Restriction Endonucleases EcoRI Research Articles

Analysis of lactoferin gene polymophism and its association to milk quality and mammary gland health in Holstein-Friesian cows

Lactoferrin (LTF) is a glycoprotein, a member of transferrin gene family which plays an important role in immune mechanisms in the mammary glands of cows. The amount of lactoferrin increases during inflammatory processes and viral infections. The aim of this investigation was to monitor the distribution of lactoferrin gene genotypes and its connection to milk quality and the occurrence of mammary gland diseases in 46 Holstein-Freisian cows of different age (2-7 years) on a farm near Belgrade. DNA was isolated from blood samples, and the polymorphism of lactoferrin gene was deterimined by PCR-RFLP method using the restriction enzyme Eco RI. We found two alelic forms of this gene in cows included in these experiments (A and B) and two genotypes (AA and AB) in a ratio 71.7% to 28.3%. The genotype BB was not found in this sample. In order to determine the degree of differences between genotypes we used discriminant analysis which has shown that there is a statistically significant difference between genotypes AA and with respect to productive parameters. When analyzed separately, the only parameter which differed significantly (p=0.021) between two genotypes was total milk production. Individuals with observed genotypes are most similar for the amount of milk fat (p=0.271). There is no statistically significant difference in the number of somatic cells in milk samples between the examined genotypes. [Projekat Ministarstva nauke Republike Srbije, br. III46002]

Progress towards Chemical Cross-Linking of Sequence-Specific Proteins to DNA Templates with Single-Molecule Sensitivity

Detecting the location of sequence-specific proteins along genetic DNA holds great promise for biomedical applications. For a number of single-molecule techniques, however, the relatively fast detachment rate (koff) of bound proteins is a limiting factor. In this study, we describe efforts to cross-link a sequence-specific protein covalently to its DNA template in order to form a stable construct for further analysis. As a model system, we study the common restriction enzyme EcoRI under non-cutting conditions. Fragments of DNA containing the recognition sequence are measured in the presence and absence of the protein under varying concentrations of chemical cross-linkers. We monitor ensemble binding stability using electophoretic mobility shift assays (EMSA) and at the single-molecule level with atomic force microscopy (AFM).

Molecular Techniques for Discrimination of Late Watergrass (Echinochloa oryzicola) and Early Watergrass (Echinochloa oryzoides) Species in Turkish Rice Production

Molecular techniques are useful tools for solving taxonomic confusion among species. Polymerase chain reaction (PCR) and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) methods were applied for the identification of barnyardgrass, early watergrass, and late watergrass. Total DNA was extracted from 266 accessions, which were collected from different rice growing areas of Turkey. The two primer sets (trn-a and trn-b1, and trn-c and trn-d) specific to a target region of the intergenic spacer between trnT (UGU) and trnL (UAA) and the entire intron region of trnL (UAA), respectively, were used in PCR amplifications. Of the 266 accessions of Echinochloa spp., only eight accessions gave a similar fragment size, which was slightly shorter than 495 bp. The PCR product obtained with the primers trn-a and trn-b1 gave two fragments when EcoRI restriction enzyme was used in barnyardgrass and early watergrass. However, not all accessions of late watergrass were digested with this enzyme. In contrast to EcoRI, the PCR product obtained using the trn-c and trn-d primer set was digested into two fragments by using AluI restriction enzyme in all accessions of late watergrass; whereas, it was not digested in barnyardgrass and early watergrass. This molecular differentiation among barnyardgrass, early watergrass, and late watergrass supports the hypothesis that late watergrass is not a synonym of early watergrass in Turkish accessions.

