endometrial ERbeta was constantly low during the estrous cycle but higher expression was found in inseminated/early pregnant sows at estrus and 70 h after ovulation. In the myometrium, high expression of ERalpha mRNA and PR mRNA was observed at proestrus and estrus in cyclic sows and at estrus in newly inseminated sows. Higher expression of myometrial ERbeta mRNA was found in inseminated/early pregnant sows compared with cyclic sows, although significant only at estrus. In conclusion, the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus differed between endometrium and myometrium as well as with stages of the estrous cycle and early pregnancy. In addition to plasma steroid levels, the differences between cyclic and inseminated/early pregnant sows suggest that other factors, e.g. insemination and/or the presence of embryos, influence the expression of these steroid receptor mRNAs in the sow uterus.

A straightforward enzymatic protocol for converting regular DNA into pseudo-complementary DNA could improve the performance of oligonucleotide microarrays by generating readily hybridizable structure-free targets. Here we screened several highly destabilizing analogs of G and C for one that could be used with 2-aminoadenine (nA) and 2-thiothymine (sT) to generate structure-free DNA that is fully accessible to complementary probes. The analogs, which included bioactive bases such as 6-thioguanine (sG), 5-nitrocytosine (NitroC), 2-pyrimidinone (P; the free base of zebularine) and 6-methylfuranopyrimidinone (MefP), were prepared as dNTPs and evaluated as substrates for T7 and Phi29 DNA polymerases that lacked editor function. Pairing properties of the analogs were characterized by solution hybridization assays using modified oligonucleotides or primer extension products. P and MeP did not support robust primer extension whereas sG and NitroC did. In hybridization assays, however, sG lacked discrimination and NitroC paired too strongly to C. The dNTPs of two other base analogs, 7-nitro-7-deazahypoxanthine (NitrocH) and 2-thiocytosine (sC), exhibited the greatest promise. Either analog could be used with nA and sT to generate DNA that was nearly structure-free. Hybridization of probes to these modified DNAs will require the development of base analogs that pair strongly to NitrocH or sC.

Electrophoretic mobility shift assays (EMSAs) are commonly used to investigate protein-DNA binding in vitro. However, EMSA can generate considerable amounts of undesirable waste, particularly when toxic compounds are examined. We therefore developed a novel in vitro protein-DNA binding assay called liquid chemiluminescent DNA pull-down assay, which is based on solution hybridization between digoxigenin-labeled DNA and glutathione S-transferase (GST)–fused DNA binding protein bound to glutathione-Sepharose beads.

The striatum is vulnerable to hypoxic-ischemic injury during development. In a rodent model of perinatal hypoxia-ischemia, it has been shown that striatal neurons are not uniformly vulnerable. Cholinergic neurons and NADPH-diaphorase-positive neurons are relatively spared. However, it is unknown what classes of striatal neurons are relatively sensitive. One of the major classes of striatal neurons uses enkephalin as a neurotransmitter. We have studied the effect of early hypoxic-ischemic injury on this class of neurons using a quantitative solution hybridization assay for preproenkephalin mRNA in conjunction with in situ hybridization. Hypoxia-ischemia results in an early (up to 24 h) decrease in striatal preproenkephalin mRNA, which is shown by in situ hybridization to occur mainly in the dorsal portion of the striatum. By 14 days, whole striatal preproenkephalin mRNA and total enkephalin-containing peptide levels are normal. However, at 14 days, in situ hybridization reveals that regions of complete preproenkephalin mRNA-positive neuron loss remain in the dorsal region. Normal whole striatal levels are due to an up-regulation of preproenkephalin mRNA expression in the ventrolateral region of the injured striatum. Given the important role that the enkephalin-containing striatal efferent projection plays in regulating motor function, its relative loss may be important in the chronic disturbances of motor control observed in brain injury due to developmental hypoxic-ischemic injury.

More than a decade of intensive use of microarray technology has flooded the scientific community with genome-wide expression data of diverse biological states. As a result, connection of the expression signatures of a relatively small number of genes related to, for example, disease states, patient responses or toxicological responses has become possible. Development of tools that enable cost- and time-efficient analysis of such signatures from large sample numbers is currently of major interest for research, drug screening and diagnostic purposes. A method named transcript analysis with aid of affinity capture (TRAC) is a novel solution hybridization and bead-based assay enabling multiplex mRNA target detection simultaneously from large sample numbers. Functionality of TRAC has been shown in a number of applications, including microbial quantification, gene expression-based monitoring of biotechnical processes, cell-based cancer marker gene screening and siRNA validation, which are reviewed here.

