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Optical DNA mapping (ODM) is based on fluorescent labeling, stretching and imaging of single DNA molecules to obtain sequence-specific fluorescence profiles, DNA barcodes. These barcodes can be mapped to theoretical counterparts obtained from DNA reference sequences, which in turn allow for DNA identification in complex samples and for detecting structural changes in individual DNA molecules. There are several types of DNA labeling schemes for ODM and for each labeling type one or several types of match scoring methods are used. By combining the information from multiple labeling schemes one can potentially improve mapping confidence; however, combining match scores from different labeling assays has not been implemented yet. In this study, we introduce two theoretical methods for dealing with analysis of DNA molecules with multiple label types. In our first method, we convert the alignment scores, given as output from the different assays, into p-values using carefully crafted null models. We then combine the p-values for different label types using standard methods to obtain a combined match score and an associated combined p-value. In the second method, we use a block bootstrap approach to check for the uniqueness of a match to a database for all barcodes matching with a combined p-value below a predefined threshold. For obtaining experimental dual-labeled DNA barcodes, we introduce a novel assay where we cut plasmid DNA molecules from bacteria with restriction enzymes and the cut sites serve as sequence-specific markers, which together with barcodes obtained using the established competitive binding labeling method, form a dual-labeled barcode. All experimental data in this study originates from this assay, but we point out that our theoretical framework can be used to combine data from all kinds of available optical DNA mapping assays. We test our multiple labeling frameworks on barcodes from two different plasmids and synthetically generated barcodes (combined competitive-binding- and nick-labeling). It is demonstrated that by simultaneously using the information from all label types, we can substantially increase the significance when we match experimental barcodes to a database consisting of theoretical barcodes for all sequenced plasmids.

Background Craniosynostosis is one of the major genetic disorders affecting 1 in 2,100–2,500 live newborn children. Environmental and genetic factors are involved in the manifestation of this disease. The suggested genetic causes of craniosynostosis are pathogenic variants in FGFR1, FGFR2, FGFR3, and TWIST1 genes. Methods In order to describe their major clinical characteristics and the presence of pathogenic variants, a sample of 36 Mexican patients with craniosynostosis diagnosed as: Crouzon (OMIM 123,500), Pfeiffer (OMIM 101,600), Apert (OMIM 101,200), Saethre‐Chotzen (OMIM 101,400), and Muenke (OMIM 602,849) was analyzed. Results In addition to craniosynostosis, most of the patients presented hypertelorism, midface hypoplasia, and abnormalities in hands and feet. To detect the pathogenic variants p.Pro252Arg FGFR1 (OMIM 136,350), p.Ser252Trp, p.Pro253Arg FGFR2 (OMIM 176,943), p.Pro250Arg, FGFR3 (OMIM 134,934), and p.Gln119Pro TWIST1 (OMIM 601,622), PCR amplification and restriction enzyme digestion were performed. Four and two patients with Apert presented the pathogenic variants p.Ser252Trp and p.Pro253Arg in FGFR2, respectively (with a frequency of 11.1% and 5.5%). The p.Pro250Arg pathogenic variant of FGFR3 was found in a patient with Muenke (with a frequency of 2.8%). The above percentages were calculated with the total number of patients. Conclusion The contribution of this work is discreet, since only 4 genes were analyzed and sample size is small. However, this strategy could be improved by sequencing the FGFR1, FGFR2, FGFR3, and TWIST1 genes, to determine different pathogenic variants. On the other hand, it would be important to include other genes, such as TCF12 (OMIM 600,480), MSX2 (OMIM 123,101), RAB23 (OMIM 606,144), and EFNB1 (OMIM 300,035), to determine their participation in craniosynostosis in the Mexican population., We describe the major clinical characteristics of Mexican patients with craniosynostosis. Analysis of FGFR1, FGFR2, FGFR3, and TWIST1 genes revealed a higher frequency of variants in Apert.

Restriction endonucleases play a central role in the microbial immune system against viruses and are widely used in DNA specific cleavage, which is called restriction digestion, for genetic engineering. Herein, we applied digital cell-free protein synthesis as an easy-to-use orthogonal readout means to assess the restriction digest efficiency, a new application of digital bioassays. The digital counting principle enabled an unprecedentedly sensitive trace analysis of undigested DNA at the single-molecule level in a PCR-free manner. Our approach can quantify the template DNA of much lower concentrations that cannot be detected by ensemble-based methods such as gold-standard DNA electrophoresis techniques. The sensitive and quantitative measurements revealed a considerable variation in the digest efficiency among restriction endonucleases, from less than 70% to more than 99%. Intriguingly, none of them showed truly complete digestion within reasonably long periods of reaction time. The same rationale was extended to a multiplexed assay and applicable to any DNA-degrading or genome-editing enzymes. The enzyme kinetic parameters and the flanking sequence-dependent digest efficiency can also be interrogated with the proposed digital counting method. The absolute number of residual intact DNA molecules per microliter was concluded to be at least 107, drawing attention to the residual issue of genetic materials associated with the interpretation of nucleases’ behaviors and functions in daily genetic engineering experiments.