Total synthesis of a gene

The method developed for the total synthesis of a given DNA containing biologically specific sequences consists of the following. The DNA in the double-stranded form is carefully divided into short single-stranded segments with suitable overlaps in the complementary strands. All the segments are chemically synthesized starting with protected nucleosides and mono-nucleotides. The 5′-OH ends of the appropriate oligonucleotides are then phosphorylated with the use of [γ-32P]ATP and polynucleotide kinase. A few to several neighboring oligonucleotides are then allowed to form bihelical complexes in aqueous solution, and the latter are joined end to end by polynucleotide ligase to form covalently linked duplexes. Subsequent head-to-tail joining of the short duplexes leads to the total DNA. The methods are described for the construction of a biologically functional suppressor transfer RNA gene. The total work involved (i) the synthesis of a 126-nucleotide-long bihelical DNA corresponding to a known precursor to the tyrosine suppressor transfer RNA, (ii) the sequencing of the promoter region and the distal region adjoining the C-C-A end, which contained a signal for the processing of the RNA transcript, (iii) total synthesis of the 207 base-pair-long DNA, which included the control elements, as well as the Eco R1 restriction endonu-clease specific sequences at the two ends, and (iv) full characterization by transcription in vitro and amber suppressor activity in vivo of the synthetic gene.

FIRST REPORT OF THE OCCURRENCE OF EMILIA YELLOW VEIN VIRUS IN CRASSOCEPHALUM CREPIDIOIDES IN CHINA

In May of 2008, the wide occurrence of Emilia sonchifolia plants with yellow vein symptoms was observed in the Chinese province of Fujian. Symptomatic plants were infected with Emilia yellow vein virus (EmYVV) (Yang et al., 2008). Subsequently, the presence of thickhead plants (Crassocephalum crepidioides) show- ing foliar yellow vein symptoms was noticed in proximity to symp- tomatic E. sonchifolia. Total DNA was extracted from symptomatic C. crepidioides leaves and tested by rolling circle amplification (Haible et al., 2006). Amplification products were digested by the restriction enzyme EcoRI for 30 min. Restriction products (2.7 kb) were then cloned and sequenced. Comparison of DNA sequences by Clustal V analysis revealed the occurrence of EmYVV and an isolate denoted Fz12 was selected for further sequence analysis. The genome of EmYVV isolate Fz12 was 2,726 nt in size and had the typical genomic organization of begomoviral DNA-A (Gen- Bank accession No. JQ247188). A BLAST analysis of the NCBI database disclosed that isolate Fz12 had the highest nucleotide se- quence identity (99.67%) with that of an EmYVV isolate from E. sonchifolia (GenBank accession No. EU377539). To our knowl- edge, this is the first report of the natural occurrence of EmYVV in C. crepidioides, a common weed species in the People’s Republic of China.

Toward detection of DNA-bound proteins using solid-state nanopores: insights from computer simulations.

Through all-atom molecular dynamics simulations, we explore the use of nanopores in thin synthetic membranes for detection and identification of DNA binding proteins. Reproducing the setup of a typical experiment, we simulate electric field driven transport of DNA-bound proteins through nanopores smaller in diameter than the proteins. As model systems, we use restriction enzymes EcoRI and BamHI specifically and nonspecifically bound to a fragment of dsDNA, and streptavidin and NeutrAvidin proteins bound to dsDNA and ssDNA via a biotin linker. Our simulations elucidate the molecular mechanics of nanopore-induced rupture of a protein-DNA complex, the effective force applied to the DNA-protein bond by the electrophoretic force in a nanopore, and the role of DNA-surface interactions in the rupture process. We evaluate the ability of the nanopore ionic current and the local electrostatic potential measured by an embedded electrode to report capture of DNA, capture of a DNA-bound protein, and rupture of the DNA-protein bond. We find that changes in the strain on dsDNA can reveal the rupture of a protein-DNA complex by altering both the nanopore ionic current and the potential of the embedded electrode. Based on the results of our simulations, we suggest a new method for detection of DNA binding proteins that utilizes peeling of a nicked double strand under the electrophoretic force in a nanopore.