A detailed protocol is presented for the targeted enrichment of whole mitochondrial genomes based on an in-solution hybridization strategy. Bait is produced in-house by sonication of two long-range PCR amplicons and ligation of biotinylated double-stranded adapters. Indexed target DNA is hybridized with the bait in a multiplex enrichment reaction and pulled down using magnetic streptavidin beads followed by subsequent post-enrichment PCR and sequencing on an Illumina MiSeq. This strategy removes the need for expensive commercial bait probes while allowing enrichment of multiple samples in a single hybridization reaction. The method is particularly suitable for degraded DNA as it is able to enrich short DNA fragments and is not susceptible to polymerase artifacts introduced during PCR-based assays.

Hybridization thermodynamics on solid supports are compared with those in solution for two types of hybridization probe, DNA and uncharged morpholino oligonucleotides of identical sequences. Trends in hybridization affinity are discussed with respect to ionic strength, temperature, and surface behavior.

Previous studies have revealed that clinically relevant changes in temperature modify clinically relevant gene expression profiles through transcriptional regulation. Temperature dependence of post-transcriptional regulation, specifically, through expression of miRNAs has been less studied. We comprehensively analyzed the effect of 24 h exposure to 32°C or 39.5°C on miRNA expression profile in primary cultured human small airway epithelial cells (hSAECs) and its impact on expression of a targeted protein, protein kinase C α (PKCα). Using microarray, and solution hybridization-based nCounter assays, with confirmation by quantitative RT-PCR, we found significant temperature-dependent changes in expression level of only five mature human miRNAs, representing only 1% of detected miRNAs. Four of these five miRNAs are the less abundant passenger (star) strands. They exhibited a similar pattern of increased expression at 32°C and reduced expression at 39.5°C relative to 37°C. As PKCα mRNA has multiple potential binding sites for three of these miRNAs, we analyzed PKCα protein expression in HEK 293T cells and hSAECs. PKCα protein levels were lowest at 32°C and highest at 39.5°C and specific miRNA inhibitors reduced these effects. Finally, we analyzed cell-cycle progression in hSAECs and found 32°C cells exhibited the greatest G1 to S transition, a process known to be inhibited by PKCα, and the effect was mitigated by specific miRNA inhibitors. These results demonstrate that exposure to clinically relevant hypothermia or hyperthermia modifies expression of a narrow subset of miRNAs and impacts expression of at least one signaling protein involved in multiple important cellular processes.

Although recent technological advances in DNA sequencing and computational biology now allow scientists to compare entire microbial genomes, comparisons of closely related bacterial species and individual isolates by whole-genome sequencing approaches remains prohibitively expensive for most laboratories. Here we report the development and testing of a biochemical approach for targeted sequencing of only those chromosomal regions that differ between two DNA preparations. The method, designated GFE (genome fragment enrichment) uses competitive solution hybridization and positive selection to obtain genomic DNA fragments that are present in one pool of fragments but not another. Repeated comparisons of the genomes of Enterococcus faecalis and E. faecium led to the identification of 225 putative genome-specific DNA fragments. Species and strain variations within these fragments were confirmed by both experimental and bioinformatic analyses. The E. faecalis genome-specific sequences identified included both a preponderance of those predicted to encode surface-exposed proteins, as well as several previously described unique marker regions embedded within highly conserved rrn operons. The GFE strategy we describe efficiently identified genomic differences between two enterococcal genomes, and will be widely applicable for studying genetic variation among closely related bacterial species.

Several PCR methods have recently been developed to identify fecal contamination in surface waters. In all cases, researchers have relied on one gene or one microorganism for selection of host-specific markers. Here we describe the application of a genome fragment enrichment (GFE) method to identify host-specific genetic markers from fecal microbial community DNA. As a proof of concept, bovine fecal DNA was challenged against a porcine fecal DNA background to select for bovine-specific DNA sequences. Bioinformatic analyses of 380 bovine enriched metagenomic sequences indicated a preponderance of Bacteroidales -like regions predicted to encode membrane-associated and secreted proteins. Oligonucleotide primers capable of annealing to select Bacteroidales -like bovine GFE sequences exhibited extremely high specificity (>99%) in PCR assays with total fecal DNAs from 279 different animal sources. These primers also demonstrated a broad distribution of corresponding genetic markers (81% positive) among 148 different bovine sources. These data demonstrate that direct metagenomic DNA analysis by the competitive solution hybridization approach described is an efficient method for identifying potentially useful fecal genetic markers and for characterizing differences between environmental microbial communities.