Sulfadoxine-pyrimethamine (SP) is the recommended drug for intermittent preventive treatment of malaria in pregnancy in most of sub-Saharan Africa. Resistance to SP is related to mutations in the dhfr and dhps gene of Plasmodium falciparum. This study determined the prevalence of Pfdhfr and Pfdhps polymorphisms found in asymptomatic pregnant women attending antenatal care in Calabar, Nigeria. From October 2013 to November 2014, asymptomatic pregnant women attending antenatal care clinics were enrolled after obtaining informed consent. Malaria diagnosis testing was done using thick and thin smears. Dried blood spot filter papers were collected. Parasite DNA was extracted from the filter papers using a chelex extraction. Extraction was followed by nested PCR and restriction enzyme digestion. P. falciparum infection was detected by microscopy in 7% (32/459) participants. Twenty-eight P. falciparum isolates were successfully genotyped. In the Pfdhfr gene, the triple mutation was almost fixed; S108N mutation was (100%), N51I (93%) and C59R mutations (93%), whereas the I164L mutation was absent. The prevalence of Pfdhps S436A, A437G, A581G and A613S mutations was 82.1% (23/28), 96.4% (27/28), 71.4% (20/28) and 71.4% (20/28) respectively. The K540E mutation was absent. The prevalence of the Pfdhfr triple mutation IRNI was 92.9% (26/28). The efficacy of SP as IPTp in Southeast Nigeria may be severely threatened. The continuous monitoring of SP molecular markers of resistance is required to assess thresholds. The evaluation of alternative preventive treatment strategies and drug options for preventing malaria in pregnancy may be necessary.

Vector integration site analysis can be important in the follow-up of patients who received gene-modified cells, but current platforms based on next-generation sequencing are expensive and relatively inaccessible. We analyzed polyclonal T cells transduced by a gammaretroviral vector, SFG.iCasp9.2A.ΔCD19, from a clinical trial. Following restriction enzyme digestion, the unknown flanking genomic sequences were amplified by inverse polymerase chain reaction (PCR) or cassette ligation PCR. Nanopore sequencing could identify thousands of unique integration sites within polyclonal samples, with cassette ligation PCR showing less bias. The assay is scalable and requires minimum capital, which together enable cost-effective and timely analysis.

This protocol is used to check if the two selected restriction enzymes can perform effective catalysis in the same solution.

β-actin gene is a kind of actin, in a variety of cells and tissues, β-actin gene expression is relatively stable [1], and β-actin gene in nuclear DNA has multiple copies, increased The detection rate of nuclear DNA [2], which is often used as an internal reference in PCR. In this experiment, the β-actin gene with good purity was obtained by PT-PCR, amplification, cloning, screening and PCR identification and restriction enzyme digestion of the RNA extracted from the mouse liver, which laid the foundation for the later experiment.

Background Group A Streptococcus strains causing wide variety of diseases, recently became noticeable in eastern India, are not amenable to standard treatment protocol thus enhancing the possibility of disease morbidity by becoming antibiotic resistance. Methods The association of Lancefield group A Streptococcal variation with degree of vir architectural diversity was evaluated using emm typing and restriction fragment length polymorphism analyses. The antibiotic sensitivity patterns were examined by modified Kirby-Bauer method of disk diffusion. Percentage calculations, 95% confidence interval and one-way ANOVA were used to assess differences in proportions. Results Our observations revealed 20 different emm types and 13 different HaeIII vir typing patterns. A 1.2 kb fragment was found in all HaeIII typing pattern. Fragments of 1.2 kb and 550 bp were conserved in majority of the isolates. HinfI digestion was found proficient in differentiating the strains of same vir typing patterns. Strong predominance of speC (85%) and speF (80%) genes have been observed encoding exotoxins production. 4 isolates were found to be erythromycin resistant and were of genotype emm49. High degree of tetracycline resistance was shown by 53.57% isolates which belonged to 12 different emm genotypes. Conclusions These findings suggested that in addition to emm typing, sequential application of HaeIII and HinfI restriction enzymes in vir typing analysis is an effective tool for group A streptococcal molecular characterization associated with antibiotic resistance. Electronic supplementary material The online version of this article (doi:10.1186/s12879-016-2079-9) contains supplementary material, which is available to authorized users.

We report a microfluidic system for automatic mitochondrial mutation diagnostics from sample purification to quantitative analysis. The system achieved direct DNA (mtDNA) mutation quantification in affected cells using a new 3D-microfluidic system, which integrated a mtDNA extraction module and a mutation detection module. Effective direct mtDNA extraction from the cells was realized using magnetic field manipulation. The obtained mtDNAs were subject to a fully automatic processing for quantitative mutation detection using integrated micropumps, micromixer and microtemperature control modules capable of mutation sensing by restriction enzyme digestion and real-time on-chip micro-PCR. Compared with traditional methods, this microfluidic system demonstrates the advantages of faster detection, requirement of fewer amount of specimens and reagents, much compact design and lower cost as well as lower risks for human errors. Thus, such system-on-chip would encourage the future translational development of rapid pathogenic mtDNA defects detection to provide more efficient clinical diagnosis and disease management strategies.