A chronocoulometric LNA sensor for amplified detection of K-ras mutation based on site-specific DNA cleavage of restriction endonuclease

An amplified chronocoulometric Locked nucleic acid (LNA) sensor (CLS) for selective electrochemical detection of K-ras mutation was developed based on site-specific DNA cleavage of restriction endonuclease EcoRI. Thiolated-hairpin LNA probe with palindrome structure of stem was immobilized on the gold nanoparticles modified gold electrode (NG/AuE). It can be cleaved by EcoRI in the absence of K-ras mutation-type DNA (complementary with the loop part of hairpin probe), but cannot be cleaved in the presence of mutation-type DNA. The difference before and after enzymatic cleavage was then monitored by chronocoulometric biosensor. Electrochemical signals are generated by chronocoulometric interrogation of Hexaammineruthenium (III) chloride (RuHex) that quantitatively binds to surface-confined hairpin LNA probe via electrostatic interactions. The results suggested this CLS had a good specificity to distinguish the K-ras mutation-type, wild-type and non-complementary sequence. There was a good linear relationship between the charge and the logarithmic function of K-ras mutation-type DNA concentration. The detection limit had been estimated as 0.5fM. It is possible to qualitatively and quantitatively detect K-ras point mutation in pancreatic cancer.

DNA-binding domains of plant-specific transcription factors: structure, function, and evolution

The families of the plant-specific transcription factors (TFs) are defined by their characteristic DNA-binding domains (DBDs), such as AP2/ERF, B3, NAC, SBP, and WRKY. Recently, three-dimensional structures of the DBDs, including those in complexes with DNA, were determined by NMR spectroscopy and X-ray crystallography. In this review we summarize the functional and evolutionary implications arising from structure analyses. The unexpected structural similarity between B3 and the noncatalytic DBD of the restriction endonuclease EcoRII allowed us to build structural models of the B3/DNA complex. Most of the DBDs of plant-specific TFs are likely to have originated from endonucleases associated with transposable elements. After the DBDs have been established in unicellular eukaryotes, they experienced extensive plant-specific expansion, by acquiring new functions.

STUDY ON THE BIOLOGICAL FUNCTIONS OF P21-ACTIVATED KINASE 5

ObjectivesPAK5 is a member of P21-activated kinase (PAK) family, which characterised by a highly conserved amino-terminal Cdc42/Rac interactive binding (CRIB) domain and a carboxyl terminal kinase domain. However, the role...

Characterization of conformation-dependent prion protein epitopes.

Whereas prion replication involves structural rearrangement of cellular prion protein (PrP(C)), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27-30), a protease-resistant counterpart of the pathogenic scrapie form (PrP(Sc)) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126-218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27-30) both renature to a common structure that reconstitutes the globular domain.

Fibroblast Growth Factor Receptor 2 Homodimerization Rapidly Reduces Transcription of the Pluripotency Gene Nanog without Dissociation of Activating Transcription Factors

Nanog or Gata6-positive cells co-exist and are convertible within the inner cell mass of murine blastocysts and embryonic stem (ES) cells. Previous studies demonstrate fibroblast growth factor receptor 2 (FGFR2) triggers Nanog gene down-regulation and differentiation to primitive endoderm (PE); however, the underlying mechanisms responsible for reversible and fluctuating cell fate are poorly understood. Using an inducible FGFR2 dimerization system in ES cells, we demonstrate that FGFR2 activation rapidly down-regulated Nanog gene transcription through activation of the Mek pathway and subsequently differentiated ES cells into PE cells. FGFR2 rather selectively repressed the Nanog gene with minimal effect on other pluripotency genes, including Oct4 and Sox2. We determined the Nanog promoter region containing minimum Oct4/Sox2 binding sites was sufficient for this transcriptional down-regulation by FGFR2, when the reporter transgenes were integrated with insulators. Of interest, FGFR2-mediated Nanog transcriptional reduction occurred without dissociation of RNA polymerase II, p300, Oct4, Sox2, and Tet1 from the Nanog proximal promoter region and with no increase in repressive histone methylation marks or DNA methylation, implying the gene repression is in the early and transient phase. Furthermore, addition of a specific FGFR inhibitor readily reversed this Nanog repression status. These findings illustrate well how FGFR2 induces rapid but reversible Nanog repression within ES cells.

