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Sample records for dna haemagglutination test

  1. Comparison of passive haemagglutination test with Widal agglutination test for serological diagnosis of typhoid fever in an endemic area.

    PubMed

    Coovadia, Y M; Singh, V; Bhana, R H; Moodley, N

    1986-06-01

    A passive haemagglutination test, using sheep red blood cells sensitised with Salmonella typhi lipopolysaccharide, was compared with the Widal test for the serological diagnosis of typhoid fever in an endemic area. The results obtained on sera from 152 patients with bacteriologically confirmed typhoid and 183 patients who did not have typhoid were analysed in terms of sensitivity, specificity, simplicity, and rapidity of the respective tests. The passive haemagglutination test was found to be more sensitive (80%) than the S typhi O antigen (71%) but marginally less sensitive than the H antigen (82%) of the Widal test. The false positive rate on control sera was 1.2% and 6.6%, respectively, for the Widal O and H antigens, and 1.6% for the passive haemagglutination test. Our findings indicate that the passive haemagglutination test is comparable with the Widal test for the serological diagnosis of typhoid fever in endemic areas, but is more simple, rapid, and economic. The passive haemagglutination test may be a useful alternative to the Widal test for the serological diagnosis of typhoid fever in busy microbiology laboratories in areas in which the disease is endemic. PMID:2424936

  2. Stable erythrocyte diagnostic preparation for passive haemagglutination test with herpes simplex virus antigen.

    PubMed

    Krichevskaya, G I; Basova, N N

    1976-10-01

    A method for the preparation of stable suspensions of erythrocytes sensitized with herpes simplex virus (HSV) antigen and for their use in the passive haemagglutination test (PHAT) was developed. Formolized sheep erythrocytes were treated with tannin and sensitized with HSV antigen prepared from infected chick embryo cell culture by ultrasonication and virus extraction with alkaline glycine buffer. Antibody titres determined in the PHAT were higher than titres of neutralizing antibody. The specificity of the results was checked by the passive haemagglutination-inhibition test (PHAIT). The sensitized erythrocytes retained their activity for 5 months (the observation period) and gave reproducible results. The availability of stable erythrocyte diagnostic preparations simplifies the detection of herpesvirus antibody and makes the method widely applicable. PMID:11670

  3. The use of the haemagglutination-inhibition test for detecting antibodies to type SAT 2 foot-and-mouth disease viruses in cattle sera.

    PubMed Central

    Booth, J. C.; Pay, T. W.; Hedger, R. S.; Barnett, I. T.

    1975-01-01

    Two strains of type SAT 2-foot-and-mouth disease virus which gave high titres of haemagglutinin activity reacted type-specifically in direct haemagglutination-inhibition tests with reference, bovine convalescent antisera. Comparisons of the haemagglutination-inhibition and the serum neutralization tests using cattle sera showed that both were equally specific and sensitive for detecting virus antibody. PMID:163274

  4. The application of the haemagglutination test to a study of the immunity to malaria in protected and unprotected population groups in Australian New Guinea*

    PubMed Central

    Desowitz, R. S.; Saave, J. J.

    1965-01-01

    The formolized tanned sheep erythrocyte haemagglutination test has been applied to an immuno-malariometric study in Australian New Guinea to determine whether the haemagglutination titre reflects a subject's immune state and to measure the effect of malaria control operations on a population's immunity. Two population groups were studied—one (unprotected) living in holoendemic malaria conditions, the other (protected) living in an area subject to malaria control measures for four years. An increase in both serological positivity rates and geometric mean titres among the unprotected group with increasing age suggests that the test does serve to assess the state of immunity; the corresponding rates were much lower in the protected population, particularly among the children. The authors foresee the possible use of the haemagglutination test as a supplement to other procedures in assessing the progress of a malaria campaign. They, note, however, that more immuno-malariometric studies on populations subject to different degrees of malaria endemicity will need to be carried out before the relationship between the immune state and serological results can be clearly established. PMID:14310901

  5. Studies on antiplague haemagglutinating antibodies

    PubMed Central

    Suzuki, Sosuke; Chikasato, Yoshio; Hotta, Susumu

    1974-01-01

    The indirect haemagglutination (IHA) test has been widely applied in the detection of antiplague antibodies in rodent sera. In the present study, acetone treatment of the test serum was tried in order to improve the specificity of the reaction. It was shown that the IHA titres of acetone-treated sera correlated well with those of untreated sera measured by the standard method recommended by WHO. Essentially the same results were obtained with sera from experimentally immunized rodents and from captured wild rats. In addition to acetone treatment, the sera were treated with 2-mercaptoethanol (ME). The results obtained indicated that the antiplague IHA antibodies produced early after the inoculation of plague bacilli were ME-sensitive, whereas those detected in the later stages or after a second inoculation were ME-resistant. The data suggest that acetone treatment of sera could be useful for the screening of antiplague antibodies, and that treatment with ME is helpful in assessing the time of past plague infections. The present survey has also shown that the positive rates of antiplague antibodies in wild rats trapped in Kobe, one of the largest sea ports in Japan, have so far been very low. PMID:4549347

  6. HPV DNA test

    MedlinePlus

    The HPV DNA test is used to check for high-risk HPV infection in women. HPV infection around the genitals is ... warts spread when you have sex. The HPV-DNA test is generally not recommended for detecting low- ...

  7. Routine DNA testing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Routine DNA testing. It’s done once you’ve Marker-Assisted Breeding Pipelined promising Qantitative Trait Loci within your own breeding program and thereby established the performance-predictive power of each DNA test for your germplasm under your conditions. By then you are ready to screen your par...

  8. Studies on marine algae for haemagglutinic activity.

    PubMed

    Alam, M T; Usmanghani, K

    1994-07-01

    Lectins (agglutinins) are important in medical and immunological applications. Phytohaemagglutinins have been found useful in blood banking. Keeping in view of these facts, the marine algae found at Karachi coastal region have been screened for agglutinic activity by using human erythrocytes of A, B, AB and 0 group. Altogether 53 algal samples were collected and subjected to extraction, fractionation serial dilution and titre determinations. The total marine algae screened for haemagglutinic activity were 44 out of these 14, 13 and 17 belonged to Chlorophyta, Phaeophyta, and Rhodophyta respectively. Among these three groups the Rhodophyta showed the highest number of lytic activity. The green marine alga Valoniopsis pachynema showed a titre value between 2(2) and 2(3), which is statistically significant. In case of brown marine algae Colpomenia sinuosa was found to be active (titre 2(3)), while Dictyota dichotoma, D. indica and Iyengaria stellata, furnished week titre value as 2(2). The red marine algae screened were 17, out of these 4 spp. showed significant activity (titre 2(3)), and these are Gelidium usmanghani, Gracilaria foliifera Hypnea pannosa and Hynea valentiae. While Scinaia fascicularis, Scinaia indica and Champia parvula were found to be weak in their onset on human erythrocytes. The results obtained were quite in agreement with those reported in the literature. PMID:16414751

  9. The serological diagnosis of Mycoplasma pneumoniae infection: a comparison of complement fixation, haemagglutination and immunofluorescence.

    PubMed Central

    Rousseau, S. A.; Tettmar, R. E.

    1985-01-01

    A total of 193 sera were examined for antibody to Mycoplasma pneumoniae by three techniques - complement fixation (CF), haemagglutination (HA) and immunofluorescence (IF), the last method being used to assess IgM, IgG and IgA antibodies. The most reliable single test for diagnosis was HA, and the most useful combination of tests was HA with IF (IgM and IgG). The IgA IF was not found to be diagnostically helpful. PMID:3934260

  10. A comparison of haemagglutination, haemagglutination inhibition and PCR for the detection of psittacine beak and feather disease virus infection and a comparison of isolates obtained from loriids.

    PubMed

    Khalesi, Bahman; Bonne, Nicolai; Stewart, Meredith; Sharp, Margaret; Raidal, Shane

    2005-11-01

    Psittacine beak and feather disease (PBFD) is recognized as a threat for endangered psittacine birds in Australia, New Zealand and South Africa. Several diagnostic methods for the detection of beak and feather disease virus (BFDV) infection have been developed but there are few studies comparing the relative merits or sensitivity and specificity of each diagnostic test. In this report, the results of PCR, haemagglutination (HA) and haemagglutination inhibition (HI) testing of diagnostic samples collected from 679 samples from a range of psittacine bird species suspected of being infected with BFDV are summarized and compared. There was a strong agreement (kappa = 0.757; P<0.0001) between PCR and HA testing of feather samples and PCR-negative birds were 12.7 times more likely to have HI antibody than PCR-positive birds. False-positive HA results with titres up to 1:320 were identified in six feather samples that were PCR negative; the haemagglutination detected in these samples was not inhibited by anti-BFDV antisera and was removed by filtration through a 0.22 microm filter. Similarly, one false-negative PCR result was detected in a feather sample that had a high HA titre (>1:40,960) and four false-positive PCR results were detected in a batch of four feather samples. Of 143 birds that were feather PCR positive, only two had detectable HI antibody, and these birds were also feather HA negative, suggesting that they were developing immunity to recent infection. All birds with HI antibody were negative on feather HA testing. The assays confirmed BFDV infection in two endangered swift parrots (Lathamus discolor) and phylogenetic analysis of the sequence data generated from ORF V1 of these isolates provide further evidence of BFDV genotypes clustering in parallel with the Loriidae, Cacatuidae and Psittacidae. PMID:16227226

  11. Future of DNA diagnostic testing

    NASA Astrophysics Data System (ADS)

    Petersen, Kurt E.

    2001-09-01

    Progress in sequencing the human genome and the DNA of other organisms is providing many opportunities for creating powerful systems for numerous and diverse applications in DNA testing. These systems and the chemical processes, such as PCR, which they are designed to carry out, have recently made great strides in miniaturization through advances in micro-fluidics and micro-optics. In addition, new techniques in biological processing, such as controlled ultrasonic lysis, are being applied to small, automated, integrated instruments designed to provide important DNA results in a timely and routine manner. These systems are bringing DNA identification out of the laboratory and into our daily lives. Instead of waiting for days or weeks for a result, we will have them in minutes. Instead of relying on the skills of molecular biologists, the average person will be able to run a DNA test. These new advances will widely impact many aspects of our medical practices, food processing, and public safety.

  12. DNA testing and domestic dogs.

    PubMed

    Mellersh, Cathryn

    2012-02-01

    There are currently about 80 different DNA tests available for mutations that are associated with inherited disease in the domestic dog, and as the tools available with which to dissect the canine genome become increasingly sophisticated, this number can be expected to rise dramatically over the next few years. With unrelenting media pressure focused firmly on the health of the purebred domestic dog, veterinarians and dog breeders are turning increasingly to DNA tests to ensure the health of their dogs. It is ultimately the responsibility of the scientists who identify disease-associated genetic variants to make sensible choices about which discoveries are appropriate to develop into commercially available DNA tests for the lay dog breeder, who needs to balance the need to improve the genetic health of their breed with the need to maintain genetic diversity. This review discusses some of the factors that should be considered along the route from mutation discovery to DNA test and some representative examples of DNA tests currently available. PMID:22071879

  13. The haemagglutination activity of equine herpesvirus type 1 glycoprotein C.

    PubMed

    Andoh, Kiyohiko; Hattori, Shiho; Mahmoud, Hassan Y A H; Takasugi, Maaya; Shimoda, Hiroshi; Bannai, Hiroshi; Tsujimura, Koji; Matsumura, Tomio; Kondo, Takashi; Kirisawa, Rikio; Mochizuki, Masami; Maeda, Ken

    2015-01-01

    Equine herpesvirus type 1 (EHV-1) has haemagglutination (HA) activity toward equine red blood cells (RBCs), but the identity of its haemagglutinin is unknown. To identify the haemagglutinin of EHV-1, the major glycoproteins of EHV-1 were expressed in 293T cells, and the cells or cell lysates were mixed with equine RBCs. The results showed that only EHV-1 glycoprotein C (gC)-producing cells adsorbed equine RBCs, and that the lysate of EHV-1 gC-expressing cells agglutinated equine RBCs. EHV-1 lacking gC did not show HA activity. HA activity was inhibited by monoclonal antibodies (MAbs) specific for gC, but not by antibodies directed against other glycoproteins. In addition, HA activity was not inhibited by the addition of heparin. These results indicate that EHV-1 gC can bind equine RBCs irrespective of heparin, in contrast to other herpesvirus gC proteins. PMID:25456403

  14. Testing the Efficacy of a Multi-Component DNA-Prime/DNA-Boost Vaccine against Trypanosoma cruzi Infection in Dogs

    PubMed Central

    Aparicio-Burgos, José E.; Ochoa-García, Laucel; Zepeda-Escobar, José Antonio; Gupta, Shivali; Dhiman, Monisha; Martínez, José Simón; de Oca-Jiménez, Roberto Montes; Arreola, Margarita Val; Barbabosa-Pliego, Alberto; Vázquez-Chagoyán, Juan C.; Garg, Nisha Jain

    2011-01-01

    Background Trypanosoma cruzi, the etiologic agent of Chagas Disease, is a major vector borne health problem in Latin America and an emerging infectious disease in the United States. Methods We tested the efficacy of a multi-component DNA-prime/DNA-boost vaccine (TcVac1) against experimental T. cruzi infection in a canine model. Dogs were immunized with antigen-encoding plasmids and cytokine adjuvants, and two weeks after the last immunization, challenged with T. cruzi trypomastigotes. We measured antibody responses by ELISA and haemagglutination assay, parasitemia and infectivity to triatomines by xenodiagnosis, and performed electrocardiography and histology to assess myocardial damage and tissue pathology. Results Vaccination with TcVac1 elicited parasite-and antigen-specific IgM and IgG (IgG2>IgG1) responses. Upon challenge infection, TcVac1-vaccinated dogs, as compared to non-vaccinated controls dogs, responded to T. cruzi with a rapid expansion of antibody response, moderately enhanced CD8+ T cell proliferation and IFN-γ production, and suppression of phagocytes’ activity evidenced by decreased myeloperoxidase and nitrite levels. Subsequently, vaccinated dogs controlled the acute parasitemia by day 37 pi (44 dpi in non-vaccinated dogs), and exhibited a moderate decline in infectivity to triatomines. TcVac1-immunized dogs did not control the myocardial parasite burden and electrocardiographic and histopatholgic cardiac alterations that are the hallmarks of acute Chagas disease. During the chronic stage, TcVac1-vaccinated dogs exhibited a moderate decline in cardiac alterations determined by EKG and anatomo-/histo-pathological analysis while chronically-infected/non-vaccinated dogs continued to exhibit severe EKG alterations. Conclusions Overall, these results demonstrated that TcVac1 provided a partial resistance to T. cruzi infection and Chagas disease, and provide an impetus to improve the vaccination strategy against Chagas disease. PMID:21625470

  15. Relationship between haemagglutination inhibition titre and immunity to influenza in ferrets.

    PubMed

    Wikramaratna, Paul S; Rambaut, Andrew

    2015-10-01

    Our understanding of the antigenic evolution of the human influenza virus is chiefly derived from experiments in which serum from influenza infected ferrets is tested against panels of virus isolates in the haemagglutination inhibition (HI) assay. The interpretation of these results has been much aided by the development of antigenic mapping techniques, which suppose that the antigenic distance between two different influenza viruses is directly proportional to their fold-difference in titre in this assay. Yet, antigenic distance is not necessarily the same as cross-protection, and high levels of protection have been observed in humans against strains to which they have low HI titres. However, no study has previously addressed the relationship between HI titre and cross-protection in ferrets: the standard animal model. This study fills this gap by analysing published data where pre-challenge HI titres are available for individual ferrets, and post-challenge outcomes have been recorded. Ultimately, this work confirms that it is the absolute, rather than relative, HI titre that determines the extent of immunity and that there is a threshold HI titre beyond which ferrets are completely protected from infection. Nevertheless, this titre is much higher in ferrets than has been suggested for humans. Further, we are consequently able to show that using distance between strains within an antigenic map to predict cross-protection between influenza viruses can be misleading. PMID:26342848

  16. Relationship between haemagglutination inhibition titre and immunity to influenza in ferrets

    PubMed Central

    Wikramaratna, Paul S.; Rambaut, Andrew

    2015-01-01

    Our understanding of the antigenic evolution of the human influenza virus is chiefly derived from experiments in which serum from influenza infected ferrets is tested against panels of virus isolates in the haemagglutination inhibition (HI) assay. The interpretation of these results has been much aided by the development of antigenic mapping techniques, which suppose that the antigenic distance between two different influenza viruses is directly proportional to their fold-difference in titre in this assay. Yet, antigenic distance is not necessarily the same as cross-protection, and high levels of protection have been observed in humans against strains to which they have low HI titres. However, no study has previously addressed the relationship between HI titre and cross-protection in ferrets: the standard animal model. This study fills this gap by analysing published data where pre-challenge HI titres are available for individual ferrets, and post-challenge outcomes have been recorded. Ultimately, this work confirms that it is the absolute, rather than relative, HI titre that determines the extent of immunity and that there is a threshold HI titre beyond which ferrets are completely protected from infection. Nevertheless, this titre is much higher in ferrets than has been suggested for humans. Further, we are consequently able to show that using distance between strains within an antigenic map to predict cross-protection between influenza viruses can be misleading. PMID:26342848

  17. Paternity testing that involves a DNA mixture.

    PubMed

    Mortera, Julia; Vecchiotti, Carla; Zoppis, Silvia; Merigioli, Sara

    2016-07-01

    Here we analyse a complex disputed paternity case, where the DNA of the putative father was extracted from his corpse that had been inhumed for over 20 years. This DNA was contaminated and appears to be a mixture of at least two individuals. Furthermore, the mother's DNA was not available. The DNA mixture was analysed so as to predict the most probable genotypes of each contributor. The major contributor's profile was then used to compute the likelihood ratio for paternity. We also show how to take into account a dropout allele and the possibility of mutation in paternity testing. PMID:27017109

  18. A survey of the nutritional and haemagglutination properties of legume seeds generally available in the UK.

    PubMed

    Grant, G; More, L J; McKenzie, N H; Stewart, J C; Pusztai, A

    1983-09-01

    Eighty-five samples from fifteen different legume seed lines generally available in the UK were examined by measurements of their net protein utilization by rats and by haemagglutination tests with erythrocytes from a number of different animal species. From these results the seeds were classified into four broad groups. Group a seeds from most varieties of kidney (Phaseolus vulgaris), runner (Phaseolus coccineus) and tepary (Phaseolus acutifolius) beans showed high reactivity with all cell types and were also highly toxic. Group b, which contained seeds from lima or butter beans (Phaseolus lunatus) and winged bean (Psophocarpus tetragonolobus), agglutinated only human and pronase-treated rat erythrocytes. These seeds did not support proper growth of the rats although the animals survived the 10 d experimental period. Group c consisted of seeds from lentils (Lens culinaris), peas (Pisum sativum), chick-peas (Cicer arietinum), blackeyed peas (Vigna sinensis), pigeon peas (Cajanus cajan), mung beans (Phaseolus aureus), field or broad beans (Vicia faba) and aduki beans (Phaseolus angularis). These generally had low reactivity with all cells and were non-toxic. Group d, represented by soya (Glycine max) and pinto (Phaseolus vulgaris) beans, generally had low reactivity with all cells but caused growth depression at certain dietary concentrations. This growth depression was probably mainly due to antinutritional factors other than lectins. Lectins from group a seeds showed many structural and immunological similarities. However the subunit composition of the lectin from the tepary bean samples was different from that of the other bean lectins in this or any other groups. PMID:6615758

  19. The relationship between colonization and haemagglutination inhibiting and B cell epitopes of Porphyromonas gingivalis

    PubMed Central

    KELLY, C G; BOOTH, V; KENDAL, H; SLANEY, J M; CURTIS, M A; LEHNER, T

    1997-01-01

    Passive immunization with the monoclonal antibody 61BG1.3 selectively prevents colonization by Porphyromonas gingivalis in humans (Booth V, Ashley FP, Lehner T. Infect Immun 1996; 64:422-7). The protective MoAb recognizes the j3 component of the RI protease of P. gingivalis which is formed by proteolytic processing of a polyprotein precursor termed PrpRl. This subunit is both a haemagglutinin and an antigen which is recognized by sera from patients with periodontitis. In this study the relationship was investigated between a colonization epitope which is recognized by the MoAb 61BG1.3, a haemagglutinating and B cell epitope which are recognized by sera from patients with periodontitis. B cell epitopes were mapped by Western blotting with a series of truncated recombinant polypeptides spanning the adhesion domain within residues 784–1130 of PrpRl and by ELISA using a panel of synthetic peptides spanning the same sequence. The epitope which is recognized by the protective MoAb was mapped within residues 907–931 of PrpRl, while serum responses of patients were directed predominantly to the adjacent carboxy-terminal sequence within residues 934–1042. The haemagglutinating epitope was mapped to residues 1073–1112. In view of our previous findings that the MoAb 61BG1.3 prevents colonization of P. gingivalis in vivo and inhibits haemagglutination, these two epitopes may be in proximity in the native protein. Active or passive immunization strategies which target the protective or haemagglutinating epitopes of the adhesion domain of PrpRl may provide a means of preventing infection with P. gingivalis. PMID:9367414

  20. Haemagglutination, haemolysin production and serum resistance of proteus and related species isolated from clinical sources.

    PubMed

    Mishra, M; Thakar, Y S; Pathak, A A

    2001-01-01

    A total of 148 strains of Proteus and related species comprising of Proteus mirabilis (116), Proteus vulgaris (24), Providentia rettgeri (4), Providentia alcalifaciens (2), Providentia stuarti (1) and Morganella morganii (1), isolated from various sources, were examined for haemagglutination (HA), haemolysin production (HL) and serum resistance (SR). Maximum isolates were obtained from urine (47.30%) and pus (40.54%) and they were multidrug resistant. The sensitivity to Ciprofloxacin was 78.38%, Gentamicin: 62.84%, Cefotaxime: 29.73%, Norfloxacin: 22.97%, Tetracycline: 20.95% and Ampicillin: 6.76%. There were four commonest resistance patterns shown by 58.62% of Proteus mirabilis and 66.67% of Proteus vulgaris strains. Haemagglutination was shown by 91 (61.49%) strains, HL production in 126 (85.14%) strains and SR by 124 (83.78%) isolates. All the three i.e. HA, HL and SR were simultaneously present in 77 (52.27%) strains, any two were present in 40 (27.03%) strains and any one was positive in 30 (20.03%) strains. Thus in as many as 147 (98.32%) isolates, any one or more virulence factors were present. The virulence in commensal pathogen like Proteus is basically a multifactorial phenomenon. The presence of more virulence factors in one strain may increase its pathogenic ability. The evaluation of multiple virulence factors instead of one single parameter will be of greater help in assessing its pathogenic potential. PMID:17664798

  1. Reverse phase passive haemagglutination and single radial immunodiffusion to detect epsilon antigen of Clostridium perfringens type D.

    PubMed

    Beh, K J; Buttery, S H

    1978-11-01

    Two in vitro immunological assays were developed for detection of the epsilon (epsilon) antigen of Cl. perfringens type D. It was found that the reverse phase passive haemagglutination assay (RPHA) was able to detect concentrations of epsilon-antigen as low as 6 x 10-7 mg/ml whereas the single radial immunodiffusion techniques (SRID) was capable of detecting concentrations of epsilon-antigen above 0.01 mg/ml. When applied to gut contents from freshly dead infected sheep the RPHA test was found to be more sensitive than mouse toxicity assay in detecting the presence of epsilon-antigen. However, very low titres were detected in gut contents from normal sheep which meant that in a diagnostic situation interpretation of RPHA titres would be difficult. No epsilon-antigen was detected by SRID in gut contents from normal sheep or in gut contents from freshly dead infected sheep. The SRID assay could detect epsilon-antigen in gut contents from infected sheep allowed to decompose for 20 h post-mortem. PMID:223537

  2. DNA nanotechnology from the test tube to the cell

    NASA Astrophysics Data System (ADS)

    Chen, Yuan-Jyue; Groves, Benjamin; Muscat, Richard A.; Seelig, Georg

    2015-09-01

    The programmability of Watson-Crick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, researchers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mechanisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology -- applications that best take advantage of the small size, biocompatibility and programmability of DNA-based systems -- lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

  3. Strategies for Implementing Cell-Free DNA Testing.

    PubMed

    Cuckle, Howard

    2016-06-01

    Maternal plasma cell-free (cf) DNA testing has higher discriminatory power for aneuploidy than any conventional multi-marker screening test. Several strategies have been suggested for introducing it into clinical practice. Secondary cfDNA, restricted only to women with positive conventional screening test, is generally cost saving and minimizes the need for invasive prenatal diagnosis but leads to a small loss in detection. Primary cfDNA, replacing conventional screening or retaining the nuchal translucency scan, is not currently cost-effective for third-party payers. Contingent cfDNA, testing about 20% of women with the highest risks based on a conventional test, is the preferred approach. PMID:27235907

  4. Effects of high hydrostatic pressure treatments on haemagglutination activity and structural conformations of phytohemagglutinin from red kidney bean (Phaseolus vulgaris).

    PubMed

    Liu, Cencen; Zhao, Mouming; Sun, Weizheng; Ren, Jiaoyan

    2013-02-15

    Red kidney beans were subjected to high hydrostatic pressure (HHP) treatment (50, 150, 250, 350, 450 MPa) and phytohaemagglutinin (PHA) was then extracted by affinity chromatography. It appeared that HHP treatment could increase crude extract yield and decrease its haemagglutination activity. For purified samples, PHA yield was not affected at pressures <450 MPa while the haemagglutination activity was noticeably reduced at 450 MPa. The structural changes were investigated using electrophoresis, size exclusion chromatography (SEC), Fourier transform infrared (FTIR) and differencial scanning calorimetry (DSC). Electrophoresis and SEC profiles revealed a new high molecular weight polymer after 450 MPa treatment. At pressures <450 MPa, FTIR showed an increase in β-sheet structure and a decrease in α-helix. At 450 MPa, the bands at 1688 cm(-1), representing aggregate strands and random coils, increased. The conclusions are that pressures <450 MPa can cause PHA unfolding and induce PHA aggregation at 450 MPa. PMID:23194535

  5. Baculovirus expression of beak and feather disease virus (BFDV) capsid protein capable of self-assembly and haemagglutination.