Rhodococcus are gram-positive bacteria, which can exist in two different shapes rod and cocci. A number of studies have been done in the past on replication and stability of small plasmids in this bacterium; however, there are no reports on spatial localization and segregation of these plasmids. In the present study, a low copy number plasmid pDS3 containing pRC4 replicon was visualized in growing cells of Rhodococcus erythropolis PR4 (NBRC100887) using P1 parS-ParB-GFP system. Cells were initially cocci and then became rod shaped in exponential phase. Cocci cells were found to be non-replicating as evident by the presence of single fluorescence focus corresponding to the plasmid and diffuse fluorescence of DnaB-GFP. Rod shaped cells contained plasmid either present as one fluorescent focus observed at the cell center or two foci localized at quarter positions. The results suggest that the plasmid is replicated at the cell center and then it goes to quarter position. In order to observe the localization of plasmid with respect to nucleoid, plasmid segregation was also studied in filaments where it was found to be replicated at the cell center in a nucleoid free region. To the best of our knowledge, this is the first report on segregation of small plasmids in R. erythropolis.

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Introduction: Glucose-6-phosphate dehydrogenase (G6PD) deficiency (Gd-) contributes to morbidity and mortality in sub-Saharan Africa but recent data on the interaction between Gd- and malaria among children is scarce. We hypothesised that, being a haemolytic factor, Gd- makes severe malarial anaemia (SMA) more common and even more severe. Methodology: We selected 930 children aged 0.5-12 years attending a reference hospital with microscopically proven falciparum malaria. G6PD and haemoglobin were typed by the fluorescent spot test and electrophoresis, respectively. Molecular typing by PCR and restriction enzyme digestion was also performed on 15% of randomly selected samples. Haematocrit (PCV) values, haemoglobin type, blood group, presence of sickle cell trait (HbAS), and parasite counts were compared between G6PD-normal and deficient children. Results: Prevalence of Gd- was 16.4% and 8.1% among boys and girls with malaria, respectively. Mean PCV was 22.8% in deficient children compared with 21.0% in normal children (p=0.041). In boys, 2.7% of Gd- had PCV ≤10%, as compared to 13.6% in Gd+ (p = 0.005). Similarly, 21.3% of Gd- had PCV ≤15% compared with 39.4% in Gd+ (p=0.003). No such difference was found among girls. Overall, HbAS was typed in 7.6% and was more common in Gd- (13.0%) than in Gd+ (6.8%), but the difference was not statistically significant (p=0.058). The mean parasite counts were significantly lower in Gd- (15477.5/μl) than in Gd+ (19784.4/μl; p=0.013), and it was independent from HbAS. Conclusion: Gd- males but not females were significantly less likely to develop severe malarial anaemia.

Background: An early initiation of antifungal therapy in invasive fungal infections (IFIs) is critical in reducing the high mortality rate. Current diagnosis of fungal infection relies on microscopy, culture, antigen, antibody specific tests and histological diagnosis. However, these tests either lack sensitivity or specificity. There is thus the need for a rapid, specific and accurate diagnostic method. Objective: The aim of our study was to establish PCR for the rapid detection of Candida and Aspergillus species in clinical specimens with improved sensitivity and specificity. Materials and Methods: A total of 71 proven cases of IFI (confirmed by culture) were collected. A total of 15 healthy, 15 patients suffering from bacterial sepsis and 15 patients with HIV, HBV viral infections were included as controls. Clinical specimens were subjected to a standardized nested amplification to produce Round I (504 bp) and Round II (150 bp) amplicons. Restriction digestion was performed on these products for further identification. Results: Analytical sensitivity was determined using 10 6 -10 CFU/ml of cell suspension. The lower detection limit of the assay was 10 CFU/ml of blood. This test was 100% sensitive and specific with a positive predictive value of 100% and a negative predictive value of 96.7%. Conclusion: The assay was found to be effective for the rapid detection of Candida and Aspergillus in clinical specimens.