The killing effect of NY-ESO-1-sensitzed dendritic cells-induced cellular toxic lymphocyte on human retinoblastoma cell

Background Cell immunologic therapy for retinoblastoma （RB）is becoming a hot research topic. Cancer-testis antigen is a human immunogenic protein and is used to treat some tumors. However,its effect on RB has not been investigated. Objective The present study was to discuss the antigen specific anti-tumor effect of cytotoxic T lymphocytes （CTL）induced by the cancer-testis antigen, NY-ESO-l-sensitized dendritic cells （DCs）, on human RB. Methods PCR was performed to amplify target gene fragments from the NY-ESO-1 plasmids,and then the target gene fragments were digested with the restriction enzymes SalI and EcoRI. Harvested fragments were inserted into the pDC316 plasmid to construct the recombinant plasmid pDC316/NY-ESO-1. The expression of NY-ESO-1 protein in human RB cells strain, HXO-RB44,was detected by immunofluoreseence and Western blot. Monoeytes wereisolated from 60 ml of peripheral blood from a healthy donor using Ficoll density-gradient centrifugation with a cell density of 1 x 107/ml. DCs isolated from blood were stimulated with recombinant human granulocyte-macrophage colony stimulating factor （rhGM-CSF） and recombinant human interleukin-4 （ rhIL-4 ）. The recombinant plasmid pDC316/NY-ESO-1 was transfected into DCs and the DCs were co-cultured with T lymphocytes. The resultant CTL were used as effector ceils. The growth of the CTL was detected by MTT assay. The CTL were then added into the growth medium used for culturing HXO-RB44 cells and the vitality of the HXO-RB44 cells was assayed by MTT assay. Results The sequence of the cloned DNA fragment of the recombinant plasmid pDC316/NY-ESO-lwas conforms with the sequence of the NY-ESO-1 gene. The expression of the NY-ESO-1 protein in HXO-RB44 cells was tested by immunofluorescence and Western blot. DCs were successfully induced with rhIL-4 and rhGM-CSF from PBMC. The recombinant expression plasmid pDC316/NY-ESO-1 was successfully transferred into DCs. These DCs had high expression of surface molecules such as HLA-DR （42. 1% ） , CD80 （54.2%） , CD83 （39.7%） and CD86 （94.8%）. The CTL that was induced by DCs-sensitized with NY-ESO-1 specifically killed HXO-RB44 cells. CTL induced by the sensitized DCs had a stronger cytotoxic effect against HXO-RB44 cells compared with un-sensitized DCs and CTL un-induced with DCs, as shown by MTT asssay（ P〈0.05 ）. The anti-tumor activity was highest when the ratio of effector to target was 75 ： 1 （ P〈0. 05 ）. Conclusions DCs transfected by the recombinant plasmid pDC316/ NY-ESO-1 can induce the proliferation of allogenic CTLs, which showed a specific anti-tunmr effect against HXORB44 cells. These results present a new type of immunotherapy for the treatment of RB. Key words: Retinoblastoma; Ddendritic cell; Cytotoxic T lymphocyte; NY-ESO-1

Cleavage and protection of locked nucleic acid–modified DNA by restriction endonucleases

Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA–modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving LNA–modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI.

Internucleosomal Interactions Mediated by Histone Tails Allow Distant Communication in Chromatin

Action across long distances on chromatin is a hallmark of eukaryotic transcriptional regulation. Although chromatin structure per se can support long-range interactions, the mechanisms of efficient communication between widely spaced DNA modules in chromatin remain a mystery. The molecular simulations described herein suggest that transient binary internucleosomal interactions can mediate distant communication in chromatin. Electrostatic interactions between the N-terminal tails of the core histones and DNA enhance the computed probability of juxtaposition of sites that lie far apart along the DNA sequence. Experimental analysis of the rates of communication in chromatin constructs confirms that long-distance communication occurs efficiently and independently of distance on tail-containing, but not on tailless, chromatin. Taken together, our data suggest that internucleosomal interactions involving the histone tails are essential for highly efficient, long-range communication between regulatory elements and their targets in eukaryotic genomes.