In order to study the effect of estradiol-17beta (E2) on estrogen (E) and progesterone (P) receptors expression in oviduct and cervix of lambs, their respective transcripts (ERalpha mRNA and PR mRNA) were determined by solution hybridization and the receptor proteins (ER and PR) by binding assays after E2 treatments. Lambs (n=4 in each group) were not treated or treated with one, two or three i.m. injections of E2 (1 microg/kg) at 24 h of interval. Tissues were obtained 12 or 24 h after the last E2 injection. Estradiol treatments increased ERalpha mRNA and PR mRNA concentrations in an organ-dependent manner: transitory in the oviduct while maintained in the cervix. The E2 effect on the oviductal and cervical ER and PR concentrations were biphasic, with an initial reduction of receptors content that was followed by restoration. The ER restoration in oviduct was earlier than in the cervix. In summary, this study shows that E2 treatments may exert an inductive effect in ERalpha mRNA and PR mRNA levels and a biphasic effect in ER and PR concentrations in oviduct and cervix of immature ewe. These E2 effects varied in timing and strength depending on the organ of the reproductive tract.

The concept of a blockade of progesterone during human pregnancy and withdrawal of this blockade at parturition remains controversial There is no sharp fall in serum progesterone before parturition, but treatment with an antiprogestin is successful for labor induction at term pregnancy. The human progesterone receptor (PR) exists in two isoforms (PR-A and PR-B), mediating different biological responses. Here, the hypothesis of a progesterone withdrawal at parturition in terms of a change in PR isoforms was tested. Cervical biopsies were obtained at term before the onset of labor, immediately after parturition and from non-pregnant women. Solution hybridization showed a tendency for the PR mRNA level to be decreased at parturition. Immunohistochemistry displayed decreased PR(A + B) and PR-B levels (p0.05) immediately after parturition. The relative importance of PR-A seemed higher immediately after parturition as compared to its importance in non-pregnant and term pregnant women. Our results are consistent with the concept of a functional progesterone blockade at the receptor level at term pregnancy, and withdrawal of this blockade at parturition. These observations may have important clinical and therapeutic implications.

In an attempt to develop a standard for ELISA-PCR detection of Shiga toxin producing Escherichia coli (STEC) O157, six published PCR tests were tested in a comparative study on a panel of 277 bacterial strains isolated from foods, animals and humans. These tests were based on the detection of the genes rfbE [J. Clin. Microbiol. 36 (1998) 1801] and rfbB [Appl. Environ. Microbiol. 65 (1999) 2954], the 3' end of the eae gene [Epidemiol. Infect. 112 (1994) 449], the region immediately flanking the 5' end of the eae gene [Int. J. Food. Microbiol. 32 (1996) 103], the flicH7 gene [J. Clin. Microbiol. 35 (1997) 656], or a part of the recently described 2634-bp Small Inserted Locus (SILO(157) locus) of STEC O157 [J. Appl. Microbiol. 93 (2002) 250]. Unlike the other PCR assays, those amplifying the rfb sequences were unable to distinguish toxigenic from nontoxigenic O157. These assays were relatively specific to STEC O157, giving essentially a cross reaction with clonally related E. coli O55 and to a lesser extent with E. coli O145, O125, O126. They also detected the Shiga toxin (stx)-negative derivatives of STEC O157. Based on these results, an ELISA-PCR assay consisting of the solution hybridization of amplicons with two probes that ensured the specificity of the amplification was developed. The ELISA-PCR assay, which used an internal control (IC) of inhibition, was able to detect 1 to 10 copies of STEC O157 in the PCR tube. Adaptation of PCR into ELISA-PCR assay format facilitates specific and sensitive detection of PCR amplification products and constitutes a method of choice for screening STEC O157.