Ureaplasma parvum serovar 3 strain, OMC-P162, was isolated from the human placenta of a preterm delivery at 26 weeks’ gestation. In this study, we sequenced the complete genome of OMC-P162 and compared it with other serovar 3 strains isolated from patients with different clinical conditions. Ten unique genes in OMC-P162, five of which encoded for hypothetical proteins, were identified. Of these, genes UPV_229 and UPV_230 formed an operon whose open reading frames were predicted to code for a DNA methyltransferase and a hypothetical protein, respectively. DNA modification analysis of the OMC-P162 genome identified N4-methylcytosine (m4C) and N6-methyladenine (m6A), but not 5-methylocytosine (m5C). UPV230 recombinant protein displayed endonuclease activity and recognized the CATG sequence, resulting in a blunt cut between A and T. This restriction enzyme activity was identical to that of the cultivated OMC-P162 strain, suggesting that this restriction enzyme was naturally expressed in OMC-P162. We designated this enzyme as UpaP162. Treatment of pT7Blue plasmid with recombinant protein UPV229 completely blocked UpaP162 restriction enzyme activity. These results suggest that the UPV_229 and UPV_230 genes act as a type II restriction-modification system in Ureaplasma OMC-P162.

Discriminating between eel species of the genus Anguilla using morphological characteristics can be problematic, particularly in the glass eel and elver stages. In this study, sequence‐characterized amplified region (SCAR) and polymerase chain reaction (PCR)‐restriction fragment length polymorphism (RFLP) markers were developed for the identification of Anguilla japonica, Anguilla bicolor bicolor, Anguilla rostrata, and Anguilla anguilla. Random amplified polymorphic DNA (RAPD) fragments from A. japonica (362 bp), A. bicolor bicolor (375 bp), A. rostrata (375 bp), and A. anguilla (375 bp) were isolated, sequenced, and converted to SCAR markers. The principal difference between the SCARs of A. japonica and the three other species is the absence of a 13 bp deletion in the A. japonica SCAR. Specific PCR primers amplified a 290 bp fragment for A. japonica and 303 bp fragments for A. bicolor bicolor, A. rostrata, and A. anguilla. Restriction enzyme digestion with TaqI, MaeI, and Tru9I yielded PCR‐RFLP...

Phage display libraries of human single-chain variable fragments (scFvs) are a reliable source of fully human antibodies for scientific and clinical applications. Frequently, scFvs form the basis of larger, bivalent formats to increase valency and avidity. A small and versatile bivalent antibody fragment is the diabody, a cross-paired scFv dimer (∼55 kDa). However, generation of diabodies from selected scFvs requires decreasing the length of the interdomain scFv linker, typically by overlap PCR. To simplify this process, we designed two scFv linkers with integrated restriction sites for easy linker length reduction (17-residue to 7-residue or 18-residue to 5-residue, respectively) and generated two fully human scFv phage display libraries. The larger library (9 × 10(9) functional members) was employed for selection against a model antigen, human N-cadherin, yielding novel scFv clones with low nanomolar monovalent affinities. ScFv clones from both libraries were reformatted into diabodies by restriction enzyme digestion and re-ligation. Size-exclusion chromatography analysis confirmed the proper dimerization of most of the diabodies. In conclusion, these specially designed scFv phage display libraries allow us to rapidly reformat the selected scFvs into diabodies, which can greatly accelerate early stage antibody development when bivalent fragments are needed for candidate screening.

Ophiopogon japonicus is a herbaceous perennial plant in Liliaceae, and its tubers are used in traditional Japanese medicine as Bakumondo, prescribed for treating cough, sputum, and thirst. Liriope is a genus of ornamental plants related to Ophiopogon, and its tubers are used in folk medicine as well. Although tubers from both genera are traded in Korean and Chinese markets, only O. japonicus is defined as the plant of origin for Bakumondo in the Japanese Pharmacopoeia [1], and Liriope tubers cannot legally be used as Bakumondo in Japan. Ophiopogon plants can be distinguished clearly from Liriope by their fruit color and by the morphological characteristics of their flowers. However, the tubers of both species are greatly similar, making it very difficult to differentiate the two genera by the appearance of their tubers. We, therefore, investigated the most appropriate DNA regions to use for practical and accurate identification of Ophiopogon and Liriope tubers. The sequence of the gene for the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (rbcL) was found to be suitable for discriminating Ophiopogon and Liriope tubers. The identification procedure was simplified using restriction enzyme digestion of the amplified rbcL fragment. The detection limit for Liriope contamination was estimated by performing the procedure using mixed samples of powdered Ophiopogon and Liriope tubers.

The Southern flying squirrel (Glaucomys volans) is a species designated at risk in Canada where its range is restricted to parts of Ontario, Quebec and Nova Scotia. Before this study, its distribution in Nova Scotia was poorly documented, with only seven site records. Based on live-trapping and intact and partial specimens provided by the public, we present data for 28 additional locations; these combined with historic records delineate a disjunct range that is more extensive than previously believed, but limited to southwest Nova Scotia. To identify specimens that were not fully intact, simple morphological and molecular techniques were employed. The latter, which consisted of PCR amplification and then restriction enzyme digestion of the cytochrome-b gene, allowed reliable species identification of tree squirrels from Nova Scotia by use of partial specimens.