    PubMed

    Stewart, Meredith E; Bonne, Nicolai; Shearer, Patrick; Khalesi, Bahman; Sharp, Margaret; Raidal, Shane

    2007-05-01

    Beak and feather disease virus (BFDV) is a common avian circovirus infection of wild Psittaciformes and is a recognised threat to endangered psittacine species. Currently, there is a requirement to develop BFDV antigen for diagnostic purposes and since efforts to propagate BFDV in vitro have so far been unsuccessful the entire coding region of BFDV ORF C1 was expressed in Sf9 insect cells using a baculovirus expression system. The entire coding region of BFDV ORF C1, the presumptive capsid, was expressed in Sf9 insect cells using baculovirus expression system. Electron microscopic examination of negatively stained material demonstrated that the recombinant protein self-assembled to produce virus-like particles (VLPs) thus confirming that ORF C1 is likely to be the sole determinant for capsid construction in vivo. BFDV VLPs also possessed haemagglutinating activity which provides further evidence that self-assembled BFDV VLPs retain receptor mediated biological activity and that the determinants for BFDV haemagglutination activity rely solely on the capsid protein. The recombinant protein reacted with anti-BFDV sera from naturally immune parrots and cockatoo and from chickens experimentally inoculated with native BFDV in both Western blots and haemagglutination inhibition (HI) assay. BFDV VLPs were also a suitable replacement antigen for serological detection of BFDV antibody by HI. PMID:17218022

  6. Coat color DNA testing in dogs: theory meets practice.

    PubMed

    Schmutz, Sheila M; Melekhovets, Yuri

    2012-12-01

    DNA tests to detect particular dog coat color alleles are in use in several DNA diagnostic laboratories. The original two genes studied were MC1R and TYRP1 and therefore these tests have been used most widely, and for the longest period of time. The original research was conducted to determine the mutation associated with a particular phenotype in one to a few dog breeds, and was subsequently expanded to include more dog breeds. The application of this testing now includes an even wider range of dog breeds, some of which would not have been expected to have some of the alleles detected. This retrospective study demonstrates that a DNA test may be designed for a particular application, but is used by clients for additional applications that were not originally anticipated. A robust protocol with DNA obtained by cheek brushes and interchanges among dog owners via the internet, have likely lead to this expanded use by clients. PMID:22507852

  7. Maternal Plasma DNA and RNA Sequencing for Prenatal Testing.

    PubMed

    Tamminga, Saskia; van Maarle, Merel; Henneman, Lidewij; Oudejans, Cees B M; Cornel, Martina C; Sistermans, Erik A

    2016-01-01

    Cell-free DNA (cfDNA) testing has recently become indispensable in diagnostic testing and screening. In the prenatal setting, this type of testing is often called noninvasive prenatal testing (NIPT). With a number of techniques, using either next-generation sequencing or single nucleotide polymorphism-based approaches, fetal cfDNA in maternal plasma can be analyzed to screen for rhesus D genotype, common chromosomal aneuploidies, and increasingly for testing other conditions, including monogenic disorders. With regard to screening for common aneuploidies, challenges arise when implementing NIPT in current prenatal settings. Depending on the method used (targeted or nontargeted), chromosomal anomalies other than trisomy 21, 18, or 13 can be detected, either of fetal or maternal origin, also referred to as unsolicited or incidental findings. For various biological reasons, there is a small chance of having either a false-positive or false-negative NIPT result, or no result, also referred to as a "no-call." Both pre- and posttest counseling for NIPT should include discussing potential discrepancies. Since NIPT remains a screening test, a positive NIPT result should be confirmed by invasive diagnostic testing (either by chorionic villus biopsy or by amniocentesis). As the scope of NIPT is widening, professional guidelines need to discuss the ethics of what to offer and how to offer. In this review, we discuss the current biochemical, clinical, and ethical challenges of cfDNA testing in the prenatal setting and its future perspectives including novel applications that target RNA instead of DNA. PMID:27117661

  8. Value of DNA tests: a decision perspective.

    PubMed

    Taroni, Franco; Bozza, Silvia; Bernard, Magali; Champod, Christophe

    2007-01-01

    Before a Court of Law testifying in DNA-evidence cases, scientists are often challenged with the idea that the more markers (loci) the better, i.e., why does the scientist not use 16 or more markers? This paper introduces a new perspective, decision analysis, to deal with the problem of the number of markers to type in a criminal context. The decision-making process, which plays a key role in the routine work of a forensic scientist, consists of the rational choice, given personal objectives, between two or more possible outcomes when the consequences of the choice are uncertain. Simulated results support the hypothesis that analytical added value does not increase with the number of markers. PMID:17209907

  9. Multi-Target Stool DNA Test: Is the Future Here?

    PubMed

    Sweetser, Seth; Ahlquist, David A

    2016-06-01

    Colorectal cancer (CRC) screening reduces CRC incidence and mortality and is widely recommended. However, despite these demonstrated benefits, a large percentage of the population remains unscreened. The multi-target stool DNA (MT-sDNA) test is a new, non-invasive option for CRC screening that has a high accuracy rate in detection of colorectal neoplasia and offers great opportunity to enhance screening uptake. This review provides the current state of the art knowledge about the use of MT-sDNA in CRC screening. PMID:27165404

  10. Opportunities for embryo transfer in the age of DNA testing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Embryo transfer (ET) has contributed to increasing selection intensity in cattle breeding for many years. Preimplantation DNA testing offers the opportunity to increase selection response further through increasing within-family selection intensity. Further increases in between-family selection inte...

  11. Genotoxicity of refinery waste assessed by some DNA damage tests.

    PubMed

    Gupta, Amit Kumar; Ahmad, Irshad; Ahmad, Masood

    2015-04-01

    Refinery waste effluent is well known to contain polycyclic aromatic hydrocarbons, phenols and heavy metals as potentially genotoxic substances. The aim of the present study was to assess the genotoxic potential of Mathura refinery wastewater (MRWW) by various in vitro tests including the single cell gel electrophoresis, plasmid nicking assay and S1 nuclease assay. Treatment of human lymphocytes to different MRWW concentrations (0.15×, 0.3×, 0.5× and 0.78×) caused the formation of comets of which the mean tail lengths increased proportionately and differed significantly from those of unexposed controls. The toxic effect of MRWW on DNA was also studied by plasmid nicking assay and S1 nuclease assay. Strand breaks formation in the MRWW treated pBR322 plasmid confirmed its genotoxic effect. Moreover, a dose dependent increase in cleavage of calf thymus DNA in S1 nuclease assay was also suggestive of the DNA damaging potential of MRWW. A higher level of ROS generation in the test water sample was recorded which might be contributing to its genotoxicity. Interaction between the constituents of MRWW and calf thymus DNA was also ascertained by UV-visible spectroscopy. PMID:24836934

  12. 78 FR 29698 - Availability of an Environmental Assessment for Field Testing a Canine Lymphoma Vaccine, DNA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-21

    ... a Canine Lymphoma Vaccine, DNA AGENCY: Animal and Plant Health Inspection Service, USDA. ACTION... testing, and then to field test, an unlicensed Canine Lymphoma Vaccine, DNA. The environmental assessment... Lymphoma Vaccine, DNA. Possible Field Test Locations: Arizona, Florida, Georgia, New York, North...

  13. An improved DNA test for bovine leucocyte adhesion deficiency.

    PubMed

    Tammen, I; Klippert, H; Kuczka, A; Treviranus, A; Pohlenz, J; Stöber, M; Simon, D; Harlizius, B

    1996-05-01

    A modified DNA test, based on the polymerase chain reaction, was developed for the monogenic recessive disease bovine leucocyte adhesion deficiency (BLAD). The test was improved by the selection of new primers which facilitated the interpretation of the results. An easily scorable banding pattern makes the test useful in cattle breeding schemes and for clinical diagnosis. A total of 2381 samples was analysed over a period of three years. The carrier rate among young bulls at artificial insemination (AI) stations decreased from 11.6 per cent in 1993 to 9.9 per cent in the first five months of 1995. Continuous screening of young bulls before entering AI is still recommended unless both parents are proven to be genetically free of BLAD. The carrier rate among clinically suspect animals was not increased, and carriers are therefore not expected to be immunodeficient. Despite all efforts to eradicate the disease, calves with BLAD were still observed in 1995. PMID:8735510

  14. Functional characterization of VC1929 of Vibrio cholerae El Tor: role in mannose-sensitive haemagglutination, virulence and utilization of sialic acid.

    PubMed

    Sharma, Sandeep K; Moe, The Su; Srivastava, Ranjana; Chandra, Deepak; Srivastava, Brahm S

    2011-11-01

    The nonadhesive mutant CD11 of Vibrio cholerae El Tor, defective in expression of mannose-sensitive haemagglutinin, lacks a protein when compared with its parent strain. Determination of the amino acid sequence revealed the identity of the protein as the product of VC1929, which is annotated to encode a protein, DctP, involved in the transport of C₄-dicarboxylates. We cloned the dctP gene in pUC19 vector and expressed it in mutant CD11. Expression of DctP in the resulting complemented strain restored virulence, adhesive and colonizing capabilities, mannose-sensitive haemagglutination (MSHA) and ability to grow in medium containing sialic acid as a sole carbon source. The mutation in CD11 was caused by insertion of an adenine nucleotide in the reading frame of dctP. Recombinant purified DctP protein showed MSHA of human red blood cells, and protected rabbits against infection by V. cholerae. The protein was localized in membrane and cell wall fractions. The mutant, recombinant CD11 expressing DctP and parent strains were grown in M9 minimal medium in the presence of various carbohydrates (glucose, malate, fumarate, succinate or N-acetylneuraminic acid). The mutant was unable to grow in minimal medium containing N-acetylneuraminic acid (sialic acid) as the sole carbon source whereas the recombinant and parent strains utilized all the sugars tested. It is concluded that DctP is a mannose-sensitive haemagglutinin and a virulence factor and is involved in the utilization of sialic acid. PMID:21873407

  15. 78 FR 58514 - Availability of an Environmental Assessment for Field Testing of a DNA Immunostimulant

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-24

    ... of a DNA Immunostimulant AGENCY: Animal and Plant Health Inspection Service, USDA. ACTION: Notice of... then to field test, an unlicensed DNA Immunostimulant recommended for reduction in morbidity and.... Product: DNA Immunostimulant. Possible Field Test Locations: Texas, Mississippi, and Georgia for...

  16. Probability of paternity in paternity testing using the DNA fingerprint procedure.

    PubMed

    Honma, M; Ishiyama, I

    1989-01-01

    For the purpose of applying DNA fingerprinting to paternity testing, we established a general formula to calculate the probability of paternity and evaluated the ability of DNA fingerprinting to determine paternity. PMID:2591980

  17. Validation of commercial DNA tests for quantitative beef quality traits.

    PubMed

    Van Eenennaam, A L; Li, J; Thallman, R M; Quaas, R L; Dikeman, M E; Gill, C A; Franke, D E; Thomas, M G

    2007-04-01

    data required to enable the integration of marker data into genetic evaluations. As DNA tests associated with more beef production traits enter the marketplace, it will become increasingly important, and likely more difficult, to find independent populations with suitable phenotypes for validation studies. PMID:17178813

  18. Verification of sex from harvested sea otters using DNA testing

    USGS Publications Warehouse

    Scribner, K.T.; Green, B.A.; Gorbics, C.; Bodkin, J.

    2005-01-01

    We used molecular genetic methods to determine the sex of 138 sea otters (Enhydra lutris) harvested from 3 regions of Alaska from 1994 to 1997, to assess the accuracy of post-harvest field-sexing. We also tested each of a series of factors associated with errors in field-sexing of sea otters, including male or female bias, age-class bias, regional bias, and bias associated with hunt characteristics. Blind control results indicated that sex was determined with 100% accuracy using polymerase chain reaction (PCR) amplification using primers that co-amplify the zinc finger-Y-X gene, located on both the mammalian Y- and X-chromosomes, and Testes Determining Factor (TDF), located on the mammalian Y-chromosome. DNA-based sexing revealed that 12.3% of the harvested sea otters were incorrectly sexed in the field, with most errors (13 of 17) occurring as males incorrectly reported as females. Thus, female harvest was overestimated. Using logistic regression analysis, we detected no statistical association of incorrect determination of sex in the field with age class, hunt region, or hunt type. The error in field-sexing appears to be random, at least with respect to the variables evaluated in this study.

  19. DNA stabilization is critical for maximizing performance of fecal DNA-based colorectal cancer tests.

    PubMed

    Olson, Jeff; Whitney, Duncan H; Durkee, Kristine; Shuber, Anthony P

    2005-09-01

    We have developed a multitarget, fecal DNA screening assay that detects the presence of gene-specific mutations and long DNA fragments associated with colorectal cancer (CRC). We continue to investigate methods that may be used to optimize clinical sensitivity. The goals of this investigation are to establish how sample handling conditions affect the stability of DNA in stool, thereby potentially limiting clinical sensitivity, and to determine conditions to ameliorate DNA degradation. A study was run comparing paired sample aliquots. Quantitative PCR data for matched aliquots was used to determine first the effect of sample incubation on total recovery and integrity of DNA, then the effect of stabilization buffer addition to stool on recoverable DNA, and finally the impact of buffer addition on assay sensitivity. Comparison of quantitative PCR data for paired aliquots shows that the amount of recoverable human DNA is negatively affected by storing stool samples (N = 43) at room temperature for > or =36 hours (P = 0.0018). However, the addition of stabilization buffer leads to a significant increase in recovery of DNA (P = 0.010), compared with samples incubated without buffer. Whereas the DNA Integrity Assay (DIA) is found to be sensitive to DNA degradation (sensitivity was reduced by 82%; P = 0.0002), point mutation marker sensitivity is more refractory. Overall, DNA can be stabilized by addition of buffer to the sample, leading to increased assay sensitivity. PMID:16106201

  20. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2013-01-01

    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  1. [Selection of suitable polypropylene tubes for DNA testing using real-time PCR].

    PubMed

    Shimizu, Eri; Futo, Satoshi; Masubuchi, Tomoko; Minegishi, Yasutaka; Kasahara, Masaki; Akiyama, Hiroshi; Teshima, Reiko; Hino, Akihiro; Mano, Jyunichi; Furui, Satoshi; Kitta, Kazumi

    2010-01-01

    Polypropylene microtubes (tubes) are generally used for bio-material tests in addition to PCR tests such as genetically modified organism (GMO) testings. However, the choice of suitable tubes is quite important, because it might influence the results: DNA binding and/or elution of chemical substances sometimes occurs. In this study, we established methods to select tubes with the most suitable characteristics for DNA testing. PMID:20208409

  2. Investigating CSI: portrayals of DNA testing on a forensic crime show and their potential effects.

    PubMed

    Ley, Barbara L; Jankowski, Natalie; Brewer, Paul R

    2012-01-01

    The popularity of forensic crime shows such as CSI has fueled debate about their potential social impact. This study considers CSI's potential effects on public understandings regarding DNA testing in the context of judicial processes, the policy debates surrounding crime laboratory procedures, and the forensic science profession, as well as an effect not discussed in previous accounts: namely, the show's potential impact on public understandings of DNA and genetics more generally. To develop a theoretical foundation for research on the "CSI effect," it draws on cultivation theory, social cognitive theory, and audience reception studies. It then uses content analysis and textual analysis to illuminate how the show depicts DNA testing. The results demonstrate that CSI tends to depict DNA testing as routine, swift, useful, and reliable and that it echoes broader discourses about genetics. At times, however, the show suggests more complex ways of thinking about DNA testing and genetics. PMID:22530487

  3. DNA methylome profiling of maternal peripheral blood and placentas reveal potential fetal DNA markers for non-invasive prenatal testing.

    PubMed

    Xiang, Yuqian; Zhang, Junyu; Li, Qiaoli; Zhou, Xinyao; Wang, Teng; Xu, Mingqing; Xia, Shihui; Xing, Qinghe; Wang, Lei; He, Lin; Zhao, Xinzhi

    2014-09-01

    Utilizing epigenetic (DNA methylation) differences to differentiate between maternal peripheral blood (PBL) and fetal (placental) DNA has been a promising strategy for non-invasive prenatal testing (NIPT). However, the differentially methylated regions (DMRs) have yet to be fully ascertained. In the present study, we performed genome-wide comparative methylome analysis between maternal PBL and placental DNA from pregnancies of first trimester by methylated DNA immunoprecipitation-sequencing (MeDIP-Seq) and Infinium HumanMethylation450 BeadChip assays. A total of 36 931 DMRs and 45 804 differentially methylated sites (DMSs) covering the whole genome, exclusive of the Y chromosome, were identified via MeDIP-Seq and Infinium 450k array, respectively, of which 3759 sites in 2188 regions were confirmed by both methods. Not only did we find the previously reported potential fetal DNA markers in our identified DMRs/DMSs but also we verified fully the identified DMRs/DMSs in the validation round by MassARRAY EpiTYPER. The screened potential fetal DNA markers may be used for NIPT on aneuploidies and other chromosomal diseases, such as cri du chat syndrome and velo-cardio-facial syndrome. In addition, these potential markers may have application in the early diagnosis of placental dysfunction, such as pre-eclampsia. PMID:24996894

  4. Prevalence, Haemolytic and Haemagglutination Activities and Antibiotic Susceptibility Profiles of Campylobacter spp. Isolated from Human Diarrhoeal Stools in Vhembe District, South Africa

    PubMed Central

    Samie, A.; Ramalivhana, J.; Igumbor, E.O.; Obi, C.L.

    2007-01-01

    Campylobacter species are increasingly being recognized as agents of gastroenteritis worldwide. However, data on the pathogenic characteristics of the organism isolated in rural communities in South Africa are lacking. In this study, the prevalence of Campylobacter spp. from diarrhoeal stools, haemolytic and haemagglutinating activities of the isolates, and antibiotic susceptibility profiles, including minimum inhibitory concentration (MIC) patterns to different antibiotics, were determined using the standard microbiological techniques. Campylobacter spp. were isolated from individuals of all age-groups; however, the infection rate was higher among individuals aged less than two years (30.4%). Of 115 Campylobacter strains isolated, polymerase chain reaction (PCR) analysis indicated that 98 (85%) were C. jejuni, while 17 (15%) were C. coli. Seventy-one (62%) of the strains showed haemolysis on human blood, and 80% agglutinated human blood, whereas 22.6% were β-lactamase-positive. Resistance to antimicrobials, such as erythromycin, ciprofloxacin, vancomycin, and fusidic acid, was high. Increased resistance to macrolide and quinolone antibiotics poses major risks for treatment failure. Haemolytic and haemagglutinating activities may be useful in preliminary characterization of pathogenic strains in settings where Campylobacter-associated infections are common. PMID:18402183

  5. Multilocus DNA fingerprinting: the independence problem in quantitative paternity testing.

    PubMed

    Krawczak, M

    1994-02-01

    A simulation study was performed in order to determine whether or not the assumption of independence, made in the quantitative analysis of multilocus DNA fingerprints, represents an inadmissible over-simplification. A total of 10,000 cases of true and false paternity, respectively, were simulated in twenty replicas of various genetic models. Log-likelihood ratios (paternity vs. non-paternity; LR) were calculated using published likelihood formulae and assuming position-wise independence. The resulting LR distributions were compared to (i) the results of a classical analysis of the underlying genotype data, and (ii) the distributions expected from the likelihood model employed in the LR calculations. Although considerable discrepancies were observed between these distributions, decision making about paternity appeared to be only marginally affected, especially when only a fraction of each multilocus DNA fingerprint was analyzed. PMID:8026429

  6. A Retrospective Approach to Testing the DNA Barcoding Method

    PubMed Central

    Chapple, David G.; Ritchie, Peter A.

    2013-01-01

    A decade ago, DNA barcoding was proposed as a standardised method for identifying existing species and speeding the discovery of new species. Yet, despite its numerous successes across a range of taxa, its frequent failures have brought into question its accuracy as a short-cut taxonomic method. We use a retrospective approach, applying the method to the classification of New Zealand skinks as it stood in 1977 (primarily based upon morphological characters), and compare it to the current taxonomy reached using both morphological and molecular approaches. For the 1977 dataset, DNA barcoding had moderate-high success in identifying specimens (78-98%), and correctly flagging specimens that have since been confirmed as distinct taxa (77-100%). But most matching methods failed to detect the species complexes that were present in 1977. For the current dataset, there was moderate-high success in identifying specimens (53-99%). For both datasets, the capacity to discover new species was dependent on the methodological approach used. Species delimitation in New Zealand skinks was hindered by the absence of either a local or global barcoding gap, a result of recent speciation events and hybridisation. Whilst DNA barcoding is potentially useful for specimen identification and species discovery in New Zealand skinks, its error rate could hinder the progress of documenting biodiversity in this group. We suggest that integrated taxonomic approaches are more effective at discovering and describing biodiversity. PMID:24244283

  7. Detection of Entamoeba histolytica DNA in the Saliva of Amoebic Liver Abscess Patients Who Received Prior Treatment with Metronidazole

    PubMed Central

    Khairnar, Krishna; Parija, Subhash Chandra

    2008-01-01

    Saliva is an easily-accessible and a non-invasive clinical specimen alternate to blood and liver pus. An attempt was made to detect Entamoeba histolytica DNA released in the saliva of amoebic liver abscess (ALA) patients by applying 16S-like rRNA gene-based nested multiplex polymerase chain reaction (NM-PCR). The NM-PCR detected E. histolytica DNA in the saliva of eight (28.6%) of 28 ALA patients. The NM-PCR result was negative for E. histolytica DNA in the saliva of all the eight ALA patients who were tested prior to treatment with metronidazole but was positive in the saliva of eight (40%) of 20 ALA patient who were tested after therapy with metronidazole. The NM-PCR detected E. histolytica DNA in liver abscess pus of all 28 (100%) patients with ALA. The TechLab E. histolytica II enzyme-linked immunosorbent assay was positive for E. histolytica Gal/GalNAc lectin antigen in the liver abscess pus of 13 (46.4%) of the 28 ALA patients. The indirect haemagglutination (IHA) test was positive for anti-amoebic antibodies in the serum of 22 (78.6%) of the 28 ALA patients and 2 (5.7%) of 35 healthy controls. The present study, for the first time, demonstrates the release of E. histolytica DNA in the saliva of ALA patients by applying NM-PCR. PMID:19069620

  8. Detection of Entamoeba histolytica DNA in the saliva of amoebic liver abscess patients who received prior treatment with metronidazole.

    PubMed

    Khairnar, Krishna; Parija, Subhash Chandra

    2008-12-01

    Saliva is an easily-accessible and a non-invasive clinical specimen alternate to blood and liver pus. An attempt was made to detect Entamoeba histolytica DNA released in the saliva of amoebic liver abscess (ALA) patients by applying 16S-like rRNA gene-based nested multiplex polymerase chain reaction (NM-PCR). The NM-PCR detected E. histolytica DNA in the saliva of eight (28.6%) of 28 ALA patients. The NM-PCR result was negative for E. histolytica DNA in the saliva of all the eight ALA patients who were tested prior to treatment with metronidazole but was positive in the saliva of eight (40%) of 20 ALA patient who were tested after therapy with metronidazole. The NM-PCR detected E. histolytica DNA in liver abscess pus of all 28 (100%) patients with ALA. The TechLab E. histolytica II enzyme-linked immunosorbent assay was positive for E. histolytica Gal/GalNAc lectin antigen in the liver abscess pus of 13 (46.4%) of the 28 ALA patients. The indirect haemagglutination (IHA) test was positive for anti-amoebic antibodies in the serum of 22 (78.6%) of the 28 ALA patients and 2 (5.7%) of 35 healthy controls. The present study, for the first time, demonstrates the release of E. histolytica DNA in the saliva of ALA patients by applying NM-PCR. PMID:19069620

  9. Testing possibilities for establishing nanodiamond-DNA-conjugates

    NASA Astrophysics Data System (ADS)

    Pohl, A.; Joch, S.; Michael, J.; Boschke, E.; Quenzel, P.; Schreiber, J.; Lapina, V.; Opitz, J.

    2011-07-01

    We report on the biofunctionalization of nanodiamond surfaces in a two step procedure: chemical modification, resulting in homogeneous and defined surface coating, with following addition of ssDNA. Carboxylation, thymidine coupling and amination methods for chemical modification of diamond surfaces for further functionalization experiments were applied. To enable the coupling process, biomolecules were also chemically modified with functional groups (-NH2). FTIR spectroscopy, fluorescence microscopy and gel electrophoresis were applied for characterizing modified ND particles and bioconjugates and for controlling the coupling success.

  10. Nanostructured SERS-electrochemical biosensors for testing of anticancer drug interactions with DNA.

    PubMed

    Ilkhani, Hoda; Hughes, Taylor; Li, Jing; Zhong, Chuan Jian; Hepel, Maria

    2016-06-15

    Widely used anti-cancer treatments involving chemotherapeutic drugs result in cancer cell damage due to their strong interaction with DNA. In this work, we have developed laboratory biosensors for screening chemotherapeutic drugs and to aid in the assessment of DNA modification/damage caused by these drugs. The sensors utilize surface-enhanced Raman scattering (SERS) spectroscopy and electrochemical methods to monitor sensory film modification and observe the drug-DNA reactivity. The self-assembled monolayer protected gold-disk electrode (AuDE) was coated with a reduced graphene oxide (rGO), decorated with plasmonic gold-coated Fe2Ni@Au magnetic nanoparticles functionalized with double-stranded DNA (dsDNA), a sequence of the breast cancer gene BRCA1. The nanobiosensors AuDE/SAM/rGO/Fe2Ni@Au/dsDNA were then subjected to the action of a model chemotherapeutic drug, doxorubicin (DOX), to assess the DNA modification and its dose dependence. The designed novel nanobiosensors offer SERS/electrochemical transduction, enabling chemically specific and highly sensitive analytical signals generation. The SERS measurements have corroborated the DOX intercalation into the DNA duplex whereas the electrochemical scans have indicated that the DNA modification by DOX proceeds in a concentration dependent manner, with limit of detection LOD=8µg/mL (S/N=3), with semilog linearity over 3 orders of magnitude. These new biosensors are sensitive to agents that interact with DNA and facilitate the analysis of functional groups for determination of the binding mode. The proposed nanobiosensors can be applied in the first stage of the drug development for testing the interactions of new drugs with DNA before the drug efficacy can be assessed in more expensive testing in vitro and in vivo. PMID:26851584

  11. Testing the feasibility of DNA typing for human identification by PCR and an oligonucleotide ligation assay

    SciTech Connect

    Delahunty, C.; Ankener, W.; Deng, Qiang

    1996-06-01

    The use of DNA typing in human genome analysis is increasing and finding widespread application in the area of forensic and paternity testing. In this report, we explore the feasibility of typing single nucleotide polymorphisms (SNPs) by using a semiautomated method for analyzing human DNA samples. In this approach, PCR is used to amplify segments of human DNA containing a common SNP. Allelic nucleotides in the amplified product are then typed by a calorimetric implementation of the oligonucleotide ligation assay (OLA). The results of the combined assay, PCR/OLA, are read directly by a spectrophotometer; the absorbances are compiled and the genotypes are automatically determined. A panel of 20 markers has been developed for DNA typing and has been tested using a sample panel from the CEPH pedigrees (CEPH parents). The results of this typing, as well as the potential to apply this method to larger populations, are discussed. 62 refs., 2 figs., 4 tabs.

  12. Estimation of the Proportion of Genetic Variation Accounted for by DNA Tests

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An increasingly relevant question in evaluating commercial DNA tests is "What proportion of the additive genetic variation in the target trait is accounted for by the test?" Therefore, several estimators of this quantity were evaluated by simulation of a population of 1000 animals with 100 sires, ea...

  13. Cell free DNA testing-interpretation of results using an online calculator.

    PubMed

    Grace, Matthew R; Hardisty, Emily; Green, Noah S; Davidson, Emily; Stuebe, Alison M; Vora, Neeta L

    2015-07-01

    All pregnant women, regardless of age, should be offered screening or invasive testing for chromosomal abnormalities at <20 weeks' gestation. Noninvasive prenatal screening for fetal aneuploidy with the use of cell-free DNA (cfDNA) is a screening method that offers high sensitivity and specificity in validation studies and has reduced the need for unnecessary invasive procedures. Laboratories often advertise and report a test's sensitivity and specificity as a means to describe the test's accuracy. The positive predictive value (PPV) of a screening test (the proportion of positive results that are truly positive) is a function of the prevalence of the condition in a population and often is not reported in direct-to-patient advertising. False-positive cfDNA screening tests have been reported, and there is evidence that some women are deciding to terminate their pregnancy without confirmatory testing. We believe that laboratories should disclose the patient-specific PPV of cfDNA screening for aneuploidy on result reports. To assist with counseling patients about the benefits, risks, and limitations of aneuploidy screening with the use of cfDNA and to demonstrate the relationship between an a priori risk and PPV, we developed a web-based calculator to estimate the PPV of the 4 commercially available cfDNA testing platforms for which data have been published. Estimates are made with the use of a patient's age and gestational age-related risk of trisomy 21, 18 and 13 or an a priori risk that is based on other findings. This web-based calculator is an aid for providers and genetic counselors to illustrate the relationship between disease prevalence and a test's PPV. It has enhanced our counseling of patients both before they elect noninvasive prenatal screening and after they receive a positive result. PMID:25957020

  14. NEXT GENERATION SEDIMENT TOXICITY TESTING VIA DNA MICROARRAYS - PHASE I

    EPA Science Inventory

    The current SBIR solicitation states that the EPA is seeking “better sampling, analysis, and monitoring technologies” to improve hazardous waste management.  Development of new methods for testing contaminated sediments is an area of particular concern because many industri...