Cervical softening is crucial for a normal parturition and corresponds to remodeling of the dominating cervical extra cellular matrix (ECM). The onset of labor as well as cervical ripening is under hormonal control. To get further information about the endocrine regulation of term cervical ripening the following study was undertaken: cervical biopsies were obtained vaginally at elective caesareans, after normal vaginal delivery and after PGE2 or antiprogestin RU486. Biopsies from non-pregnant women served as controls. The concentrations of estrogen receptor (ER) and progesterone receptor (PR) protein were quantitated by EIA and the mRNA levels by solution hybridization. The ERalpha and beta were localized by immunohistochemistry, identified by RT-PCR and quantitated by solution hybridization. The co-localizations of CD45 (leukocyte antigen) and CD68 (macrophage antigen) were studied by immunohistochemistry. The cervical concentrations of ER and PR proteins decreased at term to 15 and 25%, respectively, compared to the non-pregnant levels. A further decrease was measured in the maximal ripened cervix at parturition. The mRNA levels were unchanged but IGF-I mRNA reached a maximum at term. ERalpha mRNA was significantly decreased until delivery, whereas ERbeta mRNA, like IGF-I; was maximum at term. By immunostaining ERbeta could be co-localized with CD45 leukocyte antigen and CD68 macrophage specific antigen. Oral administration of RU486 induced a significant increase in ER protein concentration, whereas PGE2 and spontaneous ripening did not. These findings indicate that cervical ripening is related to significant local hormonal changes.

Backgrounds/Aims : In the treatment of chronic hepatitis B (CHB) with lamivudine, adequate duration of the therapy remains to be determined. In this prospective study, the authors intended to investigate whether long-term additional administration of lamivudine might enhance the durability of lamivudine-induced HBeAg seroconversion. Methods : Eighty-five CHB patients whzo achieved HBeAg seroconversion by lamivudine received additional lamivudine therapy for at least 24 months at a dose of 100 mg per day. Among them, 61 patients whose serum HBeAg and HBV-DNA (solution hybridization assay) had been negative persistently for >24 months discontinued lamivudine therapy and followed-up for >12 months. We calculated the cumulative reappearance rate of serum HBV-DNA and HBeAg and also evaluated the predictive factors for post-treatment virologic relapse. Results : The cumulative reappearance rates of serum HBV-DNA following cessation of lamivudine therapy at 6 months, 1 year and 2 years were 15%, 21%, and 31%, respectively. The cumulative reappearance rates of serum HBeAg at 6 months, 1 year and 2 years were 11%, 13% and 16%, respectively. Old age and presence of precore mutant were two independent predictive factors for viral relapse. Conclusions : These results suggested that long-term additional administration of lamivudine might enhance the durability of lamivudine-induced HBeAg seroconversion.

The steady-state mRNA levels of the NMDA receptor NR1 subunit were determined by a quantitative solution hybridization assay in selected CNS regions associated with antinociception in the rat. Tissues were obtained by microdissection from rats treated chronically with morphine alone or in combination with LY274614, a competitive NMDA receptor antagonist. Morphine treatment for 7 days resulted in the development of tolerance to morphine's analgesic effect and produced a significant decrease in the steady-state NR1 mRNA levels in the spinal cord dorsal horn (by 16%), and an elevation in nucleus raphe magnus and medial thalamus (by 26 and 38%, respectively). The NR1 mRNA levels were unchanged in the lateral paragigantocellular nucleus, locus coeruleus, periaqueductal grey, and sensorimotor cortex. NMDA receptor binding in the spinal cord measured with [3H]MK-801 was reduced approximately 50% by chronic morphine treatment. Co-administration of LY274614 (s.c. at 24 mg/kg/24 h via an osmotic pump) not only attenuated the development of morphine tolerance but also prevented the changes in the NR1 mRNA levels induced by chronic morphine administration. Neither a 7-day infusion of LY274614 nor an acute injection of morphine (10 mg/kg, s.c.) changed the NR1 mRNA levels. These results suggest that changes in the expression of the NR1 mRNA induced by chronic morphine in three CNS regions involved in antinociception are associated with the development of morphine tolerance and in the spinal cord, morphine tolerance is associated with the downregulation of NMDA receptors.