Elevated Cyclin G2 Expression Intersects with DNA Damage Checkpoint Signaling and Is Required for a Potent G2/M Checkpoint Arrest Response to Doxorubicin

To maintain genomic integrity DNA damage response (DDR), signaling pathways have evolved that restrict cellular replication and allow time for DNA repair. CCNG2 encodes an unconventional cyclin homolog, cyclin G2 (CycG2), linked to growth inhibition. Its expression is repressed by mitogens but up-regulated during cell cycle arrest responses to anti-proliferative signals. Here we investigate the potential link between elevated CycG2 expression and DDR signaling pathways. Expanding our previous finding that CycG2 overexpression induces a p53-dependent G(1)/S phase cell cycle arrest in HCT116 cells, we now demonstrate that this arrest response also requires the DDR checkpoint protein kinase Chk2. In accord with this finding we establish that ectopic CycG2 expression increases phosphorylation of Chk2 on threonine 68. We show that DNA double strand break-inducing chemotherapeutics stimulate CycG2 expression and correlate its up-regulation with checkpoint-induced cell cycle arrest and phospho-modification of proteins in the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) signaling pathways. Using pharmacological inhibitors and ATM-deficient cell lines, we delineate the DDR kinase pathway promoting CycG2 up-regulation in response to doxorubicin. Importantly, RNAi-mediated blunting of CycG2 attenuates doxorubicin-induced cell cycle checkpoint responses in multiple cell lines. Employing stable clones, we test the effect that CycG2 depletion has on DDR proteins and signals that enforce cell cycle checkpoint arrest. Our results suggest that CycG2 contributes to DNA damage-induced G(2)/M checkpoint by enforcing checkpoint inhibition of CycB1-Cdc2 complexes.

Tetracycline resistance genes in Pasteurella multocida isolates from bovine, ovine, caprine and swine pneumonic lungs originated from different Greek prefecturesa

The aim of the present work was to analyze the antibiotic resistance of Pasteurella multocida isolates from sheep, goats, cattle and pigs originated from eight different Greek prefectures, to determine the class(es) of the tet genes, to investigate any possible association with plasmids or/and chromosomes and to study the geographical distribution of the resistant isolates. Eighteen out to one hundred isolates were resistant to tetracycline with a minimal inhibitory concentration (MIC) higher than 128 μg/ml. The tetH gene was found in thirteen (72.2%) and the tetB gene was found in four isolates (22.2%). After treatment with the restriction enzymes EcoRV, EcoRI, PstI and BspHI, the tetB gene showed a new polymorphism. Partial sequencing of the tetH and tetB genes revealed five polymorphisms in the tetB gene and two polymorphisms in the tetH gene; phylogenetic and molecular evolutionary analyses were also conducted. Both tetB and tetH genes revealed patterns clearly differentiated from the rest of Pasteurella species meaning thatthe genes could be transferred from a non Pasteurella species. The results presented provide further knowledge and evidence of the role played by commensals as of resistance determinants. Key words: Pasteurella multocida, tet genes, multiple sequence analysis.

Detection of Coxsackievirus A16 by Reverse Transcription Loop-Mediated Isothermal Amplification

Aims: A one-step, single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and validated for the detection of Coxsackievirus A16 (CA16). Methods & materials: An optimized RT-LAMP assay was tested for in its sensitivity, primers specificity, products specificity and reproducibility. Results: The detection limit of the RT-LAMP assay was 106-fold dilution of stock virus or 81 copies in samples after RNA extraction, which was tenfold higher in sensitivity than the traditional reverse transcription PCR (RT-PCR) and equal to real-time RT-PCR. Digestion with a specific restriction enzyme EcoRI demonstrated that the amplified product was unique. The specificity of the assay was confirmed as it was demonstrated that the positive amplification only appeared among all CA16 stains, while no amplification was achieved in other viruses genetically related to hand, foot and mouth disease or similar clinical features. A good correlation between RT-LAMP and real-time RT-PCR was observed on the basis of the analysis of 33 clinical samples. Conclusion: RT-LAMP is a novel, alternative microbiological approach for rapid, sensitive and specific detection of CA16 in hand, foot and mouth disease.