  15. A Two-Gene Blood Test for Methylated DNA Sensitive for Colorectal Cancer

    PubMed Central

    Pedersen, Susanne K.; Baker, Rohan T.; McEvoy, Aidan; Murray, David H.; Thomas, Melissa; Molloy, Peter L.; Mitchell, Sue; Lockett, Trevor; Young, Graeme P.; LaPointe, Lawrence C.

    2015-01-01

    Background Specific genes are methylated with high frequency in colorectal neoplasia, and may leak into blood. Detection of multiple methylated DNA biomarkers in blood may improve assay sensitivity for colorectal cancer (CRC) relative to a single marker. We undertook a case-control study evaluating the presence of two methylation DNA markers, BCAT1 and IKZF1, in circulation to determine if they were complementary for detection of CRC. Methods Methylation-specific PCR assays were developed to measure the level of methylated BCAT1 and IKZF1 in DNA extracted from plasma obtained from colonoscopy-confirmed 144 healthy controls and 74 CRC cases. Results DNA yields ranged from 2 to 730 ng/mL plasma (mean 18.6ng/mL; 95% CI 11-26 ng/mL) and did not correlate with gender, age or CRC status. Methylated BCAT1 and IKZF1 DNA were detected in respectively 48 (65%) and 50 (68%) of the 74 cancers. In contrast, only 5 (4%) and 7 (5%) controls were positive for BCAT1 and IKZF1 DNA methylation, respectively. A two-gene classifier model (“either or” rule) improved segregation of CRC from controls, with 57 of 74 cancers (77%) compared to only 11 of 144 (7.6%) controls being positive for BCAT1 and/or IKZF1 DNA methylation. Increasing levels of methylated DNA were observed as CRC stage progressed. Conclusions Detection of methylated BCAT1 and/or IKZF1 DNA in plasma may have clinical application as a novel blood test for CRC. Combining the results from the two methylation-specific PCR assays improved CRC detection with minimal change in specificity. Further validation of this two-gene blood test with a view to application in screening is now indicated. PMID:25928810

  16. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  17. Fecal DNA testing for colorectal cancer screening: Molecular targets and perspectives

    PubMed Central

    Dhaliwal, Amaninder; Vlachostergios, Panagiotis J; Oikonomou, Katerina G; Moshenyat, Yitzchak

    2015-01-01

    The early detection of colorectal cancer with effective screening is essential for reduction of cancer-specific mortality. The addition of fecal DNA testing in the armamentarium of screening methods already in clinical use launches a new era in the noninvasive part of colorectal cancer screening and emanates from a large number of previous and ongoing clinical investigations and technological advancements. In this review, we discuss the molecular rational and most important genetic alterations hallmarking the early colorectal carcinogenesis process. Also, representative DNA targets-markers and key aspects of their testing at the clinical level in comparison or/and association with other screening methods are described. Finally, a critical view of the strengths and limitations of fecal DNA tests is provided, along with anticipated barriers and suggestions for further exploitation of their use. PMID:26483873

  18. Modeling and simulation of the mechanical response from nanoindentation test of DNA-filled viral capsids.

    PubMed

    Ahadi, Aylin; Johansson, Dan; Evilevitch, Alex

    2013-03-01

    Viruses can be described as biological objects composed mainly of two parts: a stiff protein shell called a capsid, and a core inside the capsid containing the nucleic acid and liquid. In many double-stranded DNA bacterial viruses (aka phage), the volume ratio between the liquid and the encapsidated DNA is approximately 1:1. Due to the dominant DNA hydration force, water strongly mediates the interaction between the packaged DNA strands. Therefore, water that hydrates the DNA plays an important role in nanoindentation experiments of DNA-filled viral capsids. Nanoindentation measurements allow us to gain further insight into the nature of the hydration and electrostatic interactions between the DNA strands. With this motivation, a continuum-based numerical model for simulating the nanoindentation response of DNA-filled viral capsids is proposed here. The viral capsid is modeled as large- strain isotropic hyper-elastic material, whereas porous elasticity is adopted to capture the mechanical response of the filled viral capsid. The voids inside the viral capsid are assumed to be filled with liquid, which is modeled as a homogenous incompressible fluid. The motion of a fluid flowing through the porous medium upon capsid indentation is modeled using Darcy's law, describing the flow of fluid through a porous medium. The nanoindentation response is simulated using three-dimensional finite element analysis and the simulations are performed using the finite element code Abaqus. Force-indentation curves for empty, partially and completely DNA-filled capsids are directly compared to the experimental data for bacteriophage λ. Material parameters such as Young's modulus, shear modulus, and bulk modulus are determined by comparing computed force-indentation curves to the data from the atomic force microscopy (AFM) experiments. Predictions are made for pressure distribution inside the capsid, as well as the fluid volume ratio variation during the indentation test. PMID:23860868

  19. Unique DNA-barcoded aerosol test particles for studying aerosol transport

    DOE PAGESBeta

    Harding, Ruth N.; Hara, Christine A.; Hall, Sara B.; Vitalis, Elizabeth A.; Thomas, Cynthia B.; Jones, A. Daniel; Day, James A.; Tur-Rojas, Vincent R.; Jorgensen, Trond; Herchert, Edwin; et al

    2016-03-22

    Data are presented for the first use of novel DNA-barcoded aerosol test particles that have been developed to track the fate of airborne contaminants in populated environments. Until DNATrax (DNA Tagged Reagents for Aerosol eXperiments) particles were developed, there was no way to rapidly validate air transport models with realistic particles in the respirable range of 1–10 μm in diameter. The DNATrax particles, developed at Lawrence Livermore National Laboratory (LLNL) and tested with the assistance of the Pentagon Force Protection Agency, are the first safe and effective materials for aerosol transport studies that are identified by DNA molecules. The usemore » of unique synthetic DNA barcodes overcomes the challenges of discerning the test material from pre-existing environmental or background contaminants (either naturally occurring or previously released). The DNATrax particle properties are demonstrated to have appropriate size range (approximately 1–4.5 μm in diameter) to accurately simulate bacterial spore transport. As a result, we describe details of the first field test of the DNATrax aerosol test particles in a large indoor facility.« less

  20. No Evidence for Interference of Hematoxylin and Eosin (HE) Staining in DNA Testing: Utility of DNA Extraction from HE-Stained Archival Tissue Sections

    PubMed Central

    Morikawa, Teppei; Shima, Kaori; Kuchiba, Aya; Yamauchi, Mai; Tanaka, Noriko; Imamura, Yu; Liao, Xiaoyun; Qian, Zhi Rong; Brahmandam, Mohan; Longtine, Janina A.; Lindeman, Neal I.; Fuchs, Charles S.; Ogino, Shuji

    2012-01-01

    Although histochemical staining has been believed to inhibit DNA amplification reaction, no previous study has systematically evaluated the influence of histochemical staining on downstream molecular assays. To evaluate an influence of hematoxylin and eosin (HE) staining on DNA testing, we isolated DNA from 10 unstained, 10 hematoxylin-stained, 10 eosin-stained or 10 HE-stained tissue sections (ie, 4 groups), from each of 5 colon cancers. Among those 4 groups, we did not observe any significant or appreciable difference in DNA fragmentation by agarose gel electrophoresis; in DNA amplification by real-time PCR; in microsatellite PCR fragment analyses; or in PCR-Pyrosequencing. As a proof-of-principle study, we successfully performed microsatellite instability analysis and sequencing of KRAS and BRAF on over 1300 colorectal cancers using DNA extracted from HE stained tissue sections. Our data provide no evidence for interfering effect of HE staining on DNA testing, suggesting that DNA from HE-stained sections can be effectively used for routine DNA testing. PMID:22706867

  1. A neutrality test for detecting selection on DNA methylation using single methylation polymorphism frequency spectrum.

    PubMed

    Wang, Jun; Fan, Chuanzhu

    2015-01-01

    Inheritable epigenetic mutations (epimutations) can contribute to transmittable phenotypic variation. Thus, epimutations can be subject to natural selection and impact the fitness and evolution of organisms. Based on the framework of the modified Tajima's D test for DNA mutations, we developed a neutrality test with the statistic "D(m)" to detect selection forces on DNA methylation mutations using single methylation polymorphisms. With computer simulation and empirical data analysis, we compared the D(m) test with the original and modified Tajima's D tests and demonstrated that the D(m) test is suitable for detecting selection on epimutations and outperforms original/modified Tajima's D tests. Due to the higher resetting rate of epimutations, the interpretation of D(m) on epimutations and Tajima's D test on DNA mutations could be different in inferring natural selection. Analyses using simulated and empirical genome-wide polymorphism data suggested that genes under genetic and epigenetic selections behaved differently. We applied the D(m) test to recently originated Arabidopsis and human genes, and showed that newly evolved genes contain higher level of rare epialleles, suggesting that epimutation may play a role in origination and evolution of genes and genomes. Overall, we demonstrate the utility of the D(m) test to detect whether the loci are under selection regarding DNA methylation. Our analytical metrics and methodology could contribute to our understanding of evolutionary processes of genes and genomes in the field of epigenetics. The Perl script for the "D(m)" test is available at http://fanlab.wayne.edu/ (last accessed December 18, 2014). PMID:25539727

  2. Demand for DNA probe testing in three genetic centres in Britain (August 1986 to July 1987).

    PubMed

    Rona, R J; Swan, A V; Beech, R; Prentice, L; Reynolds, A; Wilson, O; Mole, G; Vadera, P

    1989-04-01

    We report a preliminary analysis of the data collected during the first year of the evaluation of clinical genetics in the context of DNA probes in three genetic centres, to show the pattern of the demand for genetic services in the three centres and the services used in meeting that demand. The analysis includes information on 10,185 persons from 2852 families. The results are presented according to mode of inheritance and according to the most common disorders for which DNA probes have been used in the three centres. The results indicate that the use of DNA probes is now a major element of activity in genetic departments, and that as long as indirect DNA probe testing is the predominant manner of using recombinant technology, the clinical input will be an important element of the costs, probably more so than that of the DNA laboratories, as a large number of family members needs to be tested. In most cases centres have concentrated activity on DNA testing for common and severe genetic disorders. However, there are disorders, such as familial hypercholesterolaemia, which have not been part of the established pattern of services. Conversely, a relatively high number of families have been studied for some disorders of very low incidence. This suggests that the number of DNA laboratories should be limited. The precise arrangements will need to be established. With such services the distribution of DNA testing facilities for different disorders can be controlled to limit duplication. The model followed in Scotland based on collaboration between centres is worth considering. We have detected very large differences in take up rate of services within and between regions. Although many factors may contribute to these differences, ease of access and lay and professional awareness are probably the most important. This is supported by the fact that more patients from the same or neighbouring DHAs attend the genetic centre than from those further away. We also concluded that

  3. Estimation Of The Proportion Of Variation Accounted For By DNA Tests. II: Phenotypic Variance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The proportion of phenotypic variation accounted for (Rp2) is an important characteristic of a DNA test. Therefore, several estimators of this quantity were evaluated by simulation of 500 replicates of a population of 1000 progeny of 100 sires (3 levels of narrow sense heritability and 4 levels of ...

  4. Estimation of the Proportion of Variation Accounted for by DNA Tests. I: Genetic Variance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The proportion of genetic variation accounted for (Rg2) is an important characteristic of a DNA test. For each of 3 levels of narrow sense heritability of the observed trait (h2gy) and 4 levels of Rg2, 500 independent replicates of an observed trait and a molecular breeding value (MBV) for 1000 offs...

  5. Validation and Estimation of Additive Genetic Variation Associated with DNA Tests for Quantitative Beef Cattle Traits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The U.S. National Beef Cattle Evaluation Consortium (NBCEC) has been involved in the validation of commercial DNA tests for quantitative beef quality traits since their first appearance on the U.S. market in the early 2000s. The NBCEC Advisory Council initially requested that the NBCEC set up a syst...

  6. Effective appropriate use of dental remains and forensic DNA testing for personal identity confirmation.

    PubMed

    Ohira, Hiroshi; Yamamuro, Yoshio; Kitagawa, Yuka; Nakagawa, Kimiko; Yamamoto, Isao; Yamada, Yoshihiro

    2009-04-01

    Two severely burned human male bodies, possibly those of a parent and child, were recovered from the scene of a house fire and positive identification of the bodies was accomplished. This report describes the appropriate use of effective identification methods that made this possible. Identification of a body involves comparison of antemortem and postmortem X-ray films or dental records. In cases of poorly preserved dental remains, or in the absence of antemortem dental records, forensic DNA testing can be done. In the present case the male thought to be the son was identified from an antemortem panoramic X-ray film provided by the family dentist, which matched every significant detail in the body. On the other hand, forensic DNA testing of a sample obtained from the father's burned body was done in comparison with a swab obtained from his older brother, as the victim had no dental records for the 5 years before his death. This was able to confirm his identity. Thus positive identification was established through a combination of these methods. Although positive identification from dental records is rapid and certain, it requires antemortem dental records. If these are not available, forensic DNA testing should be attempted. In laboratories of forensic odontology, facilities for identity confirmation from both dental characteristics and forensic DNA testing should be made available. PMID:19269216

  7. USING DNA MICROARRAYS TO CHARACTERIZE GENE EXPRESSION IN TESTES OF FERTILE AND INFERTILE HUMANS AND MICE

    EPA Science Inventory

    USING DNA MICROARRAYS TO CHARACTERIZE GENE EXPRESSION
    IN TESTES OF FERTILE AND INFERTILE HUMANS AND MICE

    John C. Rockett1, J. Christopher Luft1, J. Brian Garges1, M. Stacey Ricci2, Pasquale Patrizio2, Norman B. Hecht2 and David J. Dix1
    Reproductive Toxicology Divisio...

  8. A web resource on DNA tests for canine and feline hereditary diseases

    PubMed Central

    Slutsky, Jeffrey; Raj, Karthik; Yuhnke, Scott; Bell, Jerold; Fretwell, Neale; Hedhammar, Ake; Wade, Claire; Giger, Urs

    2013-01-01

    Following the first identification of a disease-causing mutation in dogs in 1989 and the more recent completion of canine and feline genome sequences, much progress has been made in the molecular characterization of hereditary diseases in dogs and cats. To increase access to information on diagnosing hereditary diseases in dogs and cats, a web application has been developed to collect, organize and display information on available DNA tests and other supporting information, including gene and chromosomal locations, mutations, primary research citations and disease descriptions. The DNA testing information can be accessed at the URL: http://research.vet.upenn.edu/WSAVA-LabSearch. There are currently 131 molecular genetic tests available for hereditary diseases in dogs and cats offered by 43 laboratories worldwide. This tool should provide clinicians, researchers, breeders and companion animal owners with a single comprehensive, up-to-date and readily searchable webpage for information on hereditary disease testing. PMID:23582432

  9. Integrating prior knowledge in multiple testing under dependence with applications to detecting differential DNA methylation.

    PubMed

    Kuan, Pei Fen; Chiang, Derek Y

    2012-09-01

    DNA methylation has emerged as an important hallmark of epigenetics. Numerous platforms including tiling arrays and next generation sequencing, and experimental protocols are available for profiling DNA methylation. Similar to other tiling array data, DNA methylation data shares the characteristics of inherent correlation structure among nearby probes. However, unlike gene expression or protein DNA binding data, the varying CpG density which gives rise to CpG island, shore and shelf definition provides exogenous information in detecting differential methylation. This article aims to introduce a robust testing and probe ranking procedure based on a nonhomogeneous hidden Markov model that incorporates the above-mentioned features for detecting differential methylation. We revisit the seminal work of Sun and Cai (2009, Journal of the Royal Statistical Society: Series B (Statistical Methodology)71, 393-424) and propose modeling the nonnull using a nonparametric symmetric distribution in two-sided hypothesis testing. We show that this model improves probe ranking and is robust to model misspecification based on extensive simulation studies. We further illustrate that our proposed framework achieves good operating characteristics as compared to commonly used methods in real DNA methylation data that aims to detect differential methylation sites. PMID:22260651

  10. The clinical utility of HPV DNA testing in cervical cancer screening strategies.

    PubMed

    Bhatla, Neerja; Moda, Nidhi

    2009-09-01

    Cervical cancer continues to be the commonest cause of death among women in developing countries, largely due to the failure to the inability to sustain effective cytology-based screening programs. While this burden may come down following implementation of the human papillomavirus (HPV) vaccine, screening will still be required. HPV DNA testing is a promising new technology for cervical cancer prevention and is the most reproducible of all cervical cancer screening tests. Presently, the two assays most widely used for the detection of genital types are the polymerase chain reaction (PCR) and Hybrid Capture 2 assays (hc2). Rapid, affordable tests are expected to be available soon. HPV DNA testing can be used in a variety of clinical scenarios that include primary screening in women older than 30 yr; as an adjunctive test to cytology; in the triage of women with an equivocal cytologic report, e.g., ASC-US; or for follow-up post-treatment for cervical intraepithelial neoplasia (CIN). HPV DNA testing can also be performed on self-collected samples, which allows screening in remote areas and also in women who refuse gynecologic examination. PMID:19901435

  11. Pitfalls of Establishing DNA Barcoding Systems in Protists: The Cryptophyceae as a Test Case

    PubMed Central

    Hoef-Emden, Kerstin

    2012-01-01

    A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5′-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed. PMID:22970104

  12. The Sperm Chromatin Structure Assay (SCSA(®)) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility.

    PubMed

    Evenson, Donald P

    2016-06-01

    Thirty-five years ago the pioneering paper in Science (240:1131) on the relationship between sperm DNA integrity and pregnancy outcome was featured as the cover issue showing a fluorescence photomicrograph of red and green stained sperm. The flow cytometry data showed a very significant difference in sperm DNA integrity between fertile and subfertile bulls and men. This study utilized heat (100°C, 5min) to denature DNA at sites of DNA strand breaks followed by staining with acridine orange (AO) and measurements of 5000 individual sperm of green double strand (ds) DNA and red single strand (ss) DNA fluorescence. Later, the heat protocol was changed to a low pH protocol to denature the DNA at sites of strand breaks; the heat and acid procedures produced the same results. SCSA data are very advantageously dual parameter with 1024 channels (degrees) of both red and green fluorescence. Hundreds of publications on the use of the SCSA test in animals and humans have validated the SCSA as a highly useful test for determining male breeding soundness. The SCSA test is a rapid, non-biased flow cytometer machine measurement providing robust statistical data with exceptional precision and repeatability. Many genotoxic experiments showed excellent dose response data with very low coefficient of variation that further validated the SCSA as being a highly powerful assay for sperm DNA integrity. Twelve years following the introduction of the SCSA test, the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labelling (TUNEL) test (1993) for sperm was introduced as the only other flow cytometric assay for sperm DNA fragmentation. However, the TUNEL test can also be done by light microscopy with much less statistical robustness. The COMET (1998) and Sperm Chromatin Dispersion (SCD; HALO) (2003) tests were introduced as light microscope tests that don't require a flow cytometer. Since these tests measure only 50-200 sperm per sample, they suffer from the lack of

  13. Haemagglutination induced by Bordetella pertussis filamentous haemagglutinin adhesin (FHA) is inhibited by antibodies produced against FHA(430-873) fragment expressed in Lactobacillus casei.

    PubMed

    Colombi, Débora; Oliveira, Maria L S; Campos, Ivana B; Monedero, Vicente; Pérez-Martinez, Gaspar; Ho, Paulo L

    2006-12-01

    Filamentous haemagglutinin adhesin (FHA) is an important virulence factor from Bordetella pertussis related to the adhesion and spread of the bacteria through the respiratory tract. Three distinct domains have been characterized in mature FHA, and among them, the FHA(442-863) fragment was suggested to be responsible for the heparin-binding activity. In this study, we cloned the gene encoding the HEP fragment (FHA(430-873)) in a Lactobacillus casei-inducible expression vector based on the lactose operon. The recombinant bacteria, transformed with the resulting construct (L. casei-HEP), were able to express the heterologous protein depending on the sugar added to the culture. Subcutaneous inoculation of L. casei-HEP in Balb/C mice, using the cholera toxin B subunit as adjuvant, induced systemic anti-HEP antibodies that were able to inhibit in vitro erythrocyte haemagglutination induced by FHA. This is the first example of a B. pertussis antigen produced in lactic acid bacteria and opens new perspectives for alternative vaccine strategies against whooping cough. PMID:17106803

  14. Swabbing Students: Should Universities Be Allowed to Facilitate Educational DNA Testing?

    PubMed Central

    Callier, Shawneequa L.

    2012-01-01

    Recognizing the profound need for greater patient and provider familiarity with personalized genomic medicine, many university instructors are including personalized genotyping as part of their curricula. During seminars and lectures students run polymerase chain reactions on their own DNA or evaluate their experiences using direct-to-consumer genetic testing services subsidized by the university. By testing for genes that may influence behavioral or health-related traits, however, such as alcohol tolerance and cancer susceptibility, certain universities have stirred debate on the ethical concerns raised by educational genotyping. Considering the potential for psychosocial harm and medically relevant outcomes, how far should university-facilitated DNA testing be permitted to go? The analysis here distinguishes among these learning initiatives and critiques their approaches to the ethical concerns raised by educational genotyping. PMID:22452475

  15. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  16. Feasibility of cell-free circulating tumor DNA testing for lung cancer.

    PubMed

    Santarpia, Mariacarmela; Karachaliou, Niki; González-Cao, Maria; Altavilla, Giuseppe; Giovannetti, Elisa; Rosell, Rafael

    2016-04-01

    Tumor tissue genotyping is used routinely for lung cancer to identify specific targetable oncogenic alterations, including EGFR mutations and ALK rearrangements. However, tumor tissue from a single biopsy is often insufficient for molecular testing, may offer a limited evaluation because of tumor heterogeneity and can be difficult to obtain. Cell-free circulating tumor DNA has been widely investigated as a potential surrogate for tissue biopsy for noninvasive assessment of tumor-related genomic alterations. New techniques have improved EGFR mutations detection in ctDNA, thus supporting the use of this liquid biopsy for predicting response to EGFR tyrosine kinase inhibitors (TKIs) and monitoring the emergence of resistance. The serial evaluation of ctDNA during treatment is feasible and can be used to track tumor changes in real time and for a wide range of clinically useful applications. PMID:26974841

  17. Quantification of Plasmodiophora brassicae Using a DNA-Based Soil Test Facilitates Sustainable Oilseed Rape Production

    PubMed Central

    Wallenhammar, Ann-Charlotte; Gunnarson, Albin; Hansson, Fredrik; Jonsson, Anders

    2016-01-01

    Outbreaks of clubroot disease caused by the soil-borne obligate parasite Plasmodiophora brassicae are common in oilseed rape (OSR) in Sweden. A DNA-based soil testing service that identifies fields where P. brassicae poses a significant risk of clubroot infection is now commercially available. It was applied here in field surveys to monitor the prevalence of P. brassicae DNA in field soils intended for winter OSR production and winter OSR field experiments. In 2013 in Scania, prior to planting, P. brassicae DNA was detected in 60% of 45 fields on 10 of 18 farms. In 2014, P. brassicae DNA was detected in 44% of 59 fields in 14 of 36 farms, in the main winter OSR producing region in southern Sweden. P. brassicae was present indicative of a risk for >10% yield loss with susceptible cultivars (>1300 DNA copies g soil−1) in 47% and 44% of fields in 2013 and 2014 respectively. Furthermore, P. brassicae DNA was indicative of sites at risk of complete crop failure if susceptible cultivars were grown (>50 000 copies g−1 soil) in 14% and 8% of fields in 2013 and 2014, respectively. A survey of all fields at Lanna research station in western Sweden showed that P. brassicae was spread throughout the farm, as only three of the fields (20%) showed infection levels below the detection limit for P.brassicae DNA, while the level was >50,000 DNA copies g−1 soil in 20% of the fields. Soil-borne spread is of critical importance and soil scraped off footwear showed levels of up to 682 million spores g−1 soil. Soil testing is an important tool for determining the presence of P. brassicae and providing an indication of potential yield loss, e.g., in advisory work on planning for a sustainable OSR crop rotation. This soil test is gaining acceptance as a tool that increases the likelihood of success in precision agriculture and in applied research conducted in commercial oilseed fields and at research stations. The present application highlights the importance of prevention of

  18. Quantification of Plasmodiophora brassicae Using a DNA-Based Soil Test Facilitates Sustainable Oilseed Rape Production.

    PubMed

    Wallenhammar, Ann-Charlotte; Gunnarson, Albin; Hansson, Fredrik; Jonsson, Anders

    2016-01-01

    Outbreaks of clubroot disease caused by the soil-borne obligate parasite Plasmodiophora brassicae are common in oilseed rape (OSR) in Sweden. A DNA-based soil testing service that identifies fields where P. brassicae poses a significant risk of clubroot infection is now commercially available. It was applied here in field surveys to monitor the prevalence of P. brassicae DNA in field soils intended for winter OSR production and winter OSR field experiments. In 2013 in Scania, prior to planting, P. brassicae DNA was detected in 60% of 45 fields on 10 of 18 farms. In 2014, P. brassicae DNA was detected in 44% of 59 fields in 14 of 36 farms, in the main winter OSR producing region in southern Sweden. P. brassicae was present indicative of a risk for >10% yield loss with susceptible cultivars (>1300 DNA copies g soil(-1)) in 47% and 44% of fields in 2013 and 2014 respectively. Furthermore, P. brassicae DNA was indicative of sites at risk of complete crop failure if susceptible cultivars were grown (>50 000 copies g(-1) soil) in 14% and 8% of fields in 2013 and 2014, respectively. A survey of all fields at Lanna research station in western Sweden showed that P. brassicae was spread throughout the farm, as only three of the fields (20%) showed infection levels below the detection limit for P.brassicae DNA, while the level was >50,000 DNA copies g(-1) soil in 20% of the fields. Soil-borne spread is of critical importance and soil scraped off footwear showed levels of up to 682 million spores g(-1) soil. Soil testing is an important tool for determining the presence of P. brassicae and providing an indication of potential yield loss, e.g., in advisory work on planning for a sustainable OSR crop rotation. This soil test is gaining acceptance as a tool that increases the likelihood of success in precision agriculture and in applied research conducted in commercial oilseed fields and at research stations. The present application highlights the importance of prevention of

  19. Cell free fetal DNA testing in maternal blood of Romanian pregnant women

    PubMed Central

    Radoi, Viorica E; Bohiltea, Camil L; Bohiltea, Roxana E; Albu, Dragos N

    2015-01-01

    Background: The discovery of circulating fetal DNA in maternal blood led to the discovery of new strategies to perform noninvasive testing for prenatal diagnosis. Objective: The purpose of the study was to detect fetal aneuploidy at chromosomes 13, 18, 21, X, and Y by analysis of fetal cell-free DNA from maternal blood, without endangering pregnancy. Materials and Methods: This retrospective study has been performed in Bucharest at Medlife Maternal and Fetal Medicine Department between 2013-2014. In total 201 women were offered noninvasive prenatal test. Maternal plasma samples were collected from women at greater than 9 weeks of gestation after informed consent and genetics counseling. Results: From 201 patients; 28 (13.93%) had screening test with high risk for trisomy 21, 116 (57.71%) had advanced maternal age, 1 (0.49%) had second trimester ultrasound markers and the remaining 56 patients (27.86%) performed the test on request. Of those patients, 189 (94.02%) had a “low risk” result (<1/10,000). Of those who had a low risk result, 2 continued on to have amniocentesis with normal results.Five patients (2.48%) received “high risk” results (>99% risk) all for trisomy 21 (T21). T21 was confirmed by amniocentesis in 1 patient and the other 4 patients declined confirmation. The 7 remaining patients (3.48%) had a low fetal fraction of DNA. Conclusion: It is probably that prenatal diagnosis using fetal DNA in maternal blood would play an increasingly role in the future practice of prenatal testing because of high accuracy. PMID:26644790

  20. Noninvasive prenatal testing by maternal plasma DNA analysis: current practice and future applications.

    PubMed

    Chiu, Rossa W K

    2014-01-01

    Prenatal screening of fetal chromosomal aneuploidies and some common genetic diseases is an integral part of antenatal care. Definitive prenatal diagnosis is conventionally achieved by the sampling of fetal genetic material by amniocentesis or chorionic villus sampling. Due to the invasiveness of those procedures, they are associated with a 1 in 200 chance of fetal miscarriage. Hence, researchers have been exploring noninvasive ways to sample fetal genetic material. The presence of cell-free DNA released by the fetus into the circulation of its mother was demonstrated in 1997. Circulating fetal DNA is therefore obtainable through the collection of a blood sample from the pregnant woman without posing any physical harm to the fetus. By analyzing this source of fetal genetic material, researchers have succeeded in developing DNA-based noninvasive tests for the assessment of Down syndrome and single gene diseases. Since the end of 2011, tests for the noninvasive assessment of chromosomal aneuploidies have become commercially available in parts of the world. Recommendations from professional groups have since been made regarding how these tests could be incorporated into the framework of existing prenatal screening programs. More recently, cell-free circulating fetal DNA analysis have been shown to be applicable to the deciphering of the fetal molecular karyotype, genome and methylome. It is envisioned that an increasing number of the noninvasive prenatal tests will become clinically available. The ethical, social and legal implications of the introduction of some of these tests would need to be discussed in the context of different cultures, societal values and the legal framework. PMID:25083893

  1. Sex and strain differences in the hepatocyte primary culture/DNA repair test

    SciTech Connect

    McQueen, C.A.; Way, B.M. )

    1991-01-01

    The hepatocyte primary culture (HPC)/DNA repair test was developed using hepatocytes isolated from male F-344 rats. A number of genetic polymorphisms have been shown to occur in inbred strains of rats, which may lead to variation in biotransformation of xenobiotics resulting in differences in susceptibility to genotoxins. The effect of the strain utilized as a source of hepatocytes was investigated with female Lewis, F-344, and DA rats. Variation was observed when hepatocytes from the three strains were exposed to aflatoxin B{sub 1} (AFB{sub 1}). No clearcut strain differences were seen when cells were exposed to diethylnitrosamine (DEN) or 2-acetylaminofluorene. These results demonstrate that both the strain and the sex of the animal used as a source of hepatocytes can affect the HPC/DNA repair test.