Phosphodiester antisense oligodeoxynucleotides (ODNs) directed against various domains of the cloned mouse delta receptor DOR-1 reduce delta-opioid receptor binding in vivo and in vitro. The present study examines the stability of an antisense ODN (275 nM) directed against the delta-opioid receptor and its effect on DOR-1 mRNA in cultured neuroblastoma cells and in vivo. When added to NG108-15 cells, much of the antisense ODN is degraded. However, > 1% is intact, associated with cells, and stable for at least 72 h. Northern blot analysis demonstrates that treatment of NG108-15 cells with the antisense ODN reduces the levels of a species of DOR-1 mRNA by approximately 25%. Similarly, intrathecal administration of the antisense ODN results in the accumulation of intact ODN within the spinal cord, which is stable for at least 72 h, although the levels of accumulation in vivo are lower than in vitro after either 4 or 72 h. Antisense ODN treatment lowers DOR-1 mRNA levels by approximately 25%. The loss of mRNA both in vivo and in vitro corresponds quite well to the decreases in receptor binding previously observed by our laboratory and is consistent with reduction of delta-opioid receptor protein in vitro as determined by western blot with a monoclonal antibody selective for the delta-opioid receptor. In conclusion, these studies indicate that a small, but significant, proportion of ODN is taken up by cells and remains intact for up to 72 h. This appears to be sufficient to down-regulate mRNA levels of delta-opioid receptors and their expression.

Developmental changes in the levels of N-methyl-D-aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low-affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5' insert and with or without both 3' inserts. In cerebellum, however, the major NR1 variants were those containing the 5' insert and lacking both 3' inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.

Hormones released from the pituitary have been shown to regulate the expression of different proteins in the central nervous system. We wanted to examine whether peripheral administration of bovine growth hormone (bGH) regulates the expression of delta-opioid receptor (DOR) in the cerebral cortex and cerebellum. Expression of the DOR protein was quantified using Western blot densitometry. DOR mRNA was quantified with a solution hybridization RNase protection assay. Hypophysectomized (Hx) and untreated normal female rats were included in the study. All Hx rats were hormonally treated with cortisol (400 microg/kg/day) and L-thyroxine (10 microg/kg/day) for 19 days. Hypophysectomy resulted in a threefold increase in cerebral cortex and a twofold increase in cerebellum of the DOR protein compared with normal rats. One subgroup of Hx rats received bGH (1 mg/kg body weight) as a daily subcutaneous injection for 19 days. This treatment normalized the levels of DOR protein in the cerebral cortex and cerebellum. Immunohistochemical experiments showed that GH decreased DOR expression especially in layers II-VI in cerebral cortex and in stratum moleculare in cerebellum. Quantification of DOR mRNA by solution hybridization RNase protection assay corresponded to the DOR protein measurements. We conclude that the expression of DORs in cerebral cortex and cerebellum is regulated by GH.

Summary Seckel syndrome is an autosomal-recessive disorder with a frequency of less than 1/10 000 births in which there are multiple malformations including severe short stature. We report on a patient with Seckel syndrome with a current body height of −7·5 SDS. Laboratory investigations at the age of 19 months revealed high levels of IGF-I, IGF-II and IGFBP-3. These data suggested the existence of IGF-I resistance possibly caused by impairment of the IGF-I receptor (IGF-IR) or altered IGFBPs. The purpose of this investigation was to examine whether the growth retardation in a Seckel syndrome patient is related to an alteration in the IGF system. Analysis of IGF-IR mRNA of patient's and control fibroblasts by solution hybridization/RNase protection assay did not show differences of IGF-IR transcript expression or size. Affinity crosslinking studies using [125I]-IGF-I showed normal-sized IGF-IR–ligand complexes. Mutation analysis of the complete coding regions of the IGF-I and IGF-IR genes showed no evidence of genetic alterations. Ligand blot analysis of IGFBPs secreted by the patient's fibroblasts showed stronger signals than control cells. Quantitative measurement of IGFBP-3 in cell-conditioned media was performed by radioimmunoassay (RIA) and revealed a sixfold increase when compared to control fibroblasts. We conclude that in this patient with Seckel syndrome and severe growth impairment IGF-I resistance is possibly related to altered production of IGFBP-3.