Identifying the reservoirs of a pathogen is vital for control of sporadic disease and epidemics. Listeria monocytogenes is a zoonotic foodborne pathogen that is responsible for 28% of food-related deaths in the United States annually, as well as a major cause of massive product recalls worldwide. To examine the role of the dairy farm as a potential source or reservoir for L. monocytogenes subtypes shown to cause human listeriosis, we compared the pulsed-field gel electrophoresis (PFGE) restriction enzyme digestion profiles of L. monocytogenes dairy farm-associated strains (milk, environmental, and bovine) to human sporadic and epidemic disease strains. Twenty-three percent of human sporadic strains had PFGE patterns identical to that of farm isolate(s). Additionally, three farm environmental strains and one human sporadic strain had a PFGE pattern identical to a strain of L. monocytogenes responsible for the 1985 California epidemic. These data indicate that this epidemic strain continues to cause sporadic human illness and has a potential dairy farm as a reservoir.

The prevalence of Listeria monocytogenes in bulk milk from three Pacific Northwest states was assessed for 474 herds at three time points. For samples collected in November 2000 and June 2001, the L. monocytogenes prevalence levels were 4.9 and 7.0%, respectively. All isolates were subtyped by serotyping and by pulsed-field gel electrophoresis (PFGE). Forty-nine of the 55 isolates belonged to serogroup 1/2a, while 6 belonged to serogroup 4. Subtyping by PFGE revealed that isolates from 31 herds shared 10 patterns; there was a weak but significant association between PFGE subtype and geographical distance. Six herds were positive for L. monocytogenes at both time points. Of these six herds, four had indistinguishable PFGE patterns at both time points. Twenty-five of the 33 herds that were positive in June 2001 were sampled again in June 2002. L. monocytogenes was recovered from 17 of these 25 herds (68%), with the ApaI restriction enzyme digestion profiles (REDP) for 8 herds being identical to those of isolates recovered from these herds the previous year. The ApaI REDP for the bulk milk isolates were compared with those for isolates recovered from environmental and human samples that were collected by the Washington Department of Health (n = 23). Analysis of ApaI digestion profiles revealed that only two of the Washington Department of Health isolates had digestion profiles similar to those for isolates from bulk milk; however, further analysis with the use of a second enzyme (AscI) was capable of discriminating between isolates from the two sources. Thus, we found no direct REDP matches between bulk milk and clinical isolates.

Summary In this study, we have demonstrated that two independent loci are involved in the integration of the insecticidal protein gene cryIAb/cryIAc and selectable marker gene hph in the recipient genome of the elite Chinese CMS restorer line Minghui 63. We have also documented the structural organization of these transgenes in each locus by restriction enzyme digestion and Southern blot analysis. The independent locus integration of different transgenes allowed us to remove the selectable marker gene hph from the gene of interest simply by self-segregation. Not having the selectable marker gene will enhance the commercial value of our transgenic line TT51-1, which showed a consistently high level of resistance against repeated infestations of yellow stem borers and natural outbreaks of leaf-folders, without a reduction in yield potential. Received 24 June 2002; revised 21 October 2002; accepted 26 November 2002. * Correspondence (fax +632 845 0606; e-mail S.Datta@cgiar.org) Keywords

Forty-five velogenic Newcastle disease virus strains isolated in Germany between 1939 and 1995 were analysed by restriction enzyme digestion and sequencing to shed light on the relationships of past epizootics. Viruses derived from the period prior to 1970 belonged to a clade ( IVea) of genotype IV comprising the earliest isolates from Europe, and could be isolated until the late seventies from poultry. Essex' 70-like viruses, the prototype of genotype V, were already present at the beginning of the 1970-74 epizootic and in sporadic cases thereafter, indicating that these Newcastle disease outbreaks started in Western Europe. A genotype VI ( subtype VIc) isolate was obtained in the early 1980s from a single outbreak in poultry. Outbreaks between 1993-95 were again part of a Western European epizootic caused by a genotype VIIa virus that was prevalent in the Far East

Summary. The genetic variability of porcine reproductive and respiratory syndrome virus (PRRSV) was studied by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified fragments among 50 Korean isolates from open reading frame 5. All Korean PRRSVs were isolated from the field cases after the marketing of an U.S. ATCC VR2332-derived modified live PRRSV vaccine. Combining the restriction enzyme digestion patterns obtained with MluI, HincII, SacII, and HaeIII, we observed 19 distinct RFLP patterns. Seventeen out of 50 PRRSV isolates (34%) exhibited the modified live PRRSV vaccine RFLP pattern. The genomic variations that have been identified in the present study seemed to represent characteristic features of the Korean PRRSV isolates. PCR-based RFLP analysis using several restriction enzymes provides a good genetic estimate for isolate differentiation.