  2. [Paternity testing based on the analysis of DNA and its revision].

    PubMed

    Vrtĕl, R; Vodicka, R; Loyka, S; Santavý, J

    2008-01-01

    Paternity testing is nowadays mostly based on the analysis of DNA short tandem repeats (STR). Number and selection of STR loci differ according to identification kit producers and also particular laboratories. This article refers a revision of formerly excluded paternity. The cause of the mismatch was revealed by the enlargement of STR loci panel and reciprocal evaluation of samples and paternity was on the contrary confirmed. Different possibilities of failures, their consequences and preventions are also discussed. PMID:18335642

  3. Joseon funerary texts tested using ancient DNA analysis of a Korean mummy.

    PubMed

    Oh, Chang Seok; Koh, Bou-Ja; Yoo, Dong Soo; Park, Jun Bum; Min, So Ri; Kim, Yi-Suk; Lee, Sang Sup; Ge, Jianye; Seo, Seung Bum; Shin, Dong Hoon

    2015-06-01

    In Korea, ancient DNA (aDNA) analysis has been applied to investigations into the genetic affiliations of mummies found in Joseon Dynasty tombs (1392-1910 CE), becoming now indispensable tool for researches studying human remains from archaeological sites. In the course of our recent examinations on a Korean mummy of Joseon Dynasty, we discovered many teeth contained in a pouch. And in fact, the historical literature on the topic of Joseon funerals contain general accounts of pouches in which an individual's lost teeth were collected over the course of a lifetime and, after death, placed in the coffin with the body. To test the veracity of the historical texts, the present study undertook aDNA analyses and compared the results between specifically questioned (Q) samples (teeth) and known (K) samples (brain and bone) from the mummy to ensure that they came from the same individual. Although the Q-K comparison of autosomal short tandem repeat results did not show full concordance due to allelic drop-outs in some loci, our statistical calculation indicated that the teeth in the pouch are highly likely those of the mummy. Additionally, Q-K comparison of mitochondrial DNA sequence results showed 100% matches between samples. There results, in short, could not gainsay the conjecture that the teeth samples originated from the person buried in the tomb; and if so, he must have kept his teeth for a long time after their loss. As the application of aDNA analysis to Korean mummy studies develops, there will be other opportunities to test historical documents, particularly those referring to funerary rites. PMID:25998652

  4. A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care.

    PubMed

    Thierry, Alain R

    2016-01-01

    Circulating cell-free DNA (cfDNA) is a valuable source of tumor material available with a simple blood sampling enabling a noninvasive quantitative and qualitative analysis of the tumor genome. cfDNA is released by tumor cells and exhibits the genetic and epigenetic alterations of the tumor of origin. Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a noninvasive tumor molecular test for cancer patients. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure, and specific determination of cfDNA fragmentation and size, we revisited Q-PCR-based method and recently developed a the allele-specific-Q-PCR-based method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. This technique, named Intplex(®) and based on a refined Q-PCR method, derived from critical observations made on the specific structure and size of cfDNA. It enables the simultaneous determination of five parameters: the cfDNA total concentration, the presence of a previously known point mutation, the mutant (tumor) cfDNA concentration (ctDNA), the proportion of mutant cfDNA, and the cfDNA fragmentation index. Intplex(®) has enabled the first clinical validation of ctDNA analysis in oncology by detecting KRAS and BRAF point mutations in mCRC patients and has demonstrated that a blood test could replace tumor section analysis for the detection of KRAS and BRAF mutations. The Intplex(®) test can be adapted to all mutations, genes, or cancers and enables rapid, highly sensitive, cost-effective, and repetitive analysis. As regards to the determination of mutations on cfDNA Intplex(®) is limited to the mutational status of known hotspot mutation; it is a "targeted approach." However, it offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a noninvasive attractive tool potentially allowing diagnosis, prognosis, theranostics

  5. Role of intercalation and redox potential in DNA photosensitization by ruthenium(II) polypyridyl complexes: assessment using DNA repair protein tests.

    PubMed

    Gicquel, Etienne; Souchard, Jean-Pierre; Magnusson, Fay; Chemaly, Jad; Calsou, Patrick; Vicendo, Patricia

    2013-08-01

    Here we report that the photoreactivity of ruthenium(II) complexes with nucleobases may not only be modulated by their photoredox properties but also by their DNA binding mode. The damage resulting from photolysis of synthetic oligonucleotides and plasmid DNA by [Ru(bpz)3](2+), [Ru(bipy)3](2+) and the two DNA intercalating agents [Ru(bpz)2dppz](2+) and [Ru(bipy)2dppz](2+) has been monitored by polyacrylamide gel electrophoresis and by tests using proteins involved in DNA repair processes (DNA-PKCs, Ku80, Ku70, and PARP-1). The data show that intercalation controls the nature of the DNA damage photo-induced by ruthenium(II) complexes reacting with DNA via an electron transfer process. The intercalating agent [Ru(bpz)2dppz](2+) is a powerful DNA breaker inducing the formation of both single and double (DSBs) strand breaks which are recognized by the PARP-1 and DNA-PKCs proteins respectively. [Ru(bpz)2dppz](2+) is the first ruthenium(II) complex described in the literature that is able to induce DSBs by an electron transfer process. In contrast, its non-intercalating parent compound, [Ru(bpz)3](2+), is mostly an efficient DNA alkylating agent. Photoadducts are recognized by the proteins Ku70 and Ku80 as with cisplatin adducts. This result suggests that photoaddition of [Ru(bpz)2dppz](2+) is strongly affected by its DNA intercalation whereas its photonuclease activity is exalted. The data clearly show that DNA intercalation decreases drastically the photonuclease activity of ruthenium(II) complexes oxidizing guanine via the production of singlet oxygen. Interestingly, the DNA sequencing data revealed that the ligand dipyridophenazine exhibits on single-stranded oligonucleotides a preference for the 5'-TGCGT-3' sequence. Moreover the use of proteins involved in DNA repair processes to detect DNA damage was a powerful tool to examine the photoreactivity of ruthenium(II) complexes with nucleic acids. PMID:23835850

  6. Utilization of TIFF files in the analysis and interpretation of DNA tests

    SciTech Connect

    Yanamandra, K.; Krueger, S.; Thurmon, T.F.

    1994-09-01

    With the advent of DNA diagnostic testing in clinical practice, molecular geneticists in the genetic testing laboratories are routinely facing the task of interpreting electrophoretic banding patterns of DNA or RNA to diagnose genetic conditions. However, the DNA diagnosticians quite often experience difficulties in diagnoses while interpreting faint bands and smears from the blots, especially when smears are indicative of diagnosis in some conditions such as in fragile X syndrome. With the recent advances in computer imaging, we have been able to simplify this task of interpretation by converting these autoradiograms and Polaroid photographs from PCR gels into TIFF (Tagged Image Formal Files) files using HP ScanJet IIC for autoradiograms and a KODAK DCS 200 camera for PCR gels and using SigmaPlot software to analyze these TIFF files. Analysis of DNA from fragile X patients revealed poorly visible bands and smears from autoradiograms very well on TIFF files. Utilizing these files, nondiscrete bands and smears have been characterized and converted into meaningful peaks. Comparisons have been made to the respective chromosomal bands from patients and from controls and facilitated easy calculation of gene dosages. A major advantage of TIFF files is the archiving of innumerable nucleic acid blots on convenient floppy disks. These stored files could serve as a ready source of review and recall and aid in the proficiency testing and in laboratory inspections such as CAP or state inspections. They are also a ready source for publication and interlaboratory comparisons using a modem or transport by floppy without jeopardizing the quality. The TIFF patient data from our laboratory of different genetic conditions and their supplemental diagnostic value will be presented.

  7. New Statistical Tests of Neutrality for DNA Samples from a Population

    PubMed Central

    Fu, Y. X.

    1996-01-01

    The purpose of this paper is to develop statistical tests of the neutral model of evolution against a class of alternative models with the common characteristic of having an excess of mutations that occurred a long time ago or a reduction of recent mutations compared to the neutral model. This class of population genetics models include models for structured populations, models with decreasing effective population size and models of selection and mutation balance. Four statistical tests were proposed in this paper for DNA samples from a population. Two of these tests, one new and another a modification of an existing test, are based on EWENS' sampling formula, and the other two new tests make use of the frequencies of mutations of various classes. Using simulated samples and regression analyses, the critical values of these tests can be computed from regression equations. This approach for computing the critical values of a test was found to be appropriate and quite effective. We examined the powers of these four tests using simulated samples from structured populations, populations with linearly decreasing sizes and models of selection and mutation balance and found that they are more powerful than existing statistical tests of the neutral model of evolution. PMID:8722804

  8. Non-invasive, serum DNA pregnancy testing leading to incidental discovery of cancer: a good thing?

    PubMed

    Prasad, Vinay

    2015-11-01

    Cell-free DNA for perinatal screening is a growing industry. Non-invasive prenatal testing (NIPT) is based on the premise that foetal DNA is able to cross the placental barrier and enter the mother's circulation, where it can be examined for chromosomal abnormalities, such as trisomy 13, 18 or 21. Such tests are expected to be widely used by pregnant women, with the annual market expected to surpass $1 billion. Recently, a number of case reports have emerged in the haematology-oncology literature. The routine use of NIPT has led to the discovery of maternal neoplasms. Most writers have concluded that this is yet another benefit of the test; however, a closer examination of the cases reveals that this incidental detection may not improve patient outcomes. In some cases, early detection provides lead time bias, but does not change the ultimate clinical outcome, and in other cases, detection constitutes earlier knowledge of a cancer whose natural history cannot be altered. Here, we explore in detail cases where cancer was incidentally discovered among women undergoing routine non-invasive pregnancy testing, and investigate whether or not these women were benefitted by the discovery. PMID:26278647

  9. Screening Test for Shed Skin Cells by Measuring the Ratio of Human DNA to Staphylococcus epidermidis DNA.

    PubMed

    Nakanishi, Hiroaki; Ohmori, Takeshi; Hara, Masaaki; Takahashi, Shirushi; Kurosu, Akira; Takada, Aya; Saito, Kazuyuki

    2016-05-01

    A novel screening method for shed skin cells by detecting Staphylococcus epidermidis (S. epidermidis), which is a resident bacterium on skin, was developed. Staphylococcus epidermidis was detected using real-time PCR. Staphylococcus epidermidis was detected in all 20 human skin surface samples. Although not present in blood and urine samples, S. epidermidis was detected in 6 of 20 saliva samples, and 5 of 18 semen samples. The ratio of human DNA to S. epidermidisDNA was significantly smaller in human skin surface samples than in saliva and semen samples in which S. epidermidis was detected. Therefore, although skin cells could not be identified by detecting only S. epidermidis, they could be distinguished by measuring the S. epidermidis to human DNA ratio. This method could be applied to casework touch samples, which suggests that it is useful for screening whether skin cells and human DNA are present on potential evidentiary touch samples. PMID:27122397

  10. Assessing the impact of common forensic presumptive tests on the ability to obtain results using a novel rapid DNA platform.

    PubMed

    Donachie, Gillian E; Dawnay, Nick; Ahmed, Romana; Naif, Sarah; Duxbury, Nicola J; Tribble, Nicholas D

    2015-07-01

    The rise of DNA evidence to the forefront of forensic science has led to high sample numbers being submitted for profiling by investigators to casework laboratories: bottleneck effects are often seen resulting in slow turnaround times and sample backlog. The ParaDNA(®) Screening and Intelligence Tests have been designed to guide investigators on the viability of potential sources of DNA allowing them to determine which samples should be sent for full DNA analysis. Both tests are designed to augment the arsenal of available forensic tests for end users and be used concurrently to those commonly available. Therefore, assessing the impact that common forensic tests have on such novel technology is important to measure. The systems were tested against various potential inhibitors to which samples may be exposed as part of the investigative process. Presumptive test agents for biological materials (blood, semen and saliva) and those used as fingerprint enhancement agents were both used. The Screening Test showed a drop in performance following application of aluminium powder and cyanoacrylate (CNA) on fingerprints samples; however this drop in performance was not replicated with high template DNA. No significant effect was observed for any agent using the Intelligence Test. Therefore, both tests stand up well to the chemical agents applied and can be used by investigators with confidence that system performance will be maintained. PMID:25864157

  11. Evaluation of DNA from the Papanicolaou Test to Detect Ovarian and Endometrial Cancers

    PubMed Central

    Kinde, Isaac; Bettegowda, Chetan; Wang, Yuxuan; Wu, Jian; Agrawal, Nishant; Shih, Ie-Ming; Kurman, Robert; Dao, Fanny; Levine, Douglas A.; Giuntoli, Robert; Roden, Richard; Eshleman, James R.; Carvalho, Jesus Paula; Marie, Suely Kazue Nagahashi; Papadopoulos, Nickolas; Kinzler, Kenneth W.; Vogelstein, Bert; Diaz, Luis A.

    2013-01-01

    Papanicolaou (Pap) smears have revolutionized the management of patients with cervical cancers by permitting the detection of early, surgically curable tumors and their precursors. In recent years, the traditional Pap smear has been replaced by a liquid-based method, which allows not only cytologic evaluation but also collection of DNA for detection of human papillomavirus, the causative agent of cervical cancer. We reasoned that this routinely collected DNA could be exploited to detect somatic mutations present in rare tumor cells that accumulate in the cervix once shed from endometrial or ovarian cancers. A panel of genes that are commonly mutated in endometrial and ovarian cancers was assembled with new whole-exome sequencing data from 22 endometrial cancers and previously published data on other tumor types. We used this panel to search for mutations in 24 endometrial and 22 ovarian cancers and identified mutations in all 46 samples. With a sensitive massively parallel sequencing method, we were able to identify the same mutations in the DNA from liquid Pap smear specimens in 100% of endometrial cancers (24 of 24) and in 41% of ovarian cancers (9 of 22). Prompted by these findings, we developed a sequence-based method to query mutations in 12 genes in a single liquid Pap smear specimen without previous knowledge of the tumor’s genotype. When applied to 14 samples selected from the positive cases described above, the expected tumor-specific mutations were identified. These results demonstrate that DNA from most endometrial and a fraction of ovarian cancers can be detected in a standard liquid-based Pap smear specimen obtained during routine pelvic examination. Although improvements need to be made before applying this test in a routine clinical manner, it represents a promising step toward a broadly applicable screening methodology for the early detection of gynecologic malignancies. PMID:23303603

  12. Serological testing in malaria*

    PubMed Central

    1974-01-01

    The main purpose of this paper is to evaluate, in a critical manner, various serological tests with general emphasis on their value in the epidemiological assessment of malaria. Several tests have been employed in the past. However, the present memorandum will deal only with the methods that have been widely used recently—i.e., indirect immunofluorescence (IFA), passive haemagglutination (IHA), and gel-diffusion. The three immunoglobulins most commonly involved in these tests are IgG, IgM, and—to a lesser extent—IgA. PMID:4218506

  13. Is it time to sound an alarm about false-positive cell-free DNA testing for fetal aneuploidy?

    PubMed

    Mennuti, Michael T; Cherry, Athena M; Morrissette, Jennifer J D; Dugoff, Lorraine

    2013-11-01

    Testing cell-free DNA (cfDNA) in maternal blood samples has been shown to have very high sensitivity for the detection of fetal aneuploidy with very low false-positive results in high-risk patients who undergo invasive prenatal diagnosis. Recent observation in clinical practice of several cases of positive cfDNA tests for trisomy 18 and trisomy 13, which were not confirmed by cytogenetic testing of the pregnancy, may reflect a limitation of the positive predictive value of this quantitative testing, particularly when it is used to detect rare aneuploidies. Analysis of a larger number of false-positive cases is needed to evaluate whether these observations reflect the positive predictive value that should be expected. Infrequently, mechanisms (such as low percentage mosaicism or confined placental mosaicism) might also lead to positive cfDNA testing that is not concordant with standard prenatal cytogenetic diagnosis. The need to explore these and other possible causes of false-positive cfDNA testing is exemplified by 2 of these cases. Additional evaluation of cfDNA testing in clinical practice and a mechanism for the systematic reporting of false-positive and false-negative cases will be important before this test is offered widely to the general population of low-risk obstetric patients. In the meantime, incorporating information about the positive predictive value in pretest counseling and in clinical laboratory reports is recommended. These experiences reinforce the importance of offering invasive testing to confirm cfDNA results before parental decision-making. PMID:23529082

  14. DNA methylation profiling for a confirmatory test for blood, saliva, semen, vaginal fluid and menstrual blood.

    PubMed

    Lee, Hwan Young; Jung, Sang-Eun; Lee, Eun Hee; Yang, Woo Ick; Shin, Kyoung-Jin

    2016-09-01

    The ability to predict the type of tissues or cells from molecular profiles of crime scene samples has important practical implications in forensics. A previously reported multiplex assay using DNA methylation markers could only discriminate between 4 types of body fluids: blood, saliva, semen, and the body fluid which originates from female reproductive organ. In the present study, we selected 15 menstrual blood-specific CpG marker candidates based on analysis of 12 genome-wide DNA methylation profiles of vaginal fluid and menstrual blood. The menstrual blood-specificity of the candidate markers was confirmed by comparison with HumanMethylation450 BeadChip array data obtained for 58 samples including 12 blood, 12 saliva, 12 semen, 3 vaginal fluid, and 19 skin epidermis samples. Among 15CpG marker candidates, 3 were located in the promoter region of the SLC26A10 gene, and 2 of them (cg09696411 and cg18069290) showed high menstrual blood specificity. DNA methylation at the 2CpG markers was further tested by targeted bisulfite sequencing of 461 additional samples including 49 blood, 52 saliva, 34 semen, 125 vaginal fluid, and 201 menstrual blood. Because the 2 markers showed menstrual blood-specific methylation patterns, we modified our previous multiplex methylation SNaPshot reaction to include these 2 markers. In addition, a blood marker cg01543184 with cross reactivity to semen was replaced with cg08792630, and a semen-specific unmethylation marker cg17621389 was removed. The resultant multiplex methylation SNaPshot allowed positive identification of blood, saliva, semen, vaginal fluid and menstrual blood using the 9CpG markers which show a methylation signal only in the target body fluids. Because of the complexity in cell composition, menstrual bloods produced DNA methylation profiles that vary with menstrual cycle and sample collection methods, which are expected to provide more insight into forensic menstrual blood test. Moreover, because the developed

  15. Enhanced GSH synthesis by Bisphenol A exposure promoted DNA methylation process in the testes of adult rare minnow Gobiocypris rarus.

    PubMed

    Yuan, Cong; Zhang, Yingying; Liu, Yan; Zhang, Ting; Wang, Zaizhao

    2016-09-01

    DNA methylation is a commonly studied epigenetic modification. The mechanism of BPA on DNA methylation is poorly understood. The present study aims to explore whether GSH synthesis affects DNA methylation in the testes of adult male rare minnow Gobiocypris rarus in response to Bisphenol A (BPA). Male G. rarus was exposed to 1, 15 and 225μgL(-1) BPA for 7 days. The levels of global DNA methylation, hydrogen peroxide (H2O2) and glutathione (GSH) in the testes were analyzed. Meanwhile, the levels of enzymes involved in DNA methylation and de novo GSH synthesis, and the substrate contents for GSH production were measured. Furthermore, gene expression profiles of the corresponding genes of all studied enzymes were analyzed. Results indicated that BPA at 15 and 225μgL(-1) caused hypermethylation of global DNA in the testes. The 15μgL(-1) BPA resulted in significant decrease of ten-eleven translocation proteins (TETs) while 225μgL(-1) BPA caused significant increase of DNA methyltransferase proteins (DNMTs). Moreover, 225μgL(-1) BPA caused significant increase of H2O2 and GSH levels, and the de novo GSH synthesis was enhanced. These results indicated that the significant decrease of the level of TETs may be sufficient to cause the DNA hypermethylation by 15μgL(-1) BPA. However, the significantly increased of DNMTs contributed to the significant increase of DNA methylation levels by 225μgL(-1) BPA. Moreover, the elevated de novo GSH synthesis may promote the DNA methylation process. PMID:27474941

  16. Sturgeon nucleo-cytoplasmic large DNA virus phylogeny and PCR tests.

    PubMed

    Clouthier, Sharon C; VanWalleghem, Elissa; Anderson, Eric D

    2015-12-01

    Sturgeon epitheliotropic nucleo-cytoplasmic large DNA viruses (NCLDVs) can cause a lethal disease of the integumentary system. These viruses have not been assigned to any currently recognized family or genus. In this study, phylogenetic analyses using the major capsid protein (MCP) showed that the sturgeon NCLDVs formed a cohesive taxonomic group, could be identified to the species or possibly sub-species level and formed a distinct evolutionary lineage within the Megavirales. The genetic relatedness of the sturgeon virus MCP allowed design of 3 PCR diagnostic tests with analytical specificity (ASp) inclusive of this group of viruses. The conventional PCR test, C1, had broader ASp than the 2 quantitative PCR tests, Q1 and Q2, and was inclusive of the sturgeon viruses as well as some viruses belonging to the families Mimi-, Phycodna-, or Iridoviridae. Q2 had broader specificity than Q1 but both tests recognized the sturgeon NCLDVs and did not cross-react with co-localizing sturgeon herpesviruses. Analytical test performance characteristics evaluated for Q1 and Q2 revealed sensitive assays with observed 50% limits of detection between 3 and 6.25 plasmid copies and high intra- and inter-assay repeatability. Q1 was used to test for sturgeon viruses in endangered populations of lake sturgeon Acipenser fulvescens within the Winnipeg River or Nelson River drainage systems of Manitoba, Canada. Test results indicated that namao virus is endemic in the Nelson River water basin. These tests meet the analytical requirements for diagnostic testing in Canada and are useful tools for disease management in sturgeon conservation stocking programs in North America. PMID:26648102

  17. Potential of Environmental DNA to Evaluate Northern Pike (Esox lucius) Eradication Efforts: An Experimental Test and Case Study.

    PubMed

    Dunker, Kristine J; Sepulveda, Adam J; Massengill, Robert L; Olsen, Jeffrey B; Russ, Ora L; Wenburg, John K; Antonovich, Anton

    2016-01-01

    Determining the success of invasive species eradication efforts is challenging because populations at very low abundance are difficult to detect. Environmental DNA (eDNA) sampling has recently emerged as a powerful tool for detecting rare aquatic animals; however, detectable fragments of DNA can persist over time despite absence of the targeted taxa and can therefore complicate eDNA sampling after an eradication event. This complication is a large concern for fish eradication efforts in lakes since killed fish can sink to the bottom and slowly decay. DNA released from these carcasses may remain detectable for long periods. Here, we evaluated the efficacy of eDNA sampling to detect invasive Northern pike (Esox lucius) following piscicide eradication efforts in southcentral Alaskan lakes. We used field observations and experiments to test the sensitivity of our Northern pike eDNA assay and to evaluate the persistence of detectable DNA emitted from Northern pike carcasses. We then used eDNA sampling and traditional sampling (i.e., gillnets) to test for presence of Northern pike in four lakes subjected to a piscicide-treatment designed to eradicate this species. We found that our assay could detect an abundant, free-roaming population of Northern pike and could also detect low-densities of Northern pike held in cages. For these caged Northern pike, probability of detection decreased with distance from the cage. We then stocked three lakes with Northern pike carcasses and collected eDNA samples 7, 35 and 70 days post-stocking. We detected DNA at 7 and 35 days, but not at 70 days. Finally, we collected eDNA samples ~ 230 days after four lakes were subjected to piscicide-treatments and detected Northern pike DNA in 3 of 179 samples, with a single detection at each of three lakes, though we did not catch any Northern pike in gillnets. Taken together, we found that eDNA can help to inform eradication efforts if used in conjunction with multiple lines of inquiry and sampling

  18. Effect of vanillin on toxicant-induced lethality in the Drosophila melanogaster DNA repair test.

    PubMed

    Furlanetto, Magda Patrícia; Sinigaglia, Marialva; Amaral, Viviane Souza do; Dihl, Rafael Rodrigues; de Andrade, Heloísa Helena Rodrigues

    2007-01-01

    Vanillin (VA) modulates the genotoxicity of chemical and physical agents in a complex manner. Previous studies indicate that VA inhibits the mutagenicity but increases the mitotic homologous recombination caused by at least some genotoxic agents. In the present study, we have evaluated the effects of VA on the repair of lethal damage produced by three genotoxins, N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), and mitomycin C (MMC), using the DNA repair test (DRT) in Drosophila melanogaster. VA, 0.25% and 0.5% (w/v), increased the toxicity of MMC and EMS in repair-deficient flies, as measured by a decrease in the proportion of male to female progeny in the DRT; sex ratios decreased from 18-48% for MMC and 21-97% for EMS. These effects may be caused by the inhibition of nonhomologous DNA end joining caused by VA. In contrast to the results with MMC and EMS, VA protected against the lethality of ENU in repair-defective flies, as measured by a 43-207% increase in the survival of male flies in the DRT. It was inferred that the protective effect was due to VA modulating stages prior to the induction of ENU lesions in DNA, including modulating the antioxidant properties of VA and/or to its interference with the metabolic activation and/or detoxification of specific genotoxins. The results from this study indicate that the characterization of VA as a promising agent for preventing damage to genes and chromosomes should be tempered by observations that VA can increase the toxicity of chemical agents. PMID:17177210

  19. Integrating stakeholder perspectives into the translation of cell-free fetal DNA testing for aneuploidy

    PubMed Central

    2012-01-01

    Background The translation of novel genomic technologies from bench to bedside enjoins the comprehensive consideration of the perspectives of all stakeholders who stand to influence, or be influenced by, the translational course. Non-invasive prenatal aneuploidy testing that utilizes cell-free fetal DNA (cffDNA) circulating in maternal blood is one example of an innovative technology that promises significant benefits for its intended end users; however, it is currently uncertain whether it will achieve widespread clinical implementation. We conducted qualitative interviews with 18 diverse stakeholders in this domain, including prospective users of the technology and healthcare personnel, researchers and developers, and experts in social, legal, and regulatory aspects of genetic technology, and a pilot survey of 62 obstetric healthcare providers. Analysis of interview and survey data was combined with a review of the proceedings of a full-day, multidisciplinary conference on the topic and published scientific and ethics literature surrounding this and other relevant technologies. Discussion We constructed potential pathways for technological implementation, identified broad stakeholder classes party to these translational processes, and performed a preliminary assessment of the viewpoints and interrelations among these diverse stakeholders. Some of the stakeholders whose priorities are critical to understand and integrate into translation include pregnant women and their families; healthcare providers; scientists, their institutions or companies, and the funding agencies that support them; regulatory and judicial bodies; third-party payers; professional societies; educational systems; disability rights communities; and other representatives from civil society. Stakeholder interviews, survey findings, and conference proceedings add complexity to these envisioned pathways and also demonstrate a paramount need to incorporate an iterative stakeholder analysis early and

  20. Evaluation of patient education materials: the example of circulating cell free DNA testing for aneuploidy.

    PubMed

    Kloza, Edward M; Haddow, Paula K; Halliday, Jacquelyn V; O'Brien, Barbara M; Lambert-Messerlian, Geralyn M; Palomaki, Glenn E

    2015-04-01

    Informed consent is the process by which the treating health care provider discloses appropriate information to a competent patient so that the patient may make a voluntary choice to accept or refuse treatment. When the analysis of circulating cell free DNA (ccfDNA) became commercially available in 2011 through the Prenatal Diagnostic Laboratory at Women & Infants Hospital of Providence, Rhode Island to "high-risk" women, it provided an opportunity to examine how commercial laboratories informed potential consumers. We identified, via an internet search, four laboratories offering such testing in the United States and one in Europe. We evaluated patient educational materials (PEMs) from each using the Flesch Reading Ease method and a modified version of the Suitability Assessment of Materials (SAM) criteria. Pamphlets were also reviewed for their inclusion of content recommendations from the International Society for Prenatal Diagnosis, the National Society of Genetic Counselors, the American College of Obstetricians and Gynecologists jointly with the Society of Maternal Fetal Medicine, and the American College of Genetics and Genomics. Reading levels were typically high (10th-12th grade). None of the pamphlets met all SAM criteria evaluated nor did any pamphlet include all recommended content items. To comply with readability and content recommendations more closely, Women & Infants Hospital created a new pamphlet to which it applied the same criteria, and also subjected it to focus group assessment. These types of analyses can serve as a model for future evaluations of similar patient educational materials. PMID:25204423

  1. [Study of DNA extraction methods for testing for genetically modified organisms in soyproducts].