Cocaine administration increases activity at dopamine receptors, increases preprodynorphin (ppDyn) gene expression in the caudate-putamen (CPu), and activates the stress responsive hypothalamic-pituitary-adrenal (HPA) axis. To examine the hypothesis that mu-opioid receptors (MOR) may play roles in these cocaine effects, we tested the effects of acute "binge" pattern cocaine administration in mice with targeted disruption of the MOR gene. Wild-type (+/+) and homozygous MOR-deficient (-/-) mice received three injections of 15 mg/kg cocaine at 1-h intervals. Mice were sacrificed 30 min after the last injection and mRNAs for ppDyn and preproenkephalin (ppEnk) in the CPu and nucleus accumbens (NAc), and for type I corticotropin-releasing hormone receptor (CRH(1) receptor) and pro-opiomelanocortin (POMC) in the hypothalamus and pituitary, were measured by solution hybridization RNase protection assays. Cocaine elevated ppDyn mRNA in the CPu, but not NAc, of both the MOR -/- and wild-type mice. ppEnk mRNA in the CPu, but not NAc, was lower in MOR -/- mice than in wild-type mice following cocaine administration. Hypothalamic CRH(1) receptor and POMC mRNAs were expressed at similar levels in untreated and in cocaine-treated mice of each genotype. However, there were lower basal levels of CRH(1) receptor mRNA in the anterior pituitary of the MOR -/- mice than in wild-type mice and the MOR -/- mice failed to show the cocaine-induced decreases in CRH(1) receptor mRNA found in the wild-type mice. Cocaine activated the HPA axis similarly in MOR -/- and wild-type mice, as reflected in similar increases in plasma corticosterone levels in both genotypes. These results support a specific role for MORs in acute cocaine effects on striatal ppEnk gene expression and fail to support critical roles for these receptors in acute cocaine's effects on either ppDyn gene expression or HPA activation. MOR -/- mice are useful models for studying cocaine effects on ppEnk gene expression that could aid interpretation of the similar postmortem phenomena found in human cocaine addicts.

Psychostimulants such as cocaine have been shown to regulate c -fos and opioid gene expression in male rats. However, little information is available on cocaine effects in female rats or how the ovarian hormones, estrogen and progesterone, modulate these effects. In this study we used quantitative solution hybridization assays to measure c -fos and preprodynorphin (PDYN) mRNA levels after cocaine administration in the caudate/putamen of intact male and female rats or ovariectomized (OVX) female rats that were pretreated with vehicle, estrogen and/or progesterone. The c -fos mRNA levels were increased in intact male and female rats after 30 min or 3 h of one single cocaine injection and after 14 days of single daily cocaine injections. The c -fos mRNA levels were also increased after 30 min of a single cocaine injection in OVX female rats that were treated with vehicle, estrogen and/or progesterone. The PDYN mRNA levels did not change after 30 min, 3 h or 14 days in intact male or female rats. However, PDYN mRNA levels were increased in the caudate/putamen of OVX female rats pretreated with vehicle or a combination of estrogen and progesterone but not in OVX female rats that were pretreated with either estrogen or progesterone alone. Our data suggest hormonal regulation of cocaine effects on PDYN mRNA levels which may modulate cocaine-induced behaviors in female rats.

Treatment of patients with chronic hepatitis B virus (HBV) infections with potent antiviral therapy often results in dramatic reductions in the levels of viremia to very low levels. Monitoring of serum HBV DNA levels is a consistent method for the assessment of antiviral potency; however, widely used hybridization assays for the monitoring of HBV DNA levels have limited sensitivities and are not effective for the monitoring of patients whose serum HBV DNA levels have decreased to below approximately 700,000 HBV genomes/ml. The objective of the present study was to assess a PCR-based assay (the COBAS-AM assay) for quantitation of serum HBV DNA levels and to compare the results of the COBAS-AM assay with those of a solution hybridization assay with a radiolabeled probe. The precision and accuracy of the assay were determined with low-positive and high-positive controls consisting of a plasmid DNA molecule containing HBV-specific primer binding regions, and the sensitivity of the assay was determined by using serial dilutions of sera from subjects with chronic HBV infection. HBV DNA levels were quantitated in 1,695 serum samples from subjects with chronic HBV infection who were enrolled in clinical trials of lamivudine in North America or Asia. The COBAS-AM assay demonstrated high levels of inter- and intra-assay precision and accuracy, and the linear range of the COBAS-AM assay was greater than that of the solution hybridization assay. The assay is linear over a 3-log 10 range and is able to quantitate serum HBV DNA at levels 3 log 10 lower than those that can be detected by the solution hybridization assay. We found that the COBAS-AM assay is an accurate PCR-based assay for quantitation of serum HBV DNA levels in subjects with chronic HBV infection.