Organelles genome stability of wheat plantlets produced by anther culture

Plantlets derived from in vitro culture might exhibit somoclonal variation which is often heritable, and molecular variations may be generated in vitro. Since the direction of most studies is toward nuclear genome, there is a little known about the DNA of organelles. This study was conducted to test the genetic stability of wheat organelles genomes for plantlets produced by anther culture using restriction fragment length polymorphism (RFLP) analyses. One of the intergenic regions of cpDNA and one of mtDNA introns were amplified with polymerase chain reaction (PCR). The PCR products were then sequenced and digested with four restriction endonucleases (EcoR1, BamH1, NdeI and Sac1). The amplified product from cpDNA was 1000 bp in size, and digested only with NdeI into two bands with 650 and 350 bp. The amplified product from mtDNA was 1550 bp in size, and digested only with Sac1 into two bands with 1220 and 330 bp. The results obtained showed that no noticeable difference can be detected between doubled haploid plantlets and parental plants at the level of ctDNA and mtDNA organization. It can be concluded that in vitro culture by itself does not systematically generate a cytoplasmic variation in plant cells.

PCR-RFLP and genetic diversity analysis of cpDNA in some species of the genus Salvia L.

Long Accurate polymerase chain reaction restriction fragment length polymorphism (LA-PCR-RFLP) of the chloroplast DNA (cpDNA) was used to investigate phylogenetic relationships between 54 species of Salvia L. Two Ocimum L. species were used as an out group. A pair of universal primers in LA-PCR protocol resulted in long cpDNA of an expected size (∼15 kilo base pair (kb)) from each Salvia and Ocimum species, except S. x superba (hybrid; S. sylvestris x S. amplexicaulis or S. nemorosa). LA-PCR products were separately digested with each of EcoRI, HindIII, PvuII, NdeI, SmaI or MfeI restriction enzymes. S. roemeriana Scheele showed amplifi ed fragment length difference (AFLD). S. roemeriana and/or S. lyrata L. could be identifi ed with any of the enzymes used except PvuII. Identifi cation of Ocimum species from Salvia could be carried out with NdeI, SmaI or MfeI. EcoRI and MfeI exhibited more polymorphic and informative patterns than HindIII, PvuII, NdeI or SmaI. LA-PCR-RFLP analysis generated 28 polymorphic restriction fragments. Fragments were visually detected, photographed and used to build a genetic similarity data matrix based on Nei and Li (1979) method. PHYLIP software package (Felsenstein 2005) and the genetic similarity matrix were exploited to construct a phylogenetic tree. The phenogram generated by UPGMA clustering analysis separated Salvia species based on genetic distance into distinct groups obviously dependent on origin.

Stabilizing Labile DNA-Protein Complexes in Polyacrylamide Gels

The electrophoretic mobility shift assay (EMSA) is one of the most popular tools in molecular biology for measuring DNA-protein interactions. The technique uses polyacrylamide gel electrophoresis to separate DNA-protein or RNA-protein complexes from free DNA or RNA. Polyacrylamide gels stabilize DNA-protein, RNA-protein, or protein-protein complexes by a crowding or caging mechanism. Still every technique has its limitations. EMSA, as standardly practiced today, works well for complexes with association binding constants Ka>109 M−1 under normal conditions of salt and pH. Many DNA-protein complexes are not stable enough so that they dissociate while moving through the gel matrix giving smeared bands that are difficult to quantitate reliably. We take advantage of our previous observation that neutral osmolytes can strongly slow down the rate of DNA-protein complex dissociation to develop a method that uses osmotic stress to stabilize complexes in the gel matrix as well as in the solution. In this work we demonstrate that the addition of the osmolyte triethylene glycol to polyacrylamide gels dramatically stabilizes labile restriction endonuclease EcoRI complexes with nonspecific DNA sequences enabling quantitation of binding using the electrophoretic mobility shift assay. The significant improvement of the technique resulting from the addition of osmolytes to the gel matrix greatly extends the range of binding constants of protein-DNA complexes that can be investigated using this widely used assay. Extension of this approach to other techniques used for separating bound and free components such as gel chromatography and capillary electrophoresis is straightforward.