    PubMed

    Moriuchi, Rie; Monma, Kimio; Kamata, Kunihiro; Ibe, Akihiro

    2008-01-01

    In order to evaluate three different methods for DNA extraction (CTAB, DNeasy Plant Mini Kit and Wizard DNA Clean-up Resin system), the yields of DNA extracted from soyproducts and the copy numbers of lectin genes amplified by quantitative PCR were compared. Fermented foods, such as miso and nattou, gave poor yields of DNA and low copy numbers with any method. However atsu-age and kinugoshi-tofu gave high-quality results with all methods. Kinako gave a high yield of DNA, but poor amplification. Boiled soybeans and soymilk showed in poor amplification. It is important to choose the appropriate DNA extraction method for each product. PMID:19029786

  2. Does behavior reflect phylogeny in swiftlets (Aves: Apodidae)? A test using cytochrome b mitochondrial DNA sequences.

    PubMed Central

    Lee, P L; Clayton, D H; Griffiths, R; Page, R D

    1996-01-01

    Swiftlets are small insectivorous birds, many of which nest in caves and are known to echolocate. Due to a lack of distinguishing morphological characters, the taxonomy of swiftlets is primarily based on the presence or absence of echolocating ability, together with nest characters. To test the reliability of these behavioral characters, we constructed an independent phylogeny using cytochrome b mitochondrial DNA sequences from swiftlets and their relatives. This phylogeny is broadly consistent with the higher classification of swifts but does not support the monophyly of swiftlets. Echolocating swiftlets (Aerodramus) and the nonecholocating "giant swiftlet" (Hydrochous gigas) group together, but the remaining nonecholocating swiftlets belonging to Collocalia are not sister taxa to these swiftlets. While echolocation may be a synapomorphy of Aerodramus (perhaps secondarily lost in Hydrochous), no character of Aerodramus nests showed a statistically significant fit to the molecular phylogeny, indicating that nest characters are not phylogenetically reliable in this group. Images Fig. 1 PMID:8692950

  3. WHAT DNA CAN AND CANNOT SAY: PERSPECTIVES OF IMMIGRANT FAMILIES ABOUT THE USE OF GENETIC TESTING IN IMMIGRATION

    PubMed Central

    Barata, Llilda P.; Starks, Helene; Kelley, Maureen; Kuszler, Patricia; Burke, Wylie

    2016-01-01

    Genetic technologies are being implemented in areas that extend beyond the field of medicine to address social and legal problems. An emerging example is the implementation of genetic testing in the family petitioning process in immigration policy. This use of genetic testing offers the potential benefits of reducing immigration fraud and making the process more efficient and accessible for immigrants, especially those without documentation. However, little is known about the positive or negative impacts of such testing on immigrant families and their communities. This study collected empirical data through family interviews to understand the experiences and attitudes of individuals who have taken a DNA test to prove a family relationship for immigration purposes. Based on study results, we present a set of recommendations to improve the processes with which DNA testing is applied to immigration cases. We argue that DNA testing might serve as a useful tool for families who lack documentary evidence of a family relationship. However, testing might also reveal sensitive information, such as misattributed parentage, that can damage relationships and cause serious harm to beneficiaries, especially children. Petitioners should be provided with adequate information to form an understanding of the DNA test and its implementation as well as the positive and negative consequences from using it, in order to carefully assess whether DNA testing will help their case. We recommend that additional protections be put in place to safeguard children from the potential impacts of misattributed parentage or disclosure of hidden social adoptions. This research provides empirical evidence to inform policy related to the use of genetic testing in immigration. PMID:26855553

  4. An extremely sensitive species-specific ARMS PCR test for the presence of tiger bone DNA.

    PubMed

    Wetton, Jon H; Tsang, Carol S F; Roney, Chris A; Spriggs, Adrian C

    2002-04-18

    The survival of the tiger (Panthera tigris) is seriously threatened by poaching to provide raw materials for traditional Chinese medicines (TCMs). Most highly prized are the tiger's bones, which are used in combination with other animal and plant derivatives in pills and plasters for the treatment of rheumatism and other ailments. Hundreds of patent remedies have been produced which claim to contain tiger bone, but proof of its presence is needed if legislation prohibiting the trade in endangered species is to be enforced.A highly sensitive tiger-specific real-time PCR assay has been developed to address this problem. Using primers specific to the tiger mitochondrial cytochrome b gene, successful amplification has been reliably achieved from blood, hair and bone as well as from a range of TCMs spiked with 0.5% tiger bone. Although capable of detecting fewer than 10 substrate molecules, the seven varieties of TCM pills and plasters tested showed no detectable trace of tiger DNA before spiking. Furthermore, sequencing several "tiger bone" fragments seized from TCM shops has shown that they actually originated from cattle and pigs. The potential effects of traditional bone preparation methods, evidence that much lower concentrations are used than alleged on TCM packaging, and substitution of bones from other species all suggest a low likelihood of detecting tiger DNA in patent medicines. Despite this, the basic methods have been thoroughly proven and can be readily applied to derivatives from other Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) protected species, providing a rapid and highly sensitive forensic test for species of origin. Potential applications to the monitoring of wild populations are demonstrated by the successful identification of shed hairs and faecal samples. PMID:12084490

  5. Rapid diagnostic tests as a source of DNA for Plasmodium species-specific real-time PCR

    PubMed Central

    2011-01-01

    Background This study describes the use of malaria rapid diagnostic tests (RDTs) as a source of DNA for Plasmodium species-specific real-time PCR. Methods First, the best method to recover DNA from RDTs was investigated and then the applicability of this DNA extraction method was assessed on 12 different RDT brands. Finally, two RDT brands (OptiMAL Rapid Malaria Test and SDFK60 malaria Ag Plasmodium falciparum/Pan test) were comprehensively evaluated on a panel of clinical samples submitted for routine malaria diagnosis at ITM. DNA amplification was done with the 18S rRNA real-time PCR targeting the four Plasmodium species. Results of PCR on RDT were compared to those obtained by PCR on whole blood samples. Results Best results were obtained by isolating DNA from the proximal part of the nitrocellulose component of the RDT strip with a simple DNA elution method. The PCR on RDT showed a detection limit of 0.02 asexual parasites/μl, which was identical to the same PCR on whole blood. For all 12 RDT brands tested, DNA was detected except for one brand when a low parasite density sample was applied. In RDTs with a plastic seal covering the nitrocellulose strip, DNA extraction was hampered. PCR analysis on clinical RDT samples demonstrated correct identification for single species infections for all RDT samples with asexual parasites of P. falciparum (n = 60), Plasmodium vivax (n = 10), Plasmodium ovale (n = 10) and Plasmodium malariae (n = 10). Samples with only gametocytes were detected in all OptiMAL and in 10 of the 11 SDFK60 tests. None of the negative samples (n = 20) gave a signal by PCR on RDT. With PCR on RDT, higher Ct-values were observed than with PCR on whole blood, with a mean difference of 2.68 for OptiMAL and 3.53 for SDFK60. Mixed infections were correctly identified with PCR on RDT in 4/5 OptiMAL tests and 2/5 SDFK60 tests. Conclusions RDTs are a reliable source of DNA for Plasmodium real-time PCR. This study demonstrates the best method of RDT

  6. A Simple Sperm DNA Toroid Integrity Test and Risk of Miscarriage

    PubMed Central

    Chan, Philip J.; Orzylowska, Eliza M.; Corselli, Johannah U.; Jacobson, John D.; Wei, Albert K.

    2015-01-01

    Current methods of analyzing sperm chromatin competency overlook the inner sperm compartment which is inaccessible to probes and reagents. By breaking the molecular protamine disulfide bridges, the DNA toroids are exposed to integrity analysis. The aim was to develop a simple nuclear toroid test and determine its association with fertilization, pregnancy, and miscarriage. The approach involved treating washed sperm remaining after ICSI procedures (N = 35 cases) with acidified Triton X-100 and dithiothreitol (DTT) before Diff-Quik staining. Percentages of sperm with normal chromatin indicated by light-colored nuclei were assessed. The toroid integrity test showed more sperm with normal chromatin in the pregnant group (73.6 ± 1.7%, mean ± SEM) when compared with the miscarriage (51.2 ± 6.6%) or nonpregnant groups (60.9 ± 4.8%). Furthermore, the toroid results were correlated with ICSI fertilization (R = 0.32, P = 0.04) and pregnancy outcome (pregnant cases 73.6 ± 1.7% versus nonpregnant 58.0 ± 3.9%, P = 0.001). ROC calculated cut-off was >70.0% for normal toroid integrity (sensitivity 0.98, specificity 0.33, and diagnostic accuracy 78.3%). An association between normal sperm toroid integrity and miscarriage was evident when the staining procedure included acidified detergent DTT pretreatment. PMID:25649376

  7. Testing methods for sensor test and evaluation using the DNA nuclear infrared clutter simulator. Technical report, 17 August 1993-16 December 1994

    SciTech Connect

    Pritchett, D.G.; Moore, D.K.

    1997-02-01

    The purpose of this interim report is to document methods that have been developed for the test and evaluation of interceptor and surveillance sensor systems using the Defense Nuclear Agency (DNA) Nuclear Infrared Clutter Simulator (NICS) system. The methods presented here have been developed on DNA contract DNA 001-93-C-0145, Applications of the Nuclear Infrared Clutter Simulator (ANICS) program. The methods have evolved as the result of experience gained over the course of multiple NICS sensor focal plane array (FPA) test campaigns and the subsequent data analysis necessary to evaluate FPA and modeled sensor performance. Methods documented here are applicable to sensor testing with either the original NICS static display optical configuration or the new dynamic display optical configuration. The original bench is referred to as the NICS Static Display Bench, while the newest optical bench is referred to as the Dynamic Display Bench. The latter configuration utilizes DNA Nuclear Optical Dynamic Display System (NODDS) technology, which provides the sensor under test with dynamic infrared optical scenarios.

  8. Origin-Dependent Inverted-Repeat Amplification: Tests of a Model for Inverted DNA Amplification

    PubMed Central

    Brewer, Bonita J.; Payen, Celia; Di Rienzi, Sara C.; Higgins, Megan M.; Ong, Giang; Dunham, Maitreya J.; Raghuraman, M. K.

    2015-01-01

    DNA replication errors are a major driver of evolution—from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model—Origin-Dependent Inverted-Repeat Amplification (ODIRA)—proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error—the ligation of leading and lagging nascent strands to create “closed” forks—can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent—a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of

  9. Detection of the thermostable direct hemolysin gene and related DNA sequences in Vibrio parahaemolyticus and other vibrio species by the DNA colony hybridization test.

    PubMed Central

    Nishibuchi, M; Ishibashi, M; Takeda, Y; Kaper, J B

    1985-01-01

    A specific gene probe for the Vibrio parahaemolyticus thermostable direct hemolysin gene was constructed and used to examine the presence or absence of the thermostable direct hemolysin gene or related DNA sequences in V. parahaemolyticus and other vibrios by the DNA colony hybridization method. The gene probe consisted of a 406-base-pair, completely internal fragment covering 71% of the structural gene with PstI linkers added to the ends. Six copies of this 415-base-pair PstI fragment were cloned into plasmid pBR322, which yielded large amounts of the probe DNA. One hundred forty-one V. parahaemolyticus strains were tested with the gene probe, and the results were compared with those of phenotypic assays for the thermostable direct hemolysin. All Kanagawa phenomenon-positive strains were gene positive. However, 86% of the strains that exhibited weak Kanagawa phenomenon and 16% of Kanagawa phenomenon-negative strains also reacted with the gene probe. Immunological methods for the detection of the thermostable direct hemolysin (modified Elek test, enzyme-linked immunosorbent assay) showed better correlation with gene probe results. All gene-positive strains produced hemolysin detectable in the enzyme-linked immunosorbent assay, although occasional strains showed weak reaction. The modified Elek test was slightly less sensitive than the enzyme-linked immunosorbent assay. All gene-negative strains were also negative in these immunological assays. One hundred twenty-one strains of Vibrio spp. other than V. parahaemolyticus were tested with the gene probe; only Vibrio hollisae strains reacted with the probe under stringent conditions. PMID:4030087

  10. Single molecule measurements of DNA helicase activity with magnetic tweezers and t-test based step-finding analysis.

    PubMed

    Seol, Yeonee; Strub, Marie-Paule; Neuman, Keir C

    2016-08-01

    Magnetic tweezers is a versatile and easy to implement single-molecule technique that has become increasingly prevalent in the study of nucleic acid based molecular motors. Here, we provide a description of the magnetic tweezers instrument and guidelines for measuring and analyzing DNA helicase activity. Along with experimental methods, we describe a robust method of single-molecule trajectory analysis based on the Student's t-test that accommodates continuous transitions in addition to the discrete transitions assumed in most widely employed analysis routines. To illustrate the single-molecule unwinding assay and the analysis routine, we provide DNA unwinding measurements of Escherichia coli RecQ helicase under a variety of conditions (Na+, ATP, temperature, and DNA substrate geometry). These examples reveal that DNA unwinding measurements under various conditions can aid in elucidating the unwinding mechanism of DNA helicase but also emphasize that environmental effects on DNA helicase activity must be considered in relation to in vivo activity and mechanism. PMID:27131595

  11. Identification of irradiated wheat by germination test, DNA comet assay and electron spin resonance

    NASA Astrophysics Data System (ADS)

    Barros, Adilson C.; Freund, Maria Teresa L.; Villavicencio, Ana Lúcia C. H.; Delincée, Henry; Arthur, Valter

    2002-03-01

    In several countries, there has been an increase in the use of radiation for food processing thus improving the quality and sanitary conditions, inhibiting pathogenic microorganisms, delaying the natural aging process and so extending product lifetime. The need to develop analytical methods to detect these irradiated products is also increasing. The goal of this research was to identify wheat irradiated using different radiation doses. Seeds were irradiated with a gamma 60Co source (Gammacell 220 GC) in the Centro de Energia Nuclear na Agricultura and the Instituto de Pesquisas Energéticas e Nucleares. Dose rate used were 1.6 and 5.8kGy/h. Applied doses were 0.0, 0.10, 0.25, 0.50, 0.75, 1.0, and 2.0kGy. After irradiation, seeds were analysed over a 6 month period. Three different detection methods were employed to determine how irradiation had modified the samples. Screening methods consisted of a germination test measuring the inhibition of shooting and rooting and analysis of DNA fragmentation. The method of electron spin resonance spectroscopy allowed a better dosimetric evaluation. These techniques make the identification of irradiated wheat with different doses possible.

  12. DNA Yield From Tissue Samples in Surgical Pathology and Minimum Tissue Requirements for Molecular Testing.

    PubMed

    Austin, Melissa C; Smith, Christina; Pritchard, Colin C; Tait, Jonathan F

    2016-02-01

    Context .- Complex molecular assays are increasingly used to direct therapy and provide diagnostic and prognostic information but can require relatively large amounts of DNA. Objectives .- To provide data to pathologists to help them assess tissue adequacy and provide prospective guidance on the amount of tissue that should be procured. Design .- We used slide-based measurements to establish a relationship between processed tissue volume and DNA yield by A260 from 366 formalin-fixed, paraffin-embedded tissue samples submitted for the 3 most common molecular assays performed in our laboratory (EGFR, KRAS, and BRAF). We determined the average DNA yield per unit of tissue volume, and we used the distribution of DNA yields to calculate the minimum volume of tissue that should yield sufficient DNA 99% of the time. Results .- All samples with a volume greater than 8 mm(3) yielded at least 1 μg of DNA, and more than 80% of samples producing less than 1 μg were extracted from less than 4 mm(3) of tissue. Nine square millimeters of tissue should produce more than 1 μg of DNA 99% of the time. Conclusions .- We conclude that 2 tissue cores, each 1 cm long and obtained with an 18-gauge needle, will almost always provide enough DNA for complex multigene assays, and our methodology may be readily extrapolated to individual institutional practice. PMID:26098132

  13. The genotoxicities of N-nitrosamines in Drosophila melanogaster in vivo: the correlation of mutagenicity in the wing spot test with the DNA damages detected by the DNA-repair test.

    PubMed

    Negishi, T; Shiotani, T; Fujikawa, K; Hayatsu, H

    1991-04-01

    The genotoxicities of a series of N-nitrosamines were assayed in the wing spot test and a new short-term test of Drosophila melanogaster. In the spot test, larval flies trans-heterozygous for the somatic cell markers mwh and flr3 were fed the test reagents and the wing hairs in adults were inspected for clones expressing the phenotypes of the markers. In the other test, larval stock consisting of meiotic recombination-deficient (Rec-) double mutant mei-9a and mei-41D5 males and repair-proficient Rec+ females were grown on feed containing the reagents and the DNA damages were detected with the preferential killing of the Rec- larvae as an endpoint. The carcinogenic nitrosamines tested, N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodi-n-butylamine (NDBA), N-nitrosomorpholine (NMOR), N-nitro-sopiperidine (NPIP) and N-nitrosopyrrolidine (NPYR), all showed clearly positive activities in both tests. The activities in the wing spot test were ranked in a sequence of NDMA much greater than NMOR greater than NPIP greater than NDEA greater than NPYR greater than NDBA. A similar ranking was obtained in the repair assay. The genotoxicity of N-nitrosodiphenylamine (NDPhA), carcinogenicity studies of which are inconclusive, was marginal in the spot test. The non-carcinogenic N-nitrosoproline (NPRO) and the non-mutagenic N-nitrosothioproline (NTPRO) were negative in the spot test. NDPhA and NPRO were negative in the repair test as well. The DNA-repair test is thus a convenient technique for estimating the mutagenicity of compounds because of its simplicity compared with the wing spot test. These Drosophila tests may be useful in predicting carcinogenic potentials of compounds. PMID:1901957

  14. Development of genome-specific 5S rDNA markers in Brassica and related species for hybrid testing.

    PubMed

    La Mura, Maurizio; Norris, Carol; Sporle, Sue; Jayaweera, Dasuni; Greenland, Andy; Lee, David

    2010-08-01

    The Brassicaceae are targets for DNA manipulation to modify oil content and composition. However, any strategy for creating novel products using genetic modification or traditional breeding must take into account the potential for hybridization with other Brassica species, many of which are important sources of edible oils. In this study we have tested Brassica carinata, a possible target for oil modification, to establish whether it can cross with other Brassica species and related genera, and we have developed molecular DNA assays to confirm hybridization. PMID:20725152

  15. Detection of excretory Entamoeba histolytica DNA in the urine, and detection of E. histolytica DNA and lectin antigen in the liver abscess pus for the diagnosis of amoebic liver abscess

    PubMed Central

    Parija, Subhash C; Khairnar, Krishna

    2007-01-01

    Background Amoebic liver abscess (ALA) and pyogenic liver abscesses (PLA) appear identical by ultrasound and other imaging techniques. Collection of blood or liver abscess pus for diagnosis of liver abscesses is an invasive procedure, and the procedure requires technical expertise and disposable syringes. Collection of urine is a noninvasive procedure. Therefore, there has been much interest shown towards the use of urine as an alternative clinical specimen for the diagnosis of some parasitic infections. Here, we report for the first time the detection of E. histolytica DNA excreted in the urine for diagnosis of the cases of ALA. Results E. histolytica DNA was detected in liver abscess pus specimen of 80.4% of ALA patients by a nested multiplex polymerase chain reaction (PCR) targeting 16S-like r RNA gene. The nested PCR detected E. histolytica DNA in all 37 (100%) liver abscess pus specimens collected prior to metronidazole treatment, but were detected in only 53 of 75 (70.6%) pus specimens collected after therapy with metronidazole. Similarly, the PCR detected E. histolytica DNA in 21 of 53 (39.6%) urine specimens of ALA patients. The test detected E. histolytica DNA in only 4 of 23 (17.4%) urine specimens collected prior to metronidazole treatment, but were detected in 17 of 30 (56.7%) urine specimens collected after treatment with metronidazole. The enzyme-linked immunosorbent assay (ELISA) for the detection of lectin E. histolytica antigen in the liver abscess pus showed a sensitivity of 50% and the indirect haemagglutination (IHA) test for detection of amoebic antibodies in the serum showed a sensitivity of 76.8% for the diagnosis of the ALA. Conclusion The present study for the first time shows that the kidney barrier in ALA patients is permeable to E. histolytica DNA molecule resulting in excretion of E. histolytica DNA in urine which can be detected by PCR. The study also shows that the PCR for detection of E. histolytica DNA in urine of patients with ALA

  16. DNA intercalative potential of marketed drugs testing positive in in vitro cytogenetics assays.

    PubMed

    Snyder, Ronald D; Ewing, Douglas; Hendry, Lawrence B

    2006-10-10

    We have previously noted that the Physicians' Desk Reference (PDR) contains over 80 instances in which a drug elicited a positive genotoxic response in one or more in vitro assays, despite having no obvious structural features predictive of covalent drug/DNA interactive potential or known mechanistic basis. Furthermore, in most cases, these drugs were "missed" by computational genotoxicity-predicting models such as DEREK, MCASE and TOPKAT. We have previously reported the application of a V79 cell-based model and a 3D DNA docking model for predicting non-covalent chemical/DNA interactions. Those studies suggested that molecules that are very widely structurally diverse may be capable of intercalating into DNA. To determine whether such non-covalent drug/DNA interactions might be involved in unexpected drug genotoxicity, we evaluated, using both models where possible, 56 marketed pharmaceuticals, 40 of which were reported as being clastogenic in in vitro cytogenetics assays (chromosome aberrations/mouse lymphoma assay). As seen before, the two approaches showed good concordance (62%) and 26 of the 40 (65%) drugs exhibiting in vitro clastogenicity were predicted as intercalators by one or both methods. This finding provides support for the hypothesis that non-covalent DNA interaction may be a common mechanism of clastogenicity for many drugs having no obvious structural alerts for covalent DNA interaction. PMID:16857419

  17. Utility of Filter Paper for Preserving Insects, Bacteria, and Host Reservoir DNA for Molecular Testing

    PubMed Central

    Karimian, F; Sedaghat, MM; Oshaghi, MA; Mohtarami, F; Dehkordi, A Sanei; Koosha, M; Akbari, S; Hashemi-Aghdam, SS

    2011-01-01

    Background: Appropriate methodology for storage biological materials, extraction of DNA, and proper DNA preservation is vital for studies involving genetic analysis of insects, bacteria, and reservoir hosts as well as for molecular diagnostics of pathogens carried by vectors and reservoirs. Here we tried to evaluate the utility of a simple filter paper-based for storage of insects, bacteria, rodent, and human DNAs using PCR assays. Methods: Total body or haemolymph of individual mosquitoes, sand flies or cockroaches squashed or placed on the paper respectively. Extracted DNA of five different bacteria species as well as blood specimens of human and great gerbil Rhombomys opimus was pipetted directly onto filter paper. The papers were stored in room temperature up to 12 months during 2009 until 2011. At monthly intervals, PCR was conducted using a 1-mm disk from the DNA impregnated filter paper as target DNA. PCR amplification was performed against different target genes of the organisms including the ITS2-rDNA of mosquitoes, mtDNA-COI of the sand flies and cockroaches, 16SrRNA gene of the bacteria, and the mtDNA-CytB of the vertebrates. Results: Successful PCR amplification was observed for all of the specimens regardless of the loci, taxon, or time of storage. The PCR amplification were ranged from 462 to 1500 bp and worked well for the specified target gene/s. Time of storage did not affect the amplification up to one year. Conclusion: The filter paper method is a simple and economical way to store, to preserve, and to distribute DNA samples for PCR analysis. PMID:22808417

  18. Using the Cancer Risk Management Model to evaluate the health and economic impacts of cytology compared with human papillomavirus DNA testing for primary cervical cancer screening in Canada

    PubMed Central

    Popadiuk, C.; Gauvreau, C.L.; Bhavsar, M.; Nadeau, C.; Asakawa, K.; Flanagan, W.M.; Wolfson, M.C.; Coldman, A.J.; Memon, S.; Fitzgerald, N.; Lacombe, J.; Miller, A.B.