A polymerase chain reaction (PCR) assay based on a solution hybridization format with colorimetric end-point detection (PCR ELISA) was investigated for the specific detection of Campylobacter jejuni and Campylobacter coli in food samples following enrichment culture. One hundred fifteen samples of raw meat and offal (poultry, porcine, ovine, and bovine), raw shellfish, and artificially contaminated milk were enriched in blood-free Campylobacter Enrichment Broth for 48 h. Enrichment cultures were subcultured to Campylobacter blood-free selective agar plates, and presumptive isolates were identified by phenotypic methods. DNA was extracted from 1-ml aliquots of the enrichment cultures using a rapid extraction method, and the DNA was used as the template in a PCR ELISA, A comparison of the PCR El.ISA with the enrichment culture and subculture to selective agar method showed that the results of 112 of the 115 samples tested were in agreement by both methods. Seventy-one of the various food samples were positive in the PCR ELISA, and 70 samples were positive by culture. The PCR ELISA had a sensitivity of 99% and a specificity of 96%, with a positive predictive value of 97% and a negative predictive value of 98%. The PCR ELISA is a rapid, sensitive, and specific method for the detection of C. jejuni and C. coli in foods following enrichment culture and significantly reduces the time required for their detection. Dans cette etude, un dosage PCR (reaction en chaine par polymerase) ELISA (dosage d'immunoadsortion enzymatique) est evalue pour la detection specifique de Campylobacter jejuni et Campylobacter coli dans des echantillons alimentaires apres une culture d'enrichissement. Les resultats montrent que cette technique est aussi sensible et specifique et plus rapide que les methodes de cultures conventionnelles pour la detection de C. jejuni et C. coli dans les aliments.

An electrochemical genomagnetic hybridization assay has been developed to take advantage of a new and efficient magnetic separation/mixing process, the amplification feature of enzyme labels, and single-use thick-film carbon transducers operated in the pulse-voltammetric mode. It represents the first example of coupling a magnetic isolation with electrochemical detection of DNA hybridization. The new protocol employs an enzyme-linked sandwich solution hybridization, with a magnetic-particle labeled probe hybridizing to a biotinylated DNA target that captures a streptavidin-alkaline phosphatase (AP). The α-naphthol product of the enzymatic reaction is quantitated through its well-defined, low-potential (+0.1 V vs. Ag/AgCl) differential pulse-voltammetric peak at the disposable screen-printed electrode. The efficient magnetic isolation is particularly attractive for electrical detection of DNA hybridization which is commonly affected by the presence of non-hybridized nucleic acid adsorbates. The new biomagnetic processing combines such magnetic separation with a low-volume magnetic mixing, and allows simultaneous handling of 12 samples. The attractive bioanalytical behavior of the new enzyme-linked genomagnetic electrical assay is illustrated for the detection of DNA segments related to the breast-cancer BRCA1 gene.

Glucagon has previously been reported to increase serum levels of insulin-like growth factor binding protein-1 (IGFBP-1) in humans. The in vitro effect of glucagon and glucagon-like peptide-1 (7-36) amide (GLP-1) was investigated in Hep G2 human hepatoma cells. The expression of IGFBP-1 mRNA was determined by solution hybridization assay and IGFBP-1 secretion was measured by radioimmunoassay. In contrast to forskolin the peptides glucagon and GLP-1 had no effect on IGFBP-1 mRNA at 3, 6 and 24 h incubation or any detectable effect on the apparent half-life of IGFBP-1 mRNA. However, the exposure to glucagon (10 microg/mL, 2.87 microM) and GLP-1 (1 microM) caused a two-fold stimulation in protein levels of IGFBP-1 after 6 h incubation, declining to control levels after 24 h. This transient effect was dose dependent, remained when transcription was inhibited and required protein synthesis. The regulation of IGFBP-1 secretion by glucagon and GLP-1 appeared to be cAMP independent. In conclusion, glucagon and GPL-1 were shown to have a post-transcriptional stimulatory effect on IGFBP-1 release.