Polston, J. E., McGovern, R. J., and Brown, L. G. 1999. Introduction of tomato yellow leaf curl virus in Florida and implications for the spread of this and other geminiviruses of tomato. Plant Dis. 83:984-988. In July 1997, symptoms characteristic of tomato yellow leaf curl virus (TYLCV-Is) were observed on one tomato plant in a field in Collier C ounty, Florida, and on several tomato plants in a retail garden center in Sarasota, Florida. Amplification with three sets of primers, analysis of amplified fragments using restriction enzyme digestion, and hybridization with a clone of TYLCV-Is indicated that TYLCV-Is was present in symptomatic plants. The sequence of a 1,300-bp amplified fragment was 99% identical to TYLCV-Is from the Dominican Republic and 98% identical to an isolate from Israel. It appears that TYLCV-Is entered the United States in Dade County, Florida, in late 1996 or early 1997. Subsequently, infected tomato transplants produced for retail sale at two Dade County fac ilities were rapidly distributed via retail garden centers throughout the state. Infected plants purchased by homeowners and pl aced in and around homes appeared to be the source of TYLCV-Is for nearby commercial nurseries and production fields. It appears that transplants have played a role in the movement of this and probably other geminiviruses. A number of regulatory procedures, as well as field management practices, were implemented in the 1997-98 production season to minimize the movement of TYLCV-Is within and out of the state.

BackgroundFriedreich ataxia (FRDA) is caused by a homozygous GAA repeat expansion mutation within intron 1 of the FXN gene, which induces epigenetic changes and FXN gene silencing. Bisulfite sequencing studies have identified 5-methylcytosine (5 mC) DNA methylation as one of the epigenetic changes that may be involved in this process. However, analysis of samples by bisulfite sequencing is a time-consuming procedure. In addition, it has recently been shown that 5-hydroxymethylcytosine (5 hmC) is also present in mammalian DNA, and bisulfite sequencing cannot distinguish between 5 hmC and 5 mC.Methodology/principal findingsWe have developed specific MethylScreen restriction enzyme digestion and qPCR-based protocols to more rapidly quantify DNA methylation at four CpG sites in the FXN upstream GAA region. Increased DNA methylation was confirmed at all four CpG sites in both FRDA cerebellum and heart tissues. We have also analysed the DNA methylation status in FRDA cerebellum and heart tissues using an approach that enables distinction between 5 hmC and 5 mC. Our analysis reveals that the majority of DNA methylation in both FRDA and unaffected tissues actually comprises 5 hmC rather than 5 mC. We have also identified decreased occupancy of the chromatin insulator protein CTCF (CCCTC-binding factor) at the FXN 5' UTR region in the same FRDA cerebellum tissues.Conclusions/significanceIncreased DNA methylation at the FXN upstream GAA region, primarily 5 hmC rather than 5 mC, and decreased CTCF occupancy at the FXN 5' UTR are associated with FRDA disease-relevant human tissues. The role of such molecular mechanisms in FRDA pathogenesis has now to be determined.

A new method was developed to detect DNA methylation in the Citrus genome using random amplification coupled with restriction enzyme digestion. Genomic DNA from Citrus grandis (L.) Osb., Poncirus trifoliata (L.) Raf., and their F1hybrid was amplified using 7 individual 10-mer random primers. Prior to amplification the DNA templates were digested with 2 pairs of restriction endonucleases (HpaII–MspI and (or) Sau3AI–NdeII) with different sensitivities to cytosine methylation and after PCR amplification their amplified products were further digested with the same enzymes. Using this method, it was possible to detect 28 methylation events involving 23 amplified bands with the 7 random primers and 2 pairs of enzymes. A methylation polymorphism was found at a Sau3AI site in a 1.2-kb band amplified with one primer. One locus influencing cytosine methylation at this restriction site was identified through genetic analysis of a BC1population between C. grandis and P. trifoliata and was mapped to linkage group IV using an already developed core map. This technique for detecting methylation and methylation polymorphisms is simple and should be applicable to any eukaryotic species and to many situations where it is desirable to determine whether a sequence is methylated. Key words : Citrus grandis, Poncirus trifoliata, restriction endonuclease, polymerase chain reaction, RAPD.

High-throughput sequencing continues to produce an immense volume of information that is processed and assembled into mature sequence data. Data analysis tools are urgently needed that leverage the embedded DNA sequence polymorphisms and consequent changes to restriction sites or sequence motifs in a high-throughput manner to enable biological experimentation. CisSERS was developed as a standalone open source tool to analyze sequence datasets and provide biologists with individual or comparative genome organization information in terms of presence and frequency of patterns or motifs such as restriction enzymes. Predicted agarose gel visualization of the custom analyses results was also integrated to enhance the usefulness of the software. CisSERS offers several novel functionalities, such as handling of large and multiple datasets in parallel, multiple restriction enzyme site detection and custom motif detection features, which are seamlessly integrated with real time agarose gel visualization. Using a simple fasta-formatted file as input, CisSERS utilizes the REBASE enzyme database. Results from CisSERS enable the user to make decisions for designing genotyping by sequencing experiments, reduced representation sequencing, 3'UTR sequencing, and cleaved amplified polymorphic sequence (CAPS) molecular markers for large sample sets. CisSERS is a java based graphical user interface built around a perl backbone. Several of the applications of CisSERS including CAPS molecular marker development were successfully validated using wet-lab experimentation. Here, we present the tool CisSERS and results from in-silico and corresponding wet-lab analyses demonstrating that CisSERS is a technology platform solution that facilitates efficient data utilization in genomics and genetics studies.