    2016-01-01

    Background In Canada, discussion about changing from cytology to human papillomavirus (hpv) dna testing for primary screening in cervical cancer is ongoing. However, the Canadian Task Force on Preventive Health Care has not yet made a recommendation, concluding that the evidence is insufficient. Methods We used the cervical cancer and hpv transmission models of the Cancer Risk Management Model to study the health and economic outcomes of primary cytology compared with hpv dna testing in 14 screening scenarios with varying screening modalities and intervals. Projected cervical cancer cases, deaths, colposcopies, screens, costs, and incremental cost-effectiveness were evaluated. We performed sensitivity analyses for hpv dna test costs. Results Compared with triennial cytology from age 25, 5-yearly hpv dna screening alone from age 30 resulted in equivalent incident cases and deaths, but 55% (82,000) fewer colposcopies and 43% (1,195,000) fewer screens. At hpv dna screening intervals of 3 years, whether alone or in an age-based sequence with cytology, screening costs are greater, but at intervals of more than 5 years, they are lower. Scenarios on the cost-effectiveness frontier were hpv dna testing alone every 10, 7.5, 5, or 3 years, and triennial cytology starting at age 21 or 25 when combined with hpv dna testing every 3 years. Conclusions Changing from cytology to hpv dna testing as the primary screening test for cervical cancer would be an acceptable strategy in Canada with respect to incidence, mortality, screening and diagnostic test volumes. PMID:26985148

  19. Comparison of six methods of extracting Mycobacterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR.

    PubMed

    Aldous, Wade K; Pounder, June I; Cloud, Joann L; Woods, Gail L

    2005-05-01

    Six methods of extracting Mycobacterium tuberculosis DNA from sputum for testing by quantitative PCR were compared: Tris-EDTA (TE) buffer, PrepMan Ultra, 2% sodium dodecyl sulfate (SDS)-10% Triton X with and without sonication, Infectio Diagnostics, Inc. (IDI) lysing tubes, and QIAGEN QIAamp DNA mini kit; all included a 15-min boiling step. Pooled digested and decontaminated sputum was spiked with M. tuberculosis ATCC 27294. Each extraction method was repeated eight times. Quantitative PCR was performed on the Smart Cycler and Rotor-Gene 3000 using primers targeting an 83-bp fragment of IS6110. An minor grove binding Eclipse probe with a fluorescent label was used for detection. An internal control was included to detect amplification inhibition. The limit of detection of M. tuberculosis DNA was 0.5 fg with both instruments. Calculated DNA concentrations (picograms) extracted using IDI, PrepMan, QIAGEN, and TE were 42.8, 30.4, 28.2, and 7.4, respectively, when run on the Smart Cycler, and 51.7, 20.1, 14.9, and 8.6, respectively, when run on Rotor-Gene. All extractions using SDS/Triton X with or without sonication were inhibited. Of the extraction methods evaluated, IDI lysis tubes provided the greatest yield of mycobacterial DNA, and the procedure can be completed in less than 1 h versus 2.5-3 h for the QIAGEN extraction. PMID:15872286

  20. Evolution of genome size: a phylogenetic test of the DNA loss hypothesis.

    PubMed

    Pie, M R

    2007-03-01

    It has been recently suggested that the C-value paradox, the lack of an obvious association between organismal complexity and genome size, can result simply from biases in insertion and deletion rates--the DNA loss hypothesis. This hypothesis has been heavily criticized, particularly because its evidence, a negative relationship between genome size and DNA loss rate, is based on a highly selective use of the available data. In this study it is show that even the even the most favorable interpretation of the data favoring the DNA loss hypothesis is largely an artifact of phylogenetic nonindependence, supporting the assertion made by other authors that the mechanisms underlying genome size evolution might be more complex than envisioned by the DNA loss hypothesis. PMID:17486764

  1. Can ALS-Associated C9orf72 Repeat Expansions Be Diagnosed on a Blood DNA Test Alone?

    PubMed Central

    Pamphlett, Roger; Cheong, Pak Leng; Trent, Ronald J.; Yu, Bing

    2013-01-01

    Gene mutations that preferentially affect the CNS have been implicated in a number of neurological disorders. This leads to the possibility that a disease-causing mutation present only in CNS tissues could be missed if it were tested in a blood DNA sample only. The commonest mutation in amyotrophic lateral sclerosis (ALS) is an expansion of the hexanucleotide repeats of C9orf72. To find out if CNS-specific mutations of this gene could cause some cases of ALS we looked for differences in the size of C9orf72 repeats between DNA from the CNS and from white blood cells (WBCs) of 38 sporadic ALS patients, using a repeat-primed PCR screening test. We also looked for differences in C9orf72 repeats in WBC DNA from 6 ALS-discordant and 1 ALS-concordant monozygotic twins. Abnormally expanded C9orf72 repeats were found in 13% of the ALS CNS samples, as well as in their paired WBC DNA. The 87% of ALS CNS samples with normal-sized C9orf72 repeats had the same number of repeats in paired WBC samples. All ALS-discordant twins had the same normal numbers of WBC C9orf72 repeats. Although previous work suggests some tissue mosaicism in C9orf72 repeat size is probably present, this study indicates that this is not likely to be of sufficient magnitude to result in a normal C9orf72 repeat length in blood but an abnormally expanded repeat length in the CNS. This suggests that a blood DNA test alone will usually be sufficient to make a diagnosis of C9orf72 repeat-related ALS. PMID:23894576

  2. Quantitative Field Testing Rotylenchulus reniformis DNA from Metagenomic Samples Isolated Directly from Soil

    PubMed Central

    Showmaker, Kurt; Lawrence, Gary W.; Lu, Shien; Balbalian, Clarissa; Klink, Vincent P.

    2011-01-01

    A quantitative PCR procedure targeting the β-tubulin gene determined the number of Rotylenchulus reniformis Linford & Oliveira 1940 in metagenomic DNA samples isolated from soil. Of note, this outcome was in the presence of other soil-dwelling plant parasitic nematodes including its sister genus Helicotylenchus Steiner, 1945. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from soil. PMID:22194958

  3. A simple and cost-effective method of DNA extraction from small formalin-fixed paraffin-embedded tissue for molecular oncologic testing

    PubMed Central

    2014-01-01

    Background Extraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing. As molecular oncology testing becomes more important for prognostic and therapeutic decision making and tissue specimens become smaller due to earlier detection of suspicious lesions and the use of fine needle aspiration methods for tissue collection, it becomes more challenging for the typical molecular pathology laboratory to obtain reliable test results. We developed a DNA extraction method to obtain sufficient quantity and high quality genomic DNA from limited FFPE tissue for molecular oncology testing using a combination of H&E stained slides, a matrix capture method and the Qiagen DNA column. Methods Three DNA extraction methods were compared: our standard procedure of manually scraping tissue from unstained slides followed by DNA extraction using the QIAamp FFPE column (Qiagen, Valencia, CA), a glue capture method (Pinpoint Solution, Zymo Research Corp, Inc) on H&E stained slides followed by DNA extraction using either the QIAamp column or the column included with the Pinpoint kit (Zymo Research). The DNA extraction protocol was optimized. Statistical analysis was performed using the paired two-sample student’s t-test. Results The combination of the matrix capture method with the QIAamp column gave an equivalent amount of DNA as our standard extraction method using the unstained slides and a 4.6-fold higher DNA yield than using the Zymo column included in the Pinpoint Slide Solution kit. Several molecular tests were performed and DNA purified using the new method gave the same results as for the previous methods. Conclusions Using H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of specific tissue from the slides easier and reduced the risk of contamination and tissue loss. This DNA extraction method is simple, cost-effective, and blends with our current

  4. [Comparison of the CMV antigenemia test and CMV-DNA PCR results in solid organ transplant recipients].

    PubMed

    Özkarata, Emre; Özkarataş, Emre; Özbek, Ö Alpay; Avkan Oğuz, Vildan; Sayıner, A Arzu

    2016-01-01

    Cytomegalovirus (CMV) infection is among the most common important viral infections in solid organ transplant (SOT) recipients. Diagnostic tests for detecting CMV replication are widely used for this group of patients, however there is no clear agreement on the cut-off levels for interpretation of clinical decisions especially when the low level of viral load is detected. In this study, CMV pp65 antigenemia test results were compared with plasma CMV-DNA levels detected by quantitative real-time polymerase chain reaction (qPCR) in samples of kidney and liver transplant recipients in the Central Laboratory of Dokuz Eylul University Hospital between 2011 and 2013, and the correlation between these two tests and viral load equivalent to antigenemia positivity were determined. In the study, pp65 antigenemia and CMV-DNA qPCR results were evaluated retrospectively. The samples from the same patients were included if the time between antigenemia and CMV-DNA qPCR tests were less than 48 hours. SPSS v15.0 was used for correlation, regression and ROC curve analysis. The results of the 217 samples collected from 100 patients (59 male, 41 female; age range: 16-71, mean age: 46 ± 13 years), 36 liver and 64 kidney recipients were evaluated in the study. Of the patients 80% were CMV IgM negative, IgG positive; 1% was CMV IgG and IgM positive; 2% were CMV IgM and IgG negative, while for 17 patients serological results could not be reached. CMV pp65 antigenemia and CMV-DNA were both negative in 102 (47%) samples, while both were positive in 37 (17%) samples. The single sample from a case with CMV IgM and IgG positivity yielded negative results for both antigenemia and CMV-DNA tests. In 78 samples antigenemia were negative and CMV-DNA qPCR were positive, while there were no samples with antigenemia positive and qPCR negative. Mean values of antigenemia and qPCR tests were 23 positive cells/200.000 leukocytes (range: 1 to 230 positive cells) and 12.595 copies/ml (range: 180 to 106

  5. Two New Nuclear Isolation Buffers for Plant DNA Flow Cytometry: A Test with 37 Species

    PubMed Central

    Loureiro, João; Rodriguez, Eleazar; Doležel, Jaroslav; Santos, Conceição

    2007-01-01

    Background and Aims After the initial boom in the application of flow cytometry in plant sciences in the late 1980s and early 1990s, which was accompanied by development of many nuclear isolation buffers, only a few efforts were made to develop new buffer formulas. In this work, recent data on the performance of nuclear isolation buffers are utilized in order to develop new buffers, general purpose buffer (GPB) and woody plant buffer (WPB), for plant DNA flow cytometry. Methods GPB and WPB were used to prepare samples for flow cytometric analysis of nuclear DNA content in a set of 37 plant species that included herbaceous and woody taxa with leaf tissues differing in structure and chemical composition. The following parameters of isolated nuclei were assessed: forward and side light scatter, propidium iodide fluorescence, coefficient of variation of DNA peaks, quantity of debris background, and the number of particles released from sample tissue. The nuclear genome size of 30 selected species was also estimated using the buffer that performed better for a given species. Key Results In unproblematic species, the use of both buffers resulted in high quality samples. The analysis of samples obtained with GPB usually resulted in histograms of DNA content with higher or similar resolution than those prepared with the WPB. In more recalcitrant tissues, such as those from woody plants, WPB performed better and GPB failed to provide acceptable results in some cases. Improved resolution of DNA content histograms in comparison with previously published buffers was achieved in most of the species analysed. Conclusions WPB is a reliable buffer which is also suitable for the analysis of problematic tissues/species. Although GPB failed with some plant species, it provided high-quality DNA histograms in species from which nuclear suspensions are easy to prepare. The results indicate that even with a broad range of species, either GPB or WPB is suitable for preparation of high

  6. Concordance study between the ParaDNA® Intelligence Test, a rapid DNA profiling assay, and a conventional STR typing kit (AmpFlSTR® SGM Plus®).

    PubMed

    Ball, G; Dawnay, N; Stafford-Allen, B; Panasiuk, M; Rendell, P; Blackman, S; Duxbury, N; Wells, S

    2015-05-01

    The ParaDNA® Intelligence Test enables STR profiling directly from human biological samples and evidence items collected from crime scene in 75min. Designed for non-expert use this system allows DNA information to be available to investigators before it would typically be available from a laboratory. The ParaDNA Intelligence Test system amplifies D3S1358, D8S119, D16S539, D18S1358 and TH01 STR loci and the gender typing locus amelogenin and detects the alleles present with HyBeacon® probes. Individual DNA samples from 381 UK Caucasian individuals were analysed using AmpFlSTR® SGM Plus® and the ParaDNA Intelligence Test with the derived STR profiles compared. Here we describe the high level of concordance demonstrated between the two systems and discuss this with reference to allele frequencies and the discriminatory power offered by the ParaDNA Intelligence Test. PMID:25528026

  7. Dipstick test for DNA-based food authentication. Application to coffee authenticity assessment.

    PubMed

    Trantakis, Ioannis A; Spaniolas, Stelios; Kalaitzis, Panagiotis; Ioannou, Penelope C; Tucker, Gregory A; Christopoulos, Theodore K

    2012-01-25

    This paper reports DNA-based food authenticity assays, in which species identification is accomplished by the naked eye without the need of specialized instruments. Strongly colored nanoparticles (gold nanoparticles) are employed as reporters that enable visual detection. Furthermore, detection is performed in a low-cost, disposable, dipstick-type device that incorporates the required reagents in dry form, thereby avoiding multiple pipetting and incubation steps. Due to its simplicity, the method does not require highly qualified personnel. The procedure comprises the following steps: (i) PCR amplification of the DNA segment that flanks the unique SNP (species marker); (ii) a 15 min extension reaction in which DNA polymerase extends an allele-specific primer only if it is perfectly complementary with the target sequence; (iii) detection of the products of the extension reaction within a few minutes by the naked eye employing the dipstick. No purification is required prior to application of the extension products to the dipstick. The method is general and requires only a unique DNA sequence for species discrimination. The only instrument needed is a conventional thermocycler for PCR, which is common equipment in every DNA laboratory. As a model, the method was applied to the discrimination of Coffea robusta and arabica species in coffee authenticity assessment. As low as 5% of Robusta coffee can be detected in the presence of Arabica coffee. PMID:22225459

  8. Detection of Toxoplasma gondii DNA in horse meat from supermarkets in France and performance evaluation of two serological tests

    PubMed Central

    Aroussi, Abdelkrim; Vignoles, Philippe; Dalmay, François; Wimel, Laurence; Dardé, Marie-Laure; Mercier, Aurélien; Ajzenberg, Daniel

    2015-01-01

    In France, some cases of severe toxoplasmosis have been linked to the consumption of horse meat that had been imported from the American continent where atypical strains of Toxoplasma gondii are more common than in Europe. Many seroprevalence studies are presented in the literature but risk assessment of T. gondii infection after horse meat consumption is not possible in the absence of validated serological tests and the unknown correlation between detection of antibodies against T. gondii and presence of tissue cysts. We performed magnetic-capture polymerase chain reaction (MC-PCR) to detect T. gondii DNA in 231 horse meat samples purchased in supermarkets in France and evaluated the performance and level of agreement of the modified agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA) in the meat juices. The serological tests lacked sensitivity, specificity, and agreement between them, and there was no correlation with the presence of T. gondii DNA in horse meat, raising concerns about the reliability of T. gondii seroprevalence data in horses from the literature. T. gondii DNA was detected in 43% of horse meat samples but the absence of strain isolation in mice following inoculation of more than 100 horse meat samples suggests a low distribution of cysts in skeletal muscles and a low risk of T. gondii infection associated with horse meat consumption. However, to avoid any risk of toxoplasmosis, thorough cooking of horse meat is recommended. PMID:25809058

  9. [Lung cancer molecular testing, what role for Next Generation Sequencing and circulating tumor DNA].

    PubMed

    Pécuchet, Nicolas; Legras, Antoine; Laurent-Puig, Pierre; Blons, Hélène

    2016-01-01

    Molecular screening has become a standard of care for patients with advanced cancers and impacts on how to treat a patient. Advances in genomic technologies with the development of high throughput sequencing methods will certainly improve the possibilities to access a more accurate molecular diagnosis and to go beyond the identification of validated targets as a large number of genes can be screened for actionable changes. Moreover, accurate high throughput testing may help tumor classification in terms of prognosis and drug sensitivity. Finally, it will be possible to assess tumor heterogeneity and changes in molecular profiles during follow-up using ultra-deep sequencing technologies and circulating tumor DNA characterization. The accumulation of somatic ADN alterations is considered as the main contributing factor in carcinogenesis. The alterations can occur at different levels: mutation, copy number variations or gene translocations resulting in altered expression of the corresponding genes or impaired protein functions. Genes involved are mainly tumor suppressors, oncogenes or ADN repair genes whose modifications in tumors will impinge cell fate and proliferation from tumor initiation to metastasis. The entire genome of various tumor types, have now been sequenced. In lung cancer, the average number of mutations is very high with more than 8.9 mutations/Mb (Network TCGAR, 2014) that is to say more than 10,000 mutations/genome. These alterations need to be classified, indeed, some are true drivers that directly impact proliferation and some are passenger mutations linked to genetic instability. The development of targeted therapies relies on the identification of oncogenic drivers. The identification of genotype-phenotype associations as in the case of EGFR-TKI (Epidermal growth factor receptor-tyrosine kinase inhibitor) and EGFR mutations in lung cancer led to the restriction of drugs to patients for which tumor genotype predicts efficacy. Tumor

  10. Testing the utility of matK and ITS DNA regions for discrimination of Allium species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Molecular phylogenetic analysis of the genus Allium L. has been mainly based on the nucleotide sequences of ITS region. In 2009 matK and rbcL were accepted as a two-locus DNA barcode to classify plant species by the Consortium for the Barcode of Life (CBOL) Plant Working Group. MatK region has been ...

  11. Identifying Insects with Incomplete DNA Barcode Libraries, African Fruit Flies (Diptera: Tephritidae) as a Test Case

    PubMed Central

    Virgilio, Massimiliano; Jordaens, Kurt; Breman, Floris C.; Backeljau, Thierry; De Meyer, Marc

    2012-01-01

    We propose a general working strategy to deal with incomplete reference libraries in the DNA barcoding identification of species. Considering that (1) queries with a large genetic distance with their best DNA barcode match are more likely to be misidentified and (2) imposing a distance threshold profitably reduces identification errors, we modelled relationships between identification performances and distance thresholds in four DNA barcode libraries of Diptera (n = 4270), Lepidoptera (n = 7577), Hymenoptera (n = 2067) and Tephritidae (n = 602 DNA barcodes). In all cases, more restrictive distance thresholds produced a gradual increase in the proportion of true negatives, a gradual decrease of false positives and more abrupt variations in the proportions of true positives and false negatives. More restrictive distance thresholds improved precision, yet negatively affected accuracy due to the higher proportions of queries discarded (viz. having a distance query-best match above the threshold). Using a simple linear regression we calculated an ad hoc distance threshold for the tephritid library producing an estimated relative identification error <0.05. According to the expectations, when we used this threshold for the identification of 188 independently collected tephritids, less than 5% of queries with a distance query-best match below the threshold were misidentified. Ad hoc thresholds can be calculated for each particular reference library of DNA barcodes and should be used as cut-off mark defining whether we can proceed identifying the query with a known estimated error probability (e.g. 5%) or whether we should discard the query and consider alternative/complementary identification methods. PMID:22359600

  12. Application of the cell growth and DNA-inhibition tests for characterizing sulfate pulp mill waste waters.

    PubMed

    Cernáková, M; Slamenová, D; Golis, E; Sutý, L

    1993-01-01

    The evaluation of cytotoxic and genotoxic effects of selected technological samples from sulfate pulp mill waste waters by using the growing activity method for pseudodiploid fibroblasts V79 from lungs of the Chinese hamster and from human heteroploid fibroblasts EUE has been described along with the DNA-inhibition test for studying the synthesis of DNA after it has been influenced by the above-mentioned samples. Both the waste solution produced during the preparation of bleaching agents and the liquor generated after using hypochlorite (1st stage) as a fourth filter (after the production of paper pulp) are cytotoxic waste waters. Black liquor generated during the production of viscose pulp may have mutagenic effects and black liquor obtained from the production of paper pulp is characterized by mutagenic as well as carcinogenic effects. PMID:8262452

  13. Experimental test of connector rotation during DNA packaging into bacteriophage phi29 capsids.

    PubMed

    Hugel, Thorsten; Michaelis, Jens; Hetherington, Craig L; Jardine, Paul J; Grimes, Shelley; Walter, Jessica M; Falk, Wayne; Anderson, Dwight L; Bustamante, Carlos

    2007-03-01

    The bacteriophage phi29 generates large forces to compact its double-stranded DNA genome into a protein capsid by means of a portal motor complex. Several mechanical models for the generation of these high forces by the motor complex predict coupling of DNA translocation to rotation of the head-tail connector dodecamer. Putative connector rotation is investigated here by combining the methods of single-molecule force spectroscopy with polarization-sensitive single-molecule fluorescence. In our experiment, we observe motor function in several packaging complexes in parallel using video microscopy of bead position in a magnetic trap. At the same time, we follow the orientation of single fluorophores attached to the portal motor connector. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question. PMID:17311473

  14. Testing DNA barcodes in closely related species of Curcuma (Zingiberaceae) from Myanmar and China.

    PubMed

    Chen, Juan; Zhao, Jietang; Erickson, David L; Xia, Nianhe; Kress, W John

    2015-03-01

    The genus Curcuma L. is commonly used as spices, medicines, dyes and ornamentals. Owing to its economic significance and lack of clear-cut morphological differences between species, this genus is an ideal case for developing DNA barcodes. In this study, four chloroplast DNA regions (matK, rbcL, trnH-psbA and trnL-F) and one nuclear region (ITS2) were generated for 44 Curcuma species and five species from closely related genera, represented by 96 samples. PCR amplification success rate, intra- and inter-specific genetic distance variation and the correct identification percentage were taken into account to assess candidate barcode regions. PCR and sequence success rate were high in matK (89.7%), rbcL (100%), trnH-psbA (100%), trnL-F (95.7%) and ITS2 (82.6%) regions. The results further showed that four candidate chloroplast barcoding regions (matK, rbcL, trnH-psbA and trnL-F) yield no barcode gaps, indicating that the genus Curcuma represents a challenging group for DNA barcoding. The ITS2 region presented large interspecific variation and provided the highest correct identification rates (46.7%) based on BLASTClust method among the five regions. However, the ITS2 only provided 7.9% based on NJ tree method. An increase in discriminatory power needs the development of more variable markers. PMID:25158042

  15. Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects.

    PubMed

    Elbrecht, Vasco; Taberlet, Pierre; Dejean, Tony; Valentini, Alice; Usseglio-Polatera, Philippe; Beisel, Jean-Nicolas; Coissac, Eric; Boyer, Frederic; Leese, Florian

    2016-01-01

    Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate. PMID:27114891

  16. Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

    PubMed Central

    Elbrecht, Vasco; Taberlet, Pierre; Dejean, Tony; Valentini, Alice; Usseglio-Polatera, Philippe; Beisel, Jean-Nicolas; Coissac, Eric; Boyer, Frederic

    2016-01-01

    Cytochrome c oxidase I (COI) is a powerful marker for DNA barcoding of animals, with good taxonomic resolution and a large reference database. However, when used for DNA metabarcoding, estimation of taxa abundances and species detection are limited due to primer bias caused by highly variable primer binding sites across the COI gene. Therefore, we explored the ability of the 16S ribosomal DNA gene as an alternative metabarcoding marker for species level assessments. Ten bulk samples, each containing equal amounts of tissue from 52 freshwater invertebrate taxa, were sequenced with the Illumina NextSeq 500 system. The 16S primers amplified three more insect species than the Folmer COI primers and amplified more equally, probably due to decreased primer bias. Estimation of biomass might be less biased with 16S than with COI, although variation in read abundances of two orders of magnitudes is still observed. According to these results, the marker choice depends on the scientific question. If the goal is to obtain a taxonomic identification at the species level, then COI is more appropriate due to established reference databases and known taxonomic resolution of this marker, knowing that a greater proportion of insects will be missed using COI Folmer primers. If the goal is to obtain a more comprehensive survey the 16S marker, which requires building a local reference database, or optimised degenerated COI primers could be more appropriate. PMID:27114891

  17. Buffy coat specimens remain viable as a DNA source for highly multiplexed genome-wide genetic tests after long term storage

    PubMed Central

    2011-01-01

    Background Blood specimen collection at an early study visit is often included in observational studies or clinical trials for analysis of secondary outcome biomarkers. A common protocol is to store buffy coat specimens for future DNA isolation and these may remain in frozen storage for many years. It is uncertain if the DNA remains suitable for modern genome wide association (GWA) genotyping. Methods We isolated DNA from 120 Action to Control Cardiovascular Risk in Diabetes (ACCORD) clinical trial buffy coats sampling a range of storage times up to 9 years and other factors that could influence DNA yield. We performed TaqMan SNP and GWA genotyping to test whether the DNA retained integrity for high quality genetic analysis. Results We tested two QIAGEN automated protocols for DNA isolation, preferring the Compromised Blood Protocol despite similar yields. We isolated DNA from all 120 specimens (yield range 1.1-312 ug per 8.5 ml ACD tube of whole blood) with only 3/120 samples yielding < 10 ug DNA. Age of participant at blood draw was negatively associated with yield (mean change -2.1 ug/year). DNA quality was very good based on gel electrophoresis QC, TaqMan genotyping of 6 SNPs (genotyping no-call rate 1.1% in 702 genotypes), and excellent quality GWA genotyping data (maximum per sample genotype missing rate 0.64%). Conclusions When collected as a long term clinical trial or biobank specimen for DNA, buffy coats can be stored for up to 9 years in a -80degC frozen state and still produce high yields of DNA suitable for GWA analysis and other genetic testing. Trial Registration The Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial is registered with ClinicalTrials.gov, number NCT00000620. PMID:21663644

  18. [The development of a test-system for the quantitative and qualitative evaluation of DNA content in criminalistic objects by the real-time polymerase chain reaction].

    PubMed

    Lapenkov, M I; Plakhina, N V; Alekseev, Ia I; Varlamov, D A

    2011-01-01

    An original test-system for the preliminary quantitative and qualitative evaluation of isolated DNA is proposed by the polymerase chain reaction in real time (PCR-RT) based on the TaqMan technology. This test-system permits to simultaneously measure the amount of DNA in the sample, identify the genetic gender, and detect PCR inhibitors. The method has been approbated in the practical work of forensic medical experts. PMID:21735715

  19. Development of genomic DNA reference materials for genetic testing of disorders common in people of ashkenazi jewish descent.

    PubMed

    Kalman, Lisa; Wilson, Jean Amos; Buller, Arlene; Dixon, John; Edelmann, Lisa; Geller, Louis; Highsmith, William Edward; Holtegaard, Leonard; Kornreich, Ruth; Rohlfs, Elizabeth M; Payeur, Toby L; Sellers, Tina; Toji, Lorraine; Muralidharan, Kasinathan

    2009-11-01

    Many recessive genetic disorders are found at a higher incidence in people of Ashkenazi Jewish (AJ) descent than in the general population. The American College of Medical Genetics and the American College of Obstetricians and Gynecologists have recommended that individuals of AJ descent undergo carrier screening for Tay Sachs disease, Canavan disease, familial dysautonomia, mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia type C, Bloom syndrome, and Gaucher disease. Although these recommendations have led to increased test volumes and number of laboratories offering AJ screening, well-characterized genomic reference materials are not publicly available. The Centers for Disease Control and Prevention-based Genetic Testing Reference Materials Coordination Program, in collaboration with members of the genetic testing community and Coriell Cell Repositories, have developed a panel of characterized genomic reference materials for AJ genetic testing. DNA from 31 cell lines, representing many of the common alleles for Tay Sachs disease, Canavan disease, familial dysautonomia, mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia type C, Bloom syndrome, Gaucher disease, and glycogen storage disease, was prepared by the Repository and tested in six clinical laboratories using three different PCR-based assay platforms. A total of 33 disease alleles was assayed and 25 different alleles were identified. These characterized materials are publicly available from Coriell and may be used for quality control, proficiency testing, test development, and research. PMID:19815695

  20. Results of external quality assessment for proviral DNA testing of HIV tropism in the Maraviroc Switch collaborative study.