Retinoic acid (RA) induced differentiation of SH-SY5Y cells increases the expression of mu opioid receptors (HMOR) and inhibitory G proteins, as well as the efficacy of opioids to inhibit forskolin-induced adenylyl cyclase activity. We examined the time course of the effects of all-trans retinoic acid (RA) on HMOR and c-fos mRNA levels as determined by solution hybridization (using HMOR and rat c-fos riboprobes) in RNA extracts from SH-SY5Y cells. Electrophoretic Mobility Shift Assay (EMSA) and Western blot analysis were used to assess the changes AP-1 DNA binding and the presence of fos-related proteins in nuclear extracts from untreated, vehicle (ethanol) or RA-treated SH-SY5Y cells. Exposure to RA for 0.5 h had no effect on HMOR while after 6-18 h of exposure HMOR in mRNA levels were decreased by 50% and then after 168 h of RA exposure, HMOR mRNA levels were doubled. In contrast, c-fos mRNA levels were unchanged at 0.5 h, but increased by 50% after 18 and 168 h of RA exposure. RA increased AP-1 binding after 18 and 168 h and a pan fos-FRA antibody produced a supershift. Western analysis indicates that RA activates a 45-kDa protein corresponding to the size of the fos B protein. These results identify two signal transduction targets that are regulated by RA during differentiation.

Genetic variations have been associated with a predisposition to common diseases and individual variations in drug responses. Identification and genotyping a vast number of genetic polymorphisms in large populations are increasingly important for disease gene identification and pharmacogenetics. Commonly used gel electrophoresis-based genotyping methods for known polymorphisms include polymerase chain reaction (PCR) coupled with restriction fragment-length polymorphism analysis, allele-specific amplification, and oligonucleotide ligation assay. Fluorescent dye-based DNA fragmentation has been extensively used for high-throughput microsatellite or short tandem-repeat genotyping. TaqMan®1 and molecular beacon genotyping are commonly used homogeneous solution hybridization technologies. Because of the ease of experimental assay design, single nucleotide polymorphism (SNP) genotyping methods based on single-base extension are in rapid development, such as fluorescence homogenous assays, pyrosequencing and mass spectrometry. Non-PCR based genotyping assays such as Invader™ assays are promised to genotype directly from genomic DNA without the requirement of PCR amplification. The DNA microarray is a solid phase genotyping format that is rapidly developing for parallel genotyping of a large number of SNPs simultaneously. Advanced technologies to identify genetic polymorphisms rapidly, accurately, and cost effectively will fundamentally change the practice of medicine by allowing physicians to prescribe medicine based on a patient’s genetic make-up.

Articular and growth plate cartilage both arise from condensations of mesenchymal cells, but ultimately develop important histological and functional differences. Each is composed of three layers-the superficial, mid and deep zones of articular cartilage and the resting, proliferative and hypertrophic zones of growth plate cartilage. The bone morphogenetic protein (BMP) system plays an important role in cartilage development. A gradient in expression of BMP-related genes has been observed across growth plate cartilage, likely playing a role in zonal differentiation. To investigate the presence of a similar expression gradient in articular cartilage, we used laser capture microdissection (LCM) to separate murine growth plate and articular cartilage from the proximal tibia into their six constituent zones, and used a solution hybridization assay with color-coded probes (nCounter) to quantify mRNAs for 30 different BMP-related genes in each zone. In situ hybridization and immunohistochemistry were then used to confirm spatial expression patterns. Expression gradients for Bmp2 and 6 were observed across growth plate cartilage with highest expression in hypertrophic zone. However, intracellular BMP signaling, assessed by phospho-Smad1/5/8 immunohistochemical staining, appeared to be higher in the proliferative zone and prehypertrophic area than in hypertrophic zone, possibly due to high expression of Smad7, an inhibitory Smad, in the hypertrophic zone. We also found BMP expression gradients across the articular cartilage with BMP agonists primarily expressed in the superficial zone and BMP functional antagonists primarily expressed in the deep zone. Phospho-Smad1/5/8 immunohistochemical staining showed a similar gradient. In combination with previous evidence that BMPs regulate chondrocyte proliferation and differentiation, the current findings suggest that BMP signaling gradients exist across both growth plate and articular cartilage and that these gradients may contribute to the spatial differentiation of chondrocytes in the postnatal endochondral skeleton.