Bioinformatics has emerged as an integral part of life sciences and biomedical research. The bioinformatics tools developed so far exist individually and do not cross talk leading biologists to spend more time in formatting the output from one tool as input for another tool. This leads to huge loss of time and cost. We herein have made platform which integrates the tools in a way that the output of one program can be directly used as input of another and does not need any modifications. Tools for similarity search, primer designing, and restriction enzyme digestion are required in almost all biological research; therefore we initially tried to integrate these tools. BioParisodhana platform optimizes the time spend in browsing and downloading applications and is an interactive, effective and user friendly.The database is available for free at http://resource.ibab.ac.in/bioparishodhana.html.

slow type of an electrophoretic variant of lactate dehydrogenase (LDH)-B(H) subunit was analyzed for genetic mutation. DNA analysis of the variant alleles detected a base substitution, an A to T transition at codon 320 (GAT→GTT). The mutation resulted in the replacement of an aspartic acid by valine (D 320V), and in a change of electrophoretic charge. The change may cause the net charge of the variant subunit resulting in an electrophoretic B subunit variant of the slow type. The missense mutation created a new restriction site GTNAC for endonuclease Mae III in exon 7 of the LDH-B mutant gene. Thus, we can easily detect and confirm the missense mutation.

slow 型乳酸脱水素酵素 (LDH) B(H) サブユニットバリアントの1症例のDNA解析を行った結果, コドン320番でAからTへの点変異がみいだされた. これによりアスパラギン酸からバリンに置換され, 陰性荷電がひとつ消失し表面荷電がプラスに変位したサブユニットが合成され, 結果的に slow 型のBサブユニットバリアントの表現型をとったと考えられた. この変異によりMae IIIの認識配列が形成されるため, 容易に検出・確認が可能である.

We have recently reported the DNA analysis of ABO blood group genotypes by using polymerase chain reaction followed by restriction enzyme digestion according to the method of Yamamoto et al (Nature 345: 299-233, 1990). In this paper, we describe the distribution on ABO genotype in Nara prefecture. Genomic DNAs were obtained from 253 healthy volunteers which include 100-phenotype A, 100-phenotype B, 32-phenotype O, and 21-phenotype AB. The theoretical distribution of genotype AA was 20.8%, and that of BB was 14.3% based on the calculation of Hardy-Weinberg equation in Nara prefecture. DNA anlaysis revealed that the value of genotype AA was 17%, and that of BB 14%. This result was in good accord with the theoretical values. Then, we investigated the ABO genotype status in 5 Japanese families including 25 familial members, of which parents showed the following phenotypes; B×B, O×AB, AB×AB, B×A, and AB×A. No conflict genotype was observed in these family members, proving the accuracy of this method.

Gaps persist in our understanding of chromatin lower-order and higher-order structures. Xenopus egg extracts provide a way to study essential chromatin components which are difficult to manipulate in living cells, but nanoscale imaging of chromatin assembled in extracts poses a challenge. We describe a method for preparing chromatins assembled in extracts for atomic force microscopy (AFM) utilizing restriction enzyme digestion followed by transferring to a mica surface. Using this method, we find that buffer dilution of the chromatin assembly extract or incubation of chromatin in solutions of low ionic strength results in loosely-compacted chromatin fibers that are prone to unraveling into naked DNA. We also describe a method for direct AFM imaging of chromatin which does not utilize restriction enzymes and reveals higher-order fibers of varying widths. Due to the capability of controlling chromatin assembly conditions, we believe these methods have broad potential for studying physiologically relevant chromatin structures.

Studies were conducted to determine the host-feeding preference of Culex quinquefasciatus Say (Diptera: Culicidae) in relation to the availability of human and domestic animals in the city of Merida, Yucatan State, Mexico. Mosquitoes were collected in the backyards of houses using resting wooden boxes. Collections were made five times per week from January to December 2005. DNA was extracted from engorged females and tested by PCR using universal avian- and mammalian-specific primers. DNA extracted from avian-derived blood was further analyzed by PCR using primers that differentiate among the birds of three avian orders: Passeriformes, Columbiformes and Galliformes. PCR products obtained from mammalian-derived blood were subjected to restriction enzyme digestion to differentiate between human-, dog-, cat-, pig-, and horse-derived blood meals. Overall, 82% of engorged mosquitoes had fed on birds, and 18% had fed on mammals. The most frequent vertebrate hosts were Galliformes (47.1%), Passeriformes (23.8%), Columbiformes (11.2%) birds, and dogs (8.8%). The overall human blood index was 6.7%. The overall forage ratio for humans was 0.1, indicating that humans were not a preferred host for Cx. quinquefasciatus in Merida.