    PubMed

    Tu, Elise; Swenson, Luke C; Land, Sally; Pett, Sarah; Emery, Sean; Marks, Kat; Kelleher, Anthony D; Kaye, Steve; Kaiser, Rolf; Schuelter, Eugene; Harrigan, Richard

    2013-07-01

    The Maraviroc Switch collaborative study (MARCH) is a study in aviremic patients on stable antiretroviral therapy and utilizes population-based sequencing of proviral DNA to determine HIV tropism and susceptibility to maraviroc. An external quality assessment (EQA) program was implemented to ensure competency in assessing the tropism of clinical samples conducted by MARCH laboratories (n = 14). The MARCH EQA has three prestudy phases assessing V3 loop sequencing and tropism determination using the bioinformatic algorithm geno2pheno, which generates a false-positive rate (FPR). DNA sequences with low FPRs are more likely to be from CXCR4-using (X4) viruses. Phase 1 of the EQA involved chromatogram interpretation. Phases 2, 2/3, and 3 involved patient and clonal samples. Clinical samples used in these phases were from treatment-experienced HIV-infected volunteers; 18/20 had viral loads of <50 copies/ml, and 10/15 were CXCR4-tropic on prior phenotyping. All samples were tested in triplicate, and any replicate with a geno2pheno FPR of <10% was designated X4. Performance was deemed adequate if ≤2 R5 and ≤1 X4 specimens were miscalled. For several clinical samples in the EQA, triplicate testing revealed marked DNA variability (FPR range, 0 to 96.7%). Therefore, a consensus-based approach was employed for each sample, i.e., a median FPR across laboratories was used to define sample tropism. Further sequencing analysis showed mixed viral populations in the clinical samples, explaining the differences in tropism predictions. All laboratories passed the EQA after achieving predefined competence thresholds in either of the phase 2 rounds. The use of clinical samples from patients resembling those who were likely to be screened in the MARCH, coupled with triplicate testing, revealed inherent DNA variability that might have been missed if single or duplicate testing and/or clonal samples alone were used. These data highlight the importance of intensive EQA of tropism

  1. Construction and nonclinical testing of a Puumala virus synthetic M gene-based DNA vaccine.

    PubMed

    Brocato, R L; Josleyn, M J; Wahl-Jensen, V; Schmaljohn, C S; Hooper, J W

    2013-02-01

    Puumala virus (PUUV) is a causative agent of hemorrhagic fever with renal syndrome (HFRS). Although PUUV-associated HFRS does not result in high case-fatality rates, the social and economic impact is considerable. There is no licensed vaccine or specific therapeutic to prevent or treat HFRS. Here we report the synthesis of a codon-optimized, full-length M segment open reading frame and its cloning into a DNA vaccine vector to produce the plasmid pWRG/PUU-M(s2). pWRG/PUU-M(s2) delivered by gene gun produced high-titer neutralizing antibodies in hamsters and nonhuman primates. Vaccination with pWRG/PUU-M(s2) protected hamsters against infection with PUUV but not against infection by related HFRS-associated hantaviruses. Unexpectedly, vaccination protected hamsters in a lethal disease model of Andes virus (ANDV) in the absence of ANDV cross-neutralizing antibodies. This is the first evidence that an experimental DNA vaccine for HFRS can provide protection in a hantavirus lethal disease model. PMID:23239797

  2. Integration of Noninvasive DNA Testing for Aneuploidy into Prenatal Care: What Has Happened Since the Rubber Met the Road?

    PubMed Central

    Bianchi, Diana W.; Wilkins-Haug, Louise

    2016-01-01

    BACKGROUND Over the past 2 years, noninvasive prenatal testing (NIPT), which uses massively parallel sequencing to align and count DNA fragments floating in the plasma of pregnant women, has become integrated into prenatal care. Professional societies currently recommend offering NIPT as an advanced screen to pregnant women at high risk for fetal aneuploidy, reserving invasive diagnostic procedures for those at the very highest risk. CONTENT In this review, we summarize the available information on autosomal and sex chromosome aneuploidy detection. Clinical performance in CLIA-certified, College of American Pathology–accredited laboratories appears to be equivalent to prior clinical validation studies, with high sensitivities and specificities and very high negative predictive values. The main impact on clinical care has been a reduction in invasive procedures. Test accuracy is affected by the fetal fraction, the percentage of fetal DNA in the total amount of circulating cell-free DNA. Fetal fraction is in turn affected by maternal body mass index, gestational age, type of aneuploidy, singleton vs multiples, and mosaicism. Three studies comparing NIPT to serum or combined screening for autosomal aneuploidy all show that NIPT has significantly lower false-positive rates (approximately 0.1%), even in all-risk populations. A significant number of the discordant positive cases have underlying biological reasons, including confined placental mosaicism, maternal mosaicism, cotwin demise, or maternal malignancy. SUMMARY NIPT performs well as an advanced screen for whole chromosome aneuploidy. Economic considerations will likely dictate whether its use can be expanded to all risk populations and whether it can be applied routinely for the detection of subchromosome abnormalities. PMID:24255077

  3. Non-Invasive Prenatal Testing Using Cell Free DNA in Maternal Plasma: Recent Developments and Future Prospects

    PubMed Central

    Benn, Peter

    2014-01-01

    Recent advances in molecular genetic technologies have facilitated non-invasive prenatal testing (NIPT) through the analysis of cell-free fetal DNA in maternal plasma. NIPT can be used to identify monogenic disorders including the identification of autosomal recessive disorders where the maternally inherited mutation needs to be identified in the presence of an excess of maternal DNA that contains the same mutation. In the future, simultaneous screening for multiple monogenic disorders is anticipated. Several NIPT methods have been developed to screen for trisomy. These have been shown to be effective for fetal trisomy 21, 18 and 13. Although the testing has been extended to sex chromosome aneuploidy, robust estimates of the efficacy are not yet available and maternal mosaicism for gain or loss of an X-chromosome needs to be considered. Using methods based on the analysis of single nucleotide polymorphisms, diandric triploidy can be identified. NIPT is being developed to identify a number of microdeletion syndromes including α-globin gene deletion. NIPT is a profoundly important development in prenatal care that is substantially advancing the individual patient and public health benefits achieved through conventional prenatal screening and diagnosis. PMID:26237390

  4. Genotoxic potency in Drosophila melanogaster of selected aromatic amines and polycyclic aromatic hydrocarbons as assayed in the DNA repair test.

    PubMed

    Fujikawa, K; Fort, F L; Samejima, K; Sakamoto, Y

    1993-12-01

    Drosophila melanogaster stock consisting of meiotic recombination deficient (Rec-) double mutant mei-9a mei-41D5 males and Rec+ females was exposed at the larval stage to an aromatic amine or a polycyclic aromatic hydrocarbon. After emergence as adult flies, the males and the females were scored separately. When the treatment caused a dose-dependent reduction in the male to female ratio from the control level; the experiment was repeated with a larval stock consisting of Rec+ males and Rec+ females under comparable conditions. A preferential killing effect upon Rec- larvae was taken as evidence of DNA damaging effect of the test compound. Among 16 compounds tested, 1-AP, B(a)P, 2-AF, DAF, 4-AAF, 2-AAF, 1-AA, 2-AA, DMA, B(a)A and DMBA were registered as positive; Py and 3-MC were weakly positive; and B(e)P, Fluo and Ant were negative. The selective killing effects of the compounds in each of the pyrene, fluorene and anthracene series varied drastically as a function of structure in a way similar to that reported for the genotoxicity in Drosophila and the carcinogenicity in rodents. The Drosophila DNA repair assay will serve as a simple adjunct to the already available means for studying the genotoxic potency of aromatic amines and polycyclic aromatic hydrocarbons. PMID:7694108

  5. An Economic Analysis of Cell-Free DNA Non-Invasive Prenatal Testing in the US General Pregnancy Population

    PubMed Central

    Benn, Peter; Curnow, Kirsten J.; Chapman, Steven; Michalopoulos, Steven N.; Hornberger, John; Rabinowitz, Matthew

    2015-01-01

    Objective Analyze the economic value of replacing conventional fetal aneuploidy screening approaches with non-invasive prenatal testing (NIPT) in the general pregnancy population. Methods Using decision-analysis modeling, we compared conventional screening to NIPT with cell-free DNA (cfDNA) analysis in the annual US pregnancy population. Sensitivity and specificity for fetal aneuploidies, trisomy 21, trisomy 18, trisomy 13, and monosomy X, were estimated using published data and modeling of both first- and second trimester screening. Costs were assigned for each prenatal test component and for an affected birth. The overall cost to the healthcare system considered screening costs, the number of aneuploid cases detected, invasive procedures performed, procedure-related euploid losses, and affected pregnancies averted. Sensitivity analyses evaluated the effect of variation in parameters. Costs were reported in 2014 US Dollars. Results Replacing conventional screening with NIPT would reduce healthcare costs if it can be provided for $744 or less in the general pregnancy population. The most influential variables were timing of screening entry, screening costs, and pregnancy termination rates. Of the 13,176 affected pregnancies undergoing screening, NIPT detected 96.5% (12,717/13,176) of cases, compared with 85.9% (11,314/13,176) by conventional approaches. NIPT reduced invasive procedures by 60.0%, with NIPT and conventional methods resulting in 24,596 and 61,430 invasive procedures, respectively. The number of procedure-related euploid fetal losses was reduced by 73.5% (194/264) in the general screening population. Conclusion Based on our analysis, universal application of NIPT would increase fetal aneuploidy detection rates and can be economically justified. Offering this testing to all pregnant women is associated with substantial prenatal healthcare benefits. PMID:26158465

  6. Testing prey DNA fingerprinting on Amblyseius largoensis (Acari: Phytoseiidae) predation of Raoiella indica (Acari: Tenuipalpidae).

    PubMed

    Rivera-Rivera, Carlos; Galindo-Cardona, Alberto; Rodrigues, Jose Carlos Verle

    2012-08-01

    Molecular detection of predation by identifying prey markers in the digestive tract of predators has developed into a powerful tool to assess predator-prey systems in which diet identification is too time consuming or impossible. Here we explore its utility for detecting predation of the pest mite Raoiella indica Hirst by the predatory mite Amblyseius largoensis Muma, taking advantage of the color the predator acquires after eating this mite to cross-reference our results. For this, a ~410 bp segment of the cytochrome c oxidase subunit I (COI) mitochondrial gene marker specific for the subfamily Tetranychoidea was used. Amblyseius largoensis that had recently eaten were collected from greenhouse colonies containing both mites, and isolated from any other food source. Predator mites were taken for fingerprinting at 24, 48, 72 and 96 h of starving after collection, and the same process was repeated a second time, offering pollen as an alternative food source to see whether detection changed. Lastly, a sampling trial was conducted in the greenhouse, in which mites were collected regardless of their color and frozen immediately for fingerprinting. Raoiella indica DNA was detected for 48 h on starving predators, and for 96 h on those who had eaten pollen. The segment was detected in 26 % of the samples collected on the trial. This technique needs refinement specific for this system, but the results obtained here confirm that it could turn into a very useful tool for assessing aspects of this predator-prey system. PMID:22476445

  7. BRAF Mutation Testing in Cell-Free DNA from the Plasma of Patients with Advanced Cancers Using a Rapid, Automated Molecular Diagnostics System.

    PubMed

    Janku, Filip; Huang, Helen J; Claes, Bart; Falchook, Gerald S; Fu, Siqing; Hong, David; Ramzanali, Nishma M; Nitti, Giovanni; Cabrilo, Goran; Tsimberidou, Apostolia M; Naing, Aung; Piha-Paul, Sarina A; Wheler, Jennifer J; Karp, Daniel D; Holley, Veronica R; Zinner, Ralph G; Subbiah, Vivek; Luthra, Rajyalakshmi; Kopetz, Scott; Overman, Michael J; Kee, Bryan K; Patel, Sapna; Devogelaere, Benoit; Sablon, Erwin; Maertens, Geert; Mills, Gordon B; Kurzrock, Razelle; Meric-Bernstam, Funda

    2016-06-01

    Cell-free (cf) DNA from plasma offers an easily obtainable material for BRAF mutation analysis for diagnostics and response monitoring. In this study, plasma-derived cfDNA samples from patients with progressing advanced cancers or malignant histiocytosis with known BRAF(V600) status from formalin-fixed paraffin-embedded (FFPE) tumors were tested using a prototype version of the Idylla BRAF Mutation Test, a fully integrated real-time PCR-based test with turnaround time about 90 minutes. Of 160 patients, BRAF(V600) mutations were detected in 62 (39%) archival FFPE tumor samples and 47 (29%) plasma cfDNA samples. The two methods had overall agreement in 141 patients [88%; κ, 0.74; SE, 0.06; 95% confidence interval (CI), 0.63-0.85]. Idylla had a sensitivity of 73% (95% CI, 0.60-0.83) and specificity of 98% (95% CI, 0.93-1.00). A higher percentage, but not concentration, of BRAF(V600) cfDNA in the wild-type background (>2% vs. ≤ 2%) was associated with shorter overall survival (OS; P = 0.005) and in patients with BRAF mutations in the tissue, who were receiving BRAF/MEK inhibitors, shorter time to treatment failure (TTF; P = 0.001). Longitudinal monitoring demonstrated that decreasing levels of BRAF(V600) cfDNA were associated with longer TTF (P = 0.045). In conclusion, testing for BRAF(V600) mutations in plasma cfDNA using the Idylla BRAF Mutation Test has acceptable concordance with standard testing of tumor tissue. A higher percentage of mutant BRAF(V600) in cfDNA corresponded with shorter OS and in patients receiving BRAF/MEK inhibitors also with shorter TTF. Mol Cancer Ther; 15(6); 1397-404. ©2016 AACR. PMID:27207774

  8. Tracing the geographic origin of traded leopard body parts in the indian subcontinent with DNA-based assignment tests.

    PubMed

    Mondol, Samrat; Sridhar, Vanjulavalli; Yadav, Prasanjeet; Gubbi, Sanjay; Ramakrishnan, Uma

    2015-04-01

    Illicit trade in wildlife products is rapidly decimating many species across the globe. Such trade is often underestimated for wide-ranging species until it is too late for the survival of their remaining populations. Policing this trade could be vastly improved if one could reliably determine geographic origins of illegal wildlife products and identify areas where greater enforcement is needed. Using DNA-based assignment tests (i.e., samples are assigned to geographic locations), we addressed these factors for leopards (Panthera pardus) on the Indian subcontinent. We created geography-specific allele frequencies from a genetic reference database of 173 leopards across India to infer geographic origins of DNA samples from 40 seized leopard skins. Sensitivity analyses of samples of known geographic origins and assignments of seized skins demonstrated robust assignments for Indian leopards. We found that confiscated pelts seized in small numbers were not necessarily from local leopards. The geographic footprint of large seizures appeared to be bigger than the cumulative footprint of several smaller seizures, indicating widespread leopard poaching across the subcontinent. Our seized samples had male-biased sex ratios, especially the large seizures. From multiple seized sample assignments, we identified central India as a poaching hotspot for leopards. The techniques we applied can be used to identify origins of seized illegal wildlife products and trade routes at the subcontinent scale and beyond. PMID:25376464

  9. Accuracy and Cost-Effectiveness of Cervical Cancer Screening by High-Risk HPV DNA Testing of Self-Collected Vaginal Samples

    PubMed Central

    Balasubramanian, Akhila; Kulasingam, Shalini L.; Baer, Atar; Hughes, James P.; Myers, Evan R.; Mao, Constance; Kiviat, Nancy B.; Koutsky, Laura A.

    2010-01-01

    Objective Estimate the accuracy and cost-effectiveness of cervical cancer screening strategies based on high-risk HPV DNA testing of self-collected vaginal samples. Materials and Methods A subset of 1,665 women (18-50 years of age) participating in a cervical cancer screening study were screened by liquid-based cytology and by high-risk HPV DNA testing of both self-collected vaginal swab samples and clinician-collected cervical samples. Women with positive/abnormal screening test results and a subset of women with negative screening test results were triaged to colposcopy. Based on individual and combined test results, five screening strategies were defined. Estimates of sensitivity and specificity for cervical intraepithelial neoplasia grade 2 or worse were calculated and a Markov model was used to estimate the incremental cost-effectiveness ratios (ICERs) for each strategy. Results Compared to cytology-based screening, high-risk HPV DNA testing of self-collected vaginal samples was more sensitive (68%, 95%CI=58%-78% versus 85%, 95%CI=76%-94%) but less specific (89%, 95%CI=86%-91% versus 73%, 95%CI=67%-79%). A strategy of high-risk HPV DNA testing of self-collected vaginal samples followed by cytology triage of HPV positive women, was comparably sensitive (75%, 95%CI=64%-86%) and specific (88%, 95%CI=85%-92%) to cytology-based screening. In-home self-collection for high-risk HPV DNA detection followed by in-clinic cytology triage had a slightly lower lifetime cost and a slightly higher quality-adjusted life expectancy than did cytology-based screening (ICER of triennial screening compared to no screening was $9,871/QALY and $12,878/QALY, respectively). Conclusions Triennial screening by high-risk HPV DNA testing of in-home, self-collected vaginal samples followed by in-clinic cytology triage was cost-effective. PMID:20592553

  10. Problem-Based Test: Replication of Mitochondrial DNA during the Cell Cycle

    ERIC Educational Resources Information Center

    Setalo, Gyorgy, Jr.

    2013-01-01

    Terms to be familiar with before you start to solve the test: cell cycle, generation time, S-phase, cell culture synchronization, isotopic pulse-chase labeling, density labeling, equilibrium density-gradient centrifugation, buoyant density, rate-zonal centrifugation, nucleoside, nucleotide, kinase enzymes, polymerization of nucleic acids,…

  11. Highly polymorphic microsatellite DNA markers for sugarcane germplasm evaluation and variety identity testing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to evaluate the entire set of 221 sugarcane microsatellite (SSR) markers from the International Sugarcane Microsatellite Consortium for their utility on molecular characterization of elite U.S. germplasm. Five elite U.S. sugarcane clones were tested, including two cu...

  12. A new rapid method for Clostridium difficile DNA extraction and detection in stool: toward point-of-care diagnostic testing.

    PubMed

    Freifeld, Alison G; Simonsen, Kari A; Booth, Christine S; Zhao, Xing; Whitney, Scott E; Karre, Teresa; Iwen, Peter C; Viljoen, Hendrik J

    2012-01-01

    We describe a new method for the rapid diagnosis of Clostridium difficile infection, with stool sample preparation and DNA extraction by heat and physical disruption in a single-use lysis microreactor (LMR), followed by a rapid PCR amplification step. All steps can be accomplished in <20 minutes overall. Gel electrophoresis is currently used to detect the amplification product, pending real-time availability with an ultra-rapid thermocycler. Compared with the dual enzyme immunoassay (EIA) screening test (C. diff Quik Chek Complete; Techlab, Blacksburg, VA), the novel LMR/PCR assay showed complete concordance with all glutamate dehydrogenase (GDH) results (GDH(+)/toxin(+), n = 48; GDH(-)/toxin(-), n = 81). All 69 stool samples with discordant EIA results (GDH(+)/toxin(-)) were tested by both the LMR/PCR assay and the loop-mediated isothermal amplification test (LAMP) (Illumigene C. difficile; Meridian Bioscience, Cincinnati, OH). In 64/69 EIA-discordant samples, LAMP and LMR/PCR results matched (both positive in 29 sample and both negative in 35 samples); in the remaining 5 samples, results were discrepant between the LAMP assay (all five negative) and the LMR/PCR assay (all 5 positive). Overall, LMR/PCR testing matched the current algorithm of EIA and/or LAMP reflex testing in 193/198 (97.5%) samples. The present proof-of-concept study suggests that the novel LMR/PCR technique described here may be developed as an inexpensive, rapid, and reliable point-of-care diagnostic test for C. difficile infection and other infectious diseases. PMID:22402170

  13. Sex differences in DNA methylation and expression in zebrafish brain: a test of an extended 'male sex drive' hypothesis.

    PubMed

    Chatterjee, Aniruddha; Lagisz, Malgorzata; Rodger, Euan J; Zhen, Li; Stockwell, Peter A; Duncan, Elizabeth J; Horsfield, Julia A; Jeyakani, Justin; Mathavan, Sinnakaruppan; Ozaki, Yuichi; Nakagawa, Shinichi

    2016-09-30

    The sex drive hypothesis predicts that stronger selection on male traits has resulted in masculinization of the genome. Here we test whether such masculinizing effects can be detected at the level of the transcriptome and methylome in the adult zebrafish brain. Although methylation is globally similar, we identified 914 specific differentially methylated CpGs (DMCs) between males and females (435 were hypermethylated and 479 were hypomethylated in males compared to females). These DMCs were prevalent in gene body, intergenic regions and CpG island shores. We also discovered 15 distinct CpG clusters with striking sex-specific DNA methylation differences. In contrast, at transcriptome level, more female-biased genes than male-biased genes were expressed, giving little support for the male sex drive hypothesis. Our study provides genome-wide methylome and transcriptome assessment and sheds light on sex-specific epigenetic patterns and in zebrafish for the first time. PMID:27259666

  14. Myth and reality: practical test system for the measurement of anti-DNA antibodies in the diagnosis of systemic lupus erythematosus (SLE).

    PubMed

    McCloskey, Laura J; Christner, Paul; Jacobs-Kosmin, Dana; Jaskowski, Troy D; Hill, Harry R; Lakos, Gabriella; Teodorescu, Marius

    2010-01-01

    The myth persists that only the labor intensive Farr radioimmunoassay and Crithidia luciliae immunofluorescence (CL-IFA) are systemic lupus erythematosus (SLE)-specific tests. We compared them to ELISA with bacteriophage lambda DNA (EL-dsDNA) and denatured calf thymus DNA (EL-ssDNA). By percentile ranking, the specificity cut-off level was set both out of clinical context (SOCC) on 100 blood bank donors, and in clinical context (SICC) on 100 patients with either rheumatoid arthritis or scleroderma (50/50). Clinical sensitivity was calculated on 100 random SLE patients. At 95% SICC, the sensitivity of Farr, CL-IFA, EL-dsDNA, and EL-ssDNA was similar (95%CI): 76% (66-84), 76% (66-84), 63% (53-72), and 75% (65-83), respectively; 87% of the patients were positive by at least one method and 55%by all methods. At 99% SICC, the sensitivity was also similar (95% CI): 57% (47-67), 47% (37-57), 58% (47-67), and 43% (33-53), respectively. The areas under ROC curve were similar (95% CI) when patients were used as controls for specificity. At 99% SOCC, EL-ssDNA identified 89% positive, 2 negative but positive by another method at 95% SICC, and 9 negative (i.e. 89/2/9), followed by CL-IFA (80/6/14), Farr (76/12/12), and EL-dsDNA (64/23/13). Thus, at relatively low cost and easy automation, under the same conditions of specificity, the two ELISA tests combined were at least as good, if not superior, to CL-IFA or Farr: they showed similar clinical sensitivity and also identified more patients with anti-DNA antibodies. PMID:20333761

  15. Chlamydia Testing

    MedlinePlus

    ... Amplification Test (NAAT); Chlamydia trachomatis Culture; Chlamydia trachomatis DNA Probe Related tests: Gonorrhea Testing , HIV Antibody and HIV Antigen , Syphilis Tests , Herpes Testing , HPV Test , Trichomonas Testing All content on Lab Tests Online has ...

  16. Discrepancies in diagnosis of incident HIV infection between antibody- and DNA-based tests in a phase III prevention trial in southern Africa.

    PubMed

    Montgomery, E T; van der Pol, B; van der Straten, A; Ramjee, G; de Bruyn, G; Chipato, T; Blanchard, K; Padian, N S

    2012-09-01

    Dried blood spots (DBS) are widely used to test for HIV in a variety of research and service delivery settings; however, uniform guidelines regarding collection, storage and DNA extraction processes have neither been developed nor evaluated. Previously published reports suggested DBS may be stored at room temperature for up to 60 days, and intensive stability tests have shown that DBS can withstand high temperatures, humidity and freeze-thawing. During the implementation of a large randomized controlled trial (RCT) in southern Africa, with HIV acquisition as the primary endpoint, we observed 65 instances when DBS samples collected from the same day as a positive HIV antibody test yielded negative DNA polymerase chain reaction (PCR) results. The source of this discrepancy may have been due to inadequate specimen volume, filter paper or DNA extraction procedures, but were most likely due to storage conditions that have been reported as acceptable in other settings. PMID:23033520

  17. Sensitivity, Specificity, and Clinical Value of Human Papillomavirus (HPV) E6/E7 mRNA Assay as a Triage Test for Cervical Cytology and HPV DNA Test

    PubMed Central

    Benevolo, Maria; Vocaturo, Amina; Caraceni, Donatella; French, Deborah; Rosini, Sandra; Zappacosta, Roberta; Terrenato, Irene; Ciccocioppo, Lucia; Frega, Antonio; Rossi, Paolo Giorgi

    2011-01-01

    There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of

  18. Introducing a High-Risk HPV DNA Test Into a Public Sector Screening Program in El Salvador

    PubMed Central

    Cremer, Miriam L.; Maza, Mauricio; Alfaro, Karla M.; Kim, Jane J.; Ditzian, Lauren R.; Villalta, Sofia; Alonzo, Todd A.; Felix, Juan C.; Castle, Philip E.; Gage, Julia C.

    2016-01-01

    Objective In a primary human papillomavirus (HPV) screening program, we compared the 6-month follow-up among colposcopy and noncolposcopy-based management strategies for screen-positive women. Materials and Methods Women aged 30 to 49 years were screened with HPV DNA tests using both self-collection and provider collection of samples. Women testing positive received either (1) colposcopy management (CM) consisting of colposcopy and management per local guidelines or (2) screen-and-treat (ST) management using visual inspection with acetic acid to determine cryotherapy eligibility, with eligible women undergoing immediate cryotherapy. One thousand women were recruited in each cohort. Of these, 368 (18.4%) of 2000 women were recruited using a more intensive outreach strategy. Demographics, HPV positivity, and treatment compliance were compared across recruitment and management strategies. Results More women in the ST cohort received treatment within 6 months compared with those in the CM cohort (117/119 [98.3%] vs 64/93 [68.8%]; p < .001). Women recruited through more intensive outreach were more likely to be HPV positive, lived in urban areas, were more educated, and had higher numbers of lifetime sexual partners and fewer children. Conclusions Women in the CM arm were less likely to complete care than women in the ST arm. Targeted outreach to underscreened women successfully identified women with higher prevalence of HPV and possibly higher disease burden. PMID:26890683

  19. Distribution of Plasmodium species on the island of Grande Comore on the basis of DNA extracted from rapid diagnostic tests.

    PubMed

    Papa Mze, Nasserdine; Ahouidi, Ambroise D; Diedhiou, Cyrille K; Silai, Rahamatou; Diallo, Mouhamadou; Ndiaye, Daouda; Sembene, Mbacké; Mboup, Souleymane

    2016-01-01

    In the Union of Comoros, interventions for combating malaria have contributed to a spectacular decrease in the prevalence of the disease. We studied the current distribution of Plasmodium species on the island of Grande Comore using nested PCR. The rapid diagnostic tests (RDTs) currently used in the Comoros are able to identify Plasmodium falciparum but no other Plasmodium species. In this study, we tested 211 RDTs (158 positive and 53 negative). Among the 158 positive RDTs, 22 were positive for HRP2, 3 were positive only for pLDH, and 133 were positive for HRP2 and pLDH. DNA was extracted from a proximal part of the nitrocellulose membrane of RDTs. A total of 159 samples were positive by nested PCR. Of those, 156 (98.11%) were positive for P. falciparum, 2 (1.25%) were positive for P. vivaxI, and 1 (0.62%) was positive for P. malariae. None of the samples were positive for P. ovale. Our results show that P. falciparum is still the most dominant species on the island of Grande Comore, but P. vivax and P. malariae are present at a low prevalence. PMID:27561250

  20. Distribution of Plasmodium species on the island of Grande Comore on the basis of DNA extracted from rapid diagnostic tests

    PubMed Central

    Papa Mze, Nasserdine; Ahouidi, Ambroise D.; Diedhiou, Cyrille K.; Silai, Rahamatou; Diallo, Mouhamadou; Ndiaye, Daouda; Sembene, Mbacké; Mboup, Souleymane

    2016-01-01

    In the Union of Comoros, interventions for combating malaria have contributed to a spectacular decrease in the prevalence of the disease. We studied the current distribution of Plasmodium species on the island of Grande Comore using nested PCR. The rapid diagnostic tests (RDTs) currently used in the Comoros are able to identify Plasmodium falciparum but no other Plasmodium species. In this study, we tested 211 RDTs (158 positive and 53 negative). Among the 158 positive RDTs, 22 were positive for HRP2, 3 were positive only for pLDH, and 133 were positive for HRP2 and pLDH. DNA was extracted from a proximal part of the nitrocellulose membrane of RDTs. A total of 159 samples were positive by nested PCR. Of those, 156 (98.11%) were positive for P. falciparum, 2 (1.25%) were positive for P. vivaxI, and 1 (0.62%) was positive for P. malariae. None of the samples were positive for P. ovale. Our results show that P. falciparum is still the most dominant species on the island of Grande Comore, but P. vivax and P. malariae are present at a low prevalence. PMID:27561250

  1. Development of a dual test procedure for DNA typing and methamphetamine detection using a trace amount of stimulant-containing blood.

    PubMed

    Irii, Toshiaki; Maebashi, Kyoko; Fukui, Kenji; Sohma, Ryoko; Matsumoto, Sari; Takasu, Shojiro; Iwadate, Kimiharu

    2016-05-01

    Investigation of drug-related crimes, such as violation of the Stimulant Drug Control Law, requires identifying the used drug (mainly stimulant drugs, methamphetamine hydrochloride) from a drug solution and the DNA type of the drug user from a trace of blood left in the syringe used to inject the drug. In current standard test procedures, DNA typing and methamphetamine detection are performed as independent tests that use two separate portions of a precious sample. The sample can be entirely used up by either analysis. Therefore, we developed a new procedure involving partial lysis of a stimulant-containing blood sample followed by separation of the lysate into a precipitate for DNA typing and a liquid-phase fraction for methamphetamine detection. The method enables these two tests to be run in parallel using a single portion of sample. Samples were prepared by adding methamphetamine hydrochloride water solution to blood. Samples were lysed with Proteinase K in PBS at 56°C for 20min, cooled at -20°C after adding methanol, and then centrifuged at 15,000rpm. Based on the biopolymer-precipitating ability of alcohol, the precipitate was used for DNA typing and the liquid-phase fraction for methamphetamine detection. For DNA typing, the precipitate was dissolved and DNA was extracted, quantified, and subjected to STR analysis using the AmpFℓSTR® Identifiler® Plus PCR Amplification Kit. For methamphetamine detection, the liquid-phase fraction was evaporated with N2 gas after adding 20μL acetic acid and passed through an extraction column; the substances captured in the column were eluted with a solvent, derivatized, and quantitatively detected using gas chromatograph/mass spectrometry. This method was simple and could be completed in approximately 2h. Both DNA typing and methamphetamine detection were possible, which suggests that this method may be valuable for use in criminal investigations. PMID:27161925

  2. Gonorrhea Test

    MedlinePlus

    ... gonorrhoeae Culture; Neisseria gonorrhoeae Gram Stain; Neisseria gonorrhoeae DNA Probe Related tests: Chlamydia Testing , HIV Antibody and HIV Antigen , Syphilis Tests , Herpes Testing , HPV Test , Trichomonas Testing All content on Lab Tests Online has ...

  3. Genotypic tropism testing in proviral DNA to guide maraviroc initiation in aviremic subjects: 48-week analysis of the PROTEST study

    PubMed Central

    Garcia, Federico; Poveda, Eva; Jesús Pérez-Elías, Maria; Hernández Quero, José; Àngels Ribas, Maria; Martínez-Madrid, Onofre J; Flores, Juan; Crespo, Manel; Gutiérrez, Félix; García-Deltoro, Miguel; Imaz, Arkaitz; Ocampo, Antonio; Artero, Arturo; Blanco, Francisco; Bernal, Enrique; Pasquau, Juan; Mínguez-Gallego, Carlos; Pérez, Núria; Aiestarán, Aintzane; Paredes, Roger

    2014-01-01

    Introduction In a previous interim 24-week virological safety analysis of the PROTEST study [1], initiation of Maraviroc (MVC) plus 2 nucleoside reverse-transcriptase inhibitors (NRTIs) in aviremic subjects based on genotypic tropism testing of proviral HIV-1 DNA was associated with low rates of virological failure. Here we present the final 48-week analysis of the study. Methods PROTEST was a phase 4, prospective, single-arm clinical trial (ID: NCT01378910) carried on in 24 HIV care centres in Spain. Maraviroc-naïve HIV-1-positive adults with HIV-1 RNA (VL) <50 c/mL on stable ART during the previous 6 months, requiring an ART change due to toxicity, with no antiretroviral resistance to the ART started, and R5 HIV by proviral DNA genotypic tropism testing (defined as a G2P FPR >10% in a singleton), initiated MVC with 2 NRTIs and were followed for 48 weeks. Virological failure was defined as two consecutive VL>50 c/mL. Recent adherence was calculated as: (# pills taken/# pills prescribed during the previous week)*100. Results Tropism results were available from 141/175 (80.6%) subjects screened: 87/141 (60%) were R5 and 74/87 (85%) were finally included in the study. Their median age was 48 years, 16% were women, 31% were MSM, 36% had CDC category C at study entry, 62% were HCV+ and 10% were HBV+. Median CD4+ counts were 616 cells/mm3 at screening, and median nadir CD4+ counts were 143 cells/mm3. Previous ART included PIs in 46 (62%) subjects, NNRTIs in 27 (36%) and integrase inhibitors (INIs) in 1 (2%). The main reasons for treatment change were dyslipidemia (42%), gastrointestinal symptoms (22%), and liver toxicity (15%). MVC was given alongside TDF/FTC in 40 (54%) subjects, ABC/3TC in 30 (40%), AZT/3TC in 2 (3%) and ABC/TDF in 2 (3%). Sixty-two (84%) subjects maintained VL<50 c/mL through week 48, whereas 12 (16%) discontinued treatment: two (3%) withdrew informed consent, one (1%) had a R5→X4 shift in HIV tropism between the screening and baseline visits, one

  4. Motivations for Undertaking DNA Sequencing-Based Non-Invasive Prenatal Testing for Fetal Aneuploidy: A Qualitative Study with Early Adopter Patients in Hong Kong

    PubMed Central

    Yi, Huso; Hallowell, Nina; Griffiths, Sian; Yeung Leung, Tak

    2013-01-01

    Background A newly introduced cell-free fetal DNA sequencing based non-invasive prenatal testing (DNA-NIPT) detects Down syndrome with sensitivity of 99% at early gestational stage without risk of miscarriage. Attention has been given to its public health implications; little is known from consumer perspectives. This qualitative study aimed to explore women’s motivations for using, and perceptions of, DNA-NIPT in Hong Kong. Methods and Findings In-depth interviews were conducted with 45 women who had undertaken DNA-NIPT recruited by purposive sampling based on socio-demographic and clinical characteristics. The sample included 31 women identified as high-risk from serum and ultrasound based Down syndrome screening (SU-DSS). Thematic narrative analysis examined informed-decision making of the test and identified the benefits and needs. Women outlined a number of reasons for accessing DNA-NIPT: reducing the uncertainty associated with risk probability-based results from SU-DSS, undertaking DNA-NIPT as a comprehensive measure to counteract risk from childbearing especially at advanced age, perceived predictive accuracy and absence of risk of harm to fetus. Accounts of women deemed high-risk or not high-risk are distinctive in a number of respects. High-risk women accessed DNA-NIPT to get a clearer idea of their risk. This group perceived SU-DSS as an unnecessary and confusing procedure because of its varying, protocol-dependent detection rates. Those women not deemed high-risk, in contrast, undertook DNA-NIPT for psychological assurance and to reduce anxiety even after receiving the negative result from SU-DSS. Conclusions DNA-NIPT was regarded positively by women who chose this method of screening over the routine, less expensive testing options. Given its perceived utility, health providers need to consider whether DNA-NIPT should be offered as part of universal routine care to women at high-risk for fetal aneuploidy. If this is the case, then further development

  5. Susceptibility Testing by Polymerase Chain Reaction DNA Quantitation: A Method to Measure Drug Resistance of Human Immunodeficiency Virus Type 1 Isolates

    NASA Astrophysics Data System (ADS)

    Eron, Joseph J.; Gorczyca, Paul; Kaplan, Joan C.; D'Aquila, Richard T.

    1992-04-01

    Polymerase chain reaction (PCR) DNA quantitation (PDQ) susceptibility testing rapidly and directly measures nucleoside sensitivity of human immunodeficiency virus type 1 (HIV-1) isolates. PCR is used to quantitate the amount of HIV-1 DNA synthesized after in vitro infection of peripheral blood mononuclear cells. The relative amounts of HIV-1 DNA in cell lysates from cultures maintained at different drug concentrations reflect drug inhibition of virus replication. The results of PDQ susceptibility testing of 2- or 3-day cultures are supported by assays measuring HIV-1 p24 antigen production in supernatants of 7- or 10-day cultures. DNA sequence analyses to identify mutations in the reverse transcriptase gene that cause resistance to 3'-azido-3'-deoxythymidine also support the PDQ results. With the PDQ method, both infectivity titration and susceptibility testing can be performed on supernatants from primary cultures of peripheral blood mononuclear cells. PDQ susceptibility testing should facilitate epidemiologic studies of the clinical significance of drug-resistant HIV-1 isolates.

  6. Error rate for HLA-B antigen assignment by serology: implications for proficiency testing and utilization of DNA-based typing methods.

    PubMed

    Bozón, M V; Delgado, J C; Selvakumar, A; Clavijo, O P; Salazar, M; Ohashi, M; Alosco, S M; Russell, J; Yu, N; Dupont, B; Yunis, E J

    1997-10-01

    Until recently, the majority of HLA class I typing has been performed by serology. Expensive commercial typing trays are frequently used for testing non-Caucasian subjects and new strategies using DNA-based methods have been adopted for improving clinical histocompatibility testing results and adapted as supplements in proficiency testing. A double-blind comparison of the typing of HLA-B specificities in 40 samples was carried out between serology and two polymerase chain reaction (PCR) methods, PCR amplification with sequence-specific primers (PCR-SSP) and PCR amplification and subsequent hybridization with sequence-specific oligonucleotide probes (PCR-SSOP). The results demonstrated 22.5% misassignments of HLA-B antigens by serology. There was complete concordance between the results obtained with the two PCR based typing methods. A second panel of 20 donor samples with incomplete or ambiguous serologic results was analyzed by PCR-SSP and SSOP Both PCR methods identified correctly the HLA-B antigens. Our results suggest that more accurate typing results can be achieved by complementing serologic testing with DNA-based typing techniques. The level of resolution for HLA-B antigen assignment can be obtained by this combination of serology and limited DNA-based typing is equivalent to the HLA-B specificities defined by the WHO-HLA Committee. This level of resolution cannot routinely be achieved in clinical histocompatibility testing or in proficiency testing using serologic reagents only. PMID:9349624

  7. Non-invasive prenatal diagnostic test accuracy for fetal sex using cell-free DNA a review and meta-analysis

    PubMed Central

    2012-01-01

    Background Cell-free fetal DNA (cffDNA) can be detected in maternal blood during pregnancy, opening the possibility of early non-invasive prenatal diagnosis for a variety of genetic conditions. Since 1997, many studies have examined the accuracy of prenatal fetal sex determination using cffDNA, particularly for pregnancies at risk of an X-linked condition. Here we report a review and meta-analysis of the published literature to evaluate the use of cffDNA for prenatal determination (diagnosis) of fetal sex. We applied a sensitive search of multiple bibliographic databases including PubMed (MEDLINE), EMBASE, the Cochrane library and Web of Science. Results Ninety studies, incorporating 9,965 pregnancies and 10,587 fetal sex results met our inclusion criteria. Overall mean sensitivity was 96.6% (95% credible interval 95.2% to 97.7%) and mean specificity was 98.9% (95% CI = 98.1% to 99.4%). These results vary very little with trimester or week of testing, indicating that the performance of the test is reliably high. Conclusions Based on this review and meta-analysis we conclude that fetal sex can be determined with a high level of accuracy by analyzing cffDNA. Using cffDNA in prenatal diagnosis to replace or complement existing invasive methods can remove or reduce the risk of miscarriage. Future work should concentrate on the economic and ethical considerations of implementing an early non-invasive test for fetal sex. PMID:22937795

  8. The use of novel DNA nanotracers to determine groundwater flow paths - a test study at the Grimsel Deep Underground Geothermal (DUG) Laboratory in Switzerland

    NASA Astrophysics Data System (ADS)

    Kittilä, Anniina; Evans, Keith; Puddu, Michela; Mikutis, Gediminas; Grass, Robert N.; Deuber, Claudia; Saar, Martin O.

    2016-04-01

    Groundwater flow in fractured media is heterogeneous and takes place in structures with complex geometry and scale effects, which make the characterization and modeling of the groundwater flow technically challenging. Surface geophysical surveys have limited resolution of permeable structures, and often provide ambiguous results, whereas the interpretation of borehole flow logs to infer hydraulic flow paths within fractured reservoirs is usually non-unique. Nonetheless, knowledge of the hydraulic properties of individual fractures and the role they play in determining the larger-scale flow within the fracture network (i.e. the overall flow conditions) is required in many hydrogeological and geo-engineering situations, such as in geothermal reservoir studies. Tracer tests can overcome some of the aforementioned limitations by providing strong constraints on the geometry and characteristics of flow paths linking boreholes within both porous media and fracture-dominated types of reservoirs. In the case of geothermal reservoirs, tracer tests are often used to provide estimates of the pore/fracture volume swept by flow between injection and production wells. This in turn places constraints on the swept surface area, a parameter that is key for estimating the commercial longevity of the geothermal system. A problem with conventional tracer tests is that the solute species used as the tracer tend to persist in detectable quantities within the reservoir for a long time, thereby impeding repeat tracer tests. DNA nanotracers do not suffer from this problem as they can be designed with a unique signature for each test. DNA nanotracers are environmentally friendly, sub-micron sized silica particles encapsulating small fragments of synthetic DNA which can be fabricated to have a specified, uniquely detectable configuration. For this reason, repeat tracer tests conducted with a differently-encoded DNA fragment to that used in the original will not suffer interference from the

  9. Prevalence of Treponema pallidum DNA among blood donors with two different serologic tests profiles for syphilis in São Paulo, Brazil.

    PubMed

    Ferreira, S C; de Almeida-Neto, C; Nishiya, A S; Di-Lorenzo-Oliveira, C; Ferreira, J E; Alencar, C S; Levi, J E; Salles, N A; Mendrone-Junior, A; Sabino, E C

    2014-05-01

    The presence of Treponema pallidum DNA was assessed by real-time PCR in samples of blood donors with reactive serologic tests for syphilis. Treponema pallidum DNA was detected in two (1·02%) of 197 samples of VDRL>8, EIA+ and FTA-ABS+ donors, and in no sample from 80 VDRL−, EIA+ and FTA-ABS+ donors. Donors VDRL−, EIA+ and FTA-ABS+ lack demonstrable T. pallidum DNA in their blood and are unlike to transmit syphilis. Donors VDRL>8, EIA+ and FTA-ABS+ carry the risk of syphilis infectivity even in concomitance to antibodies detection. Serologic screening for syphilis may still play a role to prevent its transfusion transmission. PMID:24877236

  10. Testing a structural model for viral DNA packaging motor function by optical tweezers measurements, site directed mutagenesis, and molecular dynamics calculations

    NASA Astrophysics Data System (ADS)

    Keller, Nicholas A.; Migliori, Amy D.; Arya, Gaurav; Rao, Venigalla B.; Smith, Douglas E.

    2013-09-01

    Many double-stranded DNA viruses employ a molecular motor to package DNA into preformed capsid shells. Based on structures of phage T4 motor proteins determined by X-ray crystallography and cryo-electron microscopy, Rao, Rossmann and coworkers recently proposed a structural model for motor function. They proposed that DNA is ratcheted by a large conformational change driven by electrostatic interactions between charged residues at an interface between two globular domains of the motor protein. We have conducted experiments to test this model by studying the effect on packaging under applied load of site-directed changes altering these residues. We observe significant impairment of packaging activity including reductions in packaging rate, percent time packaging, and time active under high load. We show that these measured impairments correlate well with alterations in free energies associated with the conformational change predicted by molecular dynamics simulations.

  11. Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

    PubMed Central

    Leontiou, Chrysanthia A.; Hadjidaniel, Michael D.; Mina, Petros; Antoniou, Pavlos; Ioannides, Marios; Patsalis, Philippos C.

    2015-01-01

    Introduction Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Methods Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. Results The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Conclusion Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed

  12. 32P-postlabeling test for covalent DNA binding of chemicals in vivo: application to a variety of aromatic carcinogens and methylating agents.

    PubMed

    Reddy, M V; Gupta, R C; Randerath, E; Randerath, K

    1984-02-01

    Carcinogen--DNA adducts were detected and determined by 32P-postlabeling assay after exposure of mouse or rat tissues in vivo to a total of 28 compounds comprising 7 arylamines and derivatives, 3 azo compounds, 2 nitroaromatics, 12 polycyclic aromatic hydrocarbons, and 4 methylating agents. DNA was isolated from mouse skin, mouse liver, and rat liver after treatment with the individual carcinogens, then digested enzymatically to deoxyribonucleoside 3'-monophosphates, which were converted to 5'-32P-labeled deoxyribonucleoside 3',5'-bisphosphates by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. The nucleotides were resolved by anion-exchange t.l.c. on polyethyleneimine-cellulose and detected by autoradiography. The determination of low levels of DNA binding of the aromatic carcinogens entailed the removal of normal nucleotides prior to the resolution of adduct nucleotides. For this purpose, an alternative procedure employing reversed-phase t.l.c. was devised which offered advantages for the detection of quantitatively minor adducts. The procedures described enabled the detection of 1 aromatic DNA adduct in approximately 10(8) normal nucleotides, while the limit of detection of methylated adducts was 1 adduct in approximately 6 X 10(5) nucleotides. The results show that a great number of carcinogen-DNA adducts of diverse structure are substrates for 32P-labeling by polynucleotide kinase-catalyzed phosphorylation. Because covalent DNA adduct formation in vivo appears to be an essential property of the majority of chemical carcinogens, 32P-postlabeling analysis of carcinogen--DNA adducts in mammalian tissues may serve as a test for the screening of chemicals for potential carcinogenicity. PMID:6697441

  13. Enhancing capacitive DNA biosensor performance by target overhang with application on screening test of HLA-B*58:01 and HLA-B*57:01 genes.

    PubMed

    Thipmanee, Orawan; Numnuam, Apon; Limbut, Warakorn; Buranachai, Chittanon; Kanatharana, Proespichaya; Vilaivan, Tirayut; Hirankarn, Nattiya; Thavarungkul, Panote

    2016-08-15

    A highly sensitive label-free DNA biosensor based on PNA probes immobilized on a gold electrode was used to detect a hybridization event. The effect of a target DNA overhang on the hybridization efficiency was shown to enhance the detected signal and allowed detection at a very low concentration. The sensors performances were investigated with a complementary target that had the same length as the probe, and the signal was compared to the target DNAs with different lengths and overhangs. A longer target DNA overhang was found to provide a better response. When the overhang was on the electrode side the signal enhancement was greater than when the overhang was on the solution side due to the increased thickness of the sensing surface, hence produced a larger capacitance change. Using conformationally constrained acpcPNA probes, double stranded DNA was detected sensitively and specifically without any denaturing step. When two acpcPNA probes were applied for the screening test for the double stranded HLA-B*58:01 and HLA-B*57:01 genes that are highly similar, the method differentiated the two genes in all samples. Both purified and unpurified PCR products gave comparable results. This method would be potentially useful as a rapid screening test without the need for purification and denaturation of the PCR products. PMID:27054813

  14. Human papillomavirus mRNA and DNA testing in women with atypical squamous cells of undetermined significance: A prospective cohort study.

    PubMed

    Thomsen, Louise T; Dehlendorff, Christian; Junge, Jette; Waldstrøm, Marianne; Schledermann, Doris; Frederiksen, Kirsten; Kjaer, Susanne K

    2016-10-15

    In this prospective cohort study, we compared the performance of human papillomavirus (HPV) mRNA and DNA testing of women with atypical squamous cells of undetermined significance (ASC-US) during cervical cancer screening. Using a nationwide Danish pathology register, we identified women aged 30-65 years with ASC-US during 2005-2011 who were tested for HPV16/18/31/33/45 mRNA using PreTect HPV-Proofer (n = 3,226) or for high-risk HPV (hrHPV) DNA using Hybrid Capture 2 (HC2) (n = 9,405) or Linear Array HPV-Genotyping test (LA) (n = 1,533). Women with ≥1 subsequent examination in the register (n = 13,729) were followed for up to 9.5 years for high-grade cervical intraepithelial neoplasia (CIN) or cancer. After 3 years' follow-up, mRNA testing had higher specificity for CIN3 or worse (CIN3+) than HC2 testing (88.1% [95% confidence interval (CI): 86.8-89.6%] versus 59.3% [95% CI: 58.1-60.4%]) and higher positive predictive value (PPV) (38.2% [95% CI: 33.8%-43.1%] versus 19.5% [95% CI: 17.8-20.9%]). However, the sensitivity of mRNA testing was lower than that of HC2 testing (66.7% [95% CI: 59.3-74.5%] versus 97.0% [95% CI: 95.5-98.4%]), and women testing mRNA negative had higher 3-year risk for CIN3+ than those testing HC2 negative (3.2% [95% CI: 2.2-4.2%] versus 0.5% [95% CI: 0.3-0.7%]). Patterns were similar after 18 months and 5 years'; follow-up; for CIN2+ and cancer as outcomes; across all age groups; and when comparing mRNA testing to hrHPV DNA testing using LA. In conclusion, the HPV16/18/31/33/45 mRNA test is not optimal for ASC-US triage due to its low sensitivity and the substantial risk for precancer following a negative test. PMID:27004595

  15. Emerging stool-based and blood-based non-invasive DNA tests for colorectal cancer screening: the importance of cancer prevention in addition to cancer detection.

    PubMed

    Pickhardt, Perry J

    2016-08-01

    Colorectal cancer (CRC) screening can be undertaken utilizing a variety of distinct approaches, which provides both opportunities and confusion. Traditionally, there has often been a trade-off between the degree of invasiveness of a screening test and its ability to prevent cancer, with fecal occult blood testing (FOBT) and optical colonoscopy (OC) at each end of the spectrum. CT colonography (CTC), although currently underutilized for CRC screening, represents an exception since it is only minimally invasive, yet provides accurate evaluation for advanced adenomas. More recently, the FDA approved a multi-target stool DNA test (Cologuard) and a blood-based test (Epi proColon) for average-risk CRC screening. This commentary will provide an overview of these two new non-invasive tests, including the clinical indications, mechanism of action, and diagnostic performance. Relevance to radiology practice, including a comparison with CTC, will also be discussed. PMID:27259335

  16. DNA methylation based testing of 418 patients suspected of having Prader-Willi syndrome (PWS) and tentative localization of the 15q11-13 imprinting center

    SciTech Connect

    Horsthemke, B.; Dittrich, B.; Buiting, K.

    1994-09-01

    Using a test based on parent-of-origin specific DNA methylation at the D15S63 (PW71) locus, we studied 358 patients (aged 1-33 years) for diagnostic confirmation of PWS and 60 infants (aged 0-12 months) with severe hypotonia of unknown origin. 57/358 patients were examined personally. 28/57 patients showed typical clinical signs of PWS and lacked the paternal PW71 band. 29-57 patients did not fulfill the diagnostic criteria for PWS and had a normal methylation pattern. This suggests that the test detects most if not all patients with typical PWS. 301/358 samples were sent to us from outside. The test confirmed the diagnosis in 105/301 patients. Most of the other 196 patients lacked neonatal hypotonia and hypogonadism. On the other hand, 27/60 hypotonic infants tested positive for PWS. These results indicate that PWS is often not recognized in infants and wrongly diagnosed in obese children and adults. We propose to perform the PW71 methylation test in every newborn with severe hypotonia of unknown origin to avoid unnecessary diagnostic procedures. In the course of diagnostic testing we and others have identified PWS and Angelman syndrome (AS) patients with apparently normal chromosomes of biparental inheritance, but abnormal DNA methylation and gene expression. The identification of microdeletions in some of these patients suggests the existence of an imprinting center within 30 kb proximal of SNRPN, which regulates the chromatin structure, DNA methylation and gene expression. We propose that the paternal copy of 15q11-13 has a euchromatoid structure, from which the PWS genes are transcribed, and that the maternal copy has a heterochromatoid structure from which the AS gene is transcribed. Depending on the parental origin of the mutation, both chromosomes of a patient have a heterochromatoid domain, which silences the PWS genes, or a euchromatoid domain, which silences the AS gene.

  17. Long-Term Follow-up of HPV Infection Using Urine and Cervical Quantitative HPV DNA Testing

    PubMed Central

    Vorsters, Alex; Van Keer, Severien; Biesmans, Samantha; Hens, Annick; De Coster, Ilse; Goossens, Herman; Ieven, Margareta; Van Damme, Pierre

    2016-01-01

    The link between infection with high-risk human papillomavirus (hrHPV) and cervical cancer has been clearly demonstrated. Virological end-points showing the absence of persistent HPV infection are now accepted as a way of monitoring the impact of prophylactic vaccination programs and therapeutic vaccine trials. This study investigated the use of urine samples, which can be collected by self-sampling at home, instead of cervical samples for follow-up of an HPV intervention trial. Eighteen initially HPV DNA-positive women participating in an HPV therapeutic vaccine trial were monitored during a three-year follow-up period. A total of 172 urine samples and 85 cervical samples were collected. We obtained a paired urine sample for each of the 85 cervical samples by recovering urine samples from six monthly gynaecological examinations. We performed a small pilot study in which the participating women used a urine collection device at home and returned their urine sample to the laboratory by mail. All samples were analyzed using quantitative real-time HPV DNA PCR. A good association (κ value of 0.65) was found between the presence of HPV DNA in urine and a subsequent cervical sample. Comparisons of the number of HPV DNA copies in urine and paired cervical samples revealed a significant Spearman rho of 0.676. This correlation was superior in women with severe lesions. The HPV DNA results of the small pilot study based on self-collected urine samples at home are consistent with previous and subsequent urine and/or cervical results. We demonstrated that urine sampling may be a valid alternative to cervical samples for the follow-up of HPV intervention trials or programs. The potential clinical value of urine viral load monitoring should be further investigated. PMID:27196899

  18. Long-Term Follow-up of HPV Infection Using Urine and Cervical Quantitative HPV DNA Testing.

    PubMed

    Vorsters, Alex; Van Keer, Severien; Biesmans, Samantha; Hens, Annick; De Coster, Ilse; Goossens, Herman; Ieven, Margareta; Van Damme, Pierre

    2016-01-01

    The link between infection with high-risk human papillomavirus (hrHPV) and cervical cancer has been clearly demonstrated. Virological end-points showing the absence of persistent HPV infection are now accepted as a way of monitoring the impact of prophylactic vaccination programs and therapeutic vaccine trials. This study investigated the use of urine samples, which can be collected by self-sampling at home, instead of cervical samples for follow-up of an HPV intervention trial. Eighteen initially HPV DNA-positive women participating in an HPV therapeutic vaccine trial were monitored during a three-year follow-up period. A total of 172 urine samples and 85 cervical samples were collected. We obtained a paired urine sample for each of the 85 cervical samples by recovering urine samples from six monthly gynaecological examinations. We performed a small pilot study in which the participating women used a urine collection device at home and returned their urine sample to the laboratory by mail. All samples were analyzed using quantitative real-time HPV DNA PCR. A good association (κ value of 0.65) was found between the presence of HPV DNA in urine and a subsequent cervical sample. Comparisons of the number of HPV DNA copies in urine and paired cervical samples revealed a significant Spearman rho of 0.676. This correlation was superior in women with severe lesions. The HPV DNA results of the small pilot study based on self-collected urine samples at home are consistent with previous and subsequent urine and/or cervical results. We demonstrated that urine sampling may be a valid alternative to cervical samples for the follow-up of HPV intervention trials or programs. The potential clinical value of urine viral load monitoring should be further investigated. PMID:27196899

  19. Ancient DNA

    PubMed Central

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    In the past two decades, ancient DNA research has progressed from the retrieval of small fragments of mitochondrial DNA from