Biological assays at the single molecule level are crucial to fundamental studies of DNA-protein mechanisms. In order to cater for high throughput applications, one area of immense research potential is single-molecule bioassays where miniaturized devices are developed to perform rapid and effective biological reactions and analyses. With the success of various emerging technologies for engineering miniaturized structures down to the nanoscale level, supported by specialized equipment for detection, many investigations in the field of life science that were once thought impossible can now be actively explored. In this review, the significance of downscaling to the single-molecule level is firstly presented in selected examples, with the focus placed on restriction enzyme assays. To determine the effectiveness of single-molecule restriction enzyme reactions, simple and direct analytical methods based on DNA stretching have often been reliably employed. DNA stretching can be realized based on a number of working principles related to the physical forces exerted on the DNA samples. We then discuss two examples of a nanochannel system and a microchamber system where single-molecule restriction enzyme digestion and DNA stretching have been integrated, which possess prospective capabilities of developing into highly sensitive and high-throughput restriction enzyme assays. Finally, we take a brief look at the general trends in technological development in this field by comparing the advantages and disadvantages of performing assays at bulk, microscale and single-molecule levels.

Infectious clones of viruses allow elucidation of viral gene function in plants. This study presents an adaptation and expansion of the circular polymerase extension reaction that enables both cell-based and cell-free one-step assembly of a plant viral cDNA. To demonstrate its infectious nature, the generated clone was introduced into plants by Agrobacterium infiltration. This technique eliminates the cumbersome strategy of restriction enzyme digestion and ligation and instead relies on the sequence specificity of overlapping PCR fragments to assemble a complete functional infectious clone of a virus. This technique is rapid and potentially applicable for cloning virus genes which may be difficult to clone using conventional approaches due to toxicity problems that may be encountered when cloning in E. coli .

Concerns over the development of antibiotic-resistant bacteria within the food animal industry have intensified the search for natural approaches to the prevention and treatment of bacterial diseases. Competitive exclusion cultures are the foundation of a disease-management strategy based on the use of benign bacterial strains to prevent the establishment of pathogenic bacteria within a specific host. Differentiation of phenotypically ambiguous isolates is a critical step in establishing a manageable library of bacteria for use in the development of defined competitive exclusion cultures. We used automated ribotyping techniques to dereplicate a large collection of phenotypically ambiguous isolates from a continuous-flow competitive exclusion culture. A total of 157 isolates were screened following an EcoRI restriction enzyme digestion. The 157 isolates were resolved into 23 ribogroups, which represents an 85% reduction in the number of isolates in the bacterial isolate library. Seventy-six percent of the isolates fit into one of five ribogroups. This work demonstrated that automated ribotyping is an effective and efficient tool for dereplication of diverse bacterial isolate libraries.

The surveillance of prevalent Leishmania and sand fly species in endemic areas is important for prediction of the risk and expansion of leishmaniasis. In this study, we developed a polymerase chain reaction (PCR)-based method for detection of Leishmania minicircle DNA within individual sand flies. Using this method, we detected minicircle DNA in 6 (3.3%) of 183 sand flies, while 5 (3.5%) of 143 were positive for Leishmania promastigotes in the same areas by microscopic examination. The species were identified as Leishmania (Leishmania) mexicana by nucleotide sequencing of the cytochrome b gene. Additionally, all the Leishmania-positive sand flies were identified as Lutzomyia ayacuchensis by the restriction enzyme digestion of the PCR-amplified 18S ribosomal RNA gene fragments. Since this combined method is relatively easy and can process a large number of samples, it will be a powerful tool for the rapid identification of prevalent sand fly and Leishmania species as well as monitoring the infection rate in sand fly populations in endemic areas. Fil: Kato, Hirotomo. Yamaguchi University; Japón Fil: Uezato, Hiroshi. University of the Ryukyus; Japón Fil: Katakura, Ken. Hokkaido University; Japón Fil: Calvopina, Manuel. Kochi University. Kochi Medical School; Japón Fil: Marco, Jorge Diego. Kochi University. Kochi Medical School; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Barroso, Paola Andrea. Kochi University. Kochi Medical School; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Gomez, Eduardo. Universidad Católica de Guayaquil; Ecuador Fil: Mimori, Tatsuyuki. Kumamoto University; Japón Fil: Korenaga, Masataka. Kochi University. Kochi Medical School; Japón Fil: Iwata, Hiroyuki. Yamaguchi University; Japón Fil: Nonaka, Shigeo. University ok the Ryukyus; Japón Fil: Hashiguchi, Yoshihisa. Kochi University. Kochi Medical School; Japón

The determination of ApoE genotypes is a common clinical laboratory procedure. The most frequently used method involves a PCR amplification of the target region followed by a restriction enzyme digestion of the resultant amplicon to obtain a genotype-specific restriction fragment pattern. We describe an unexpected and surprising difficulty that was encountered during our routine laboratory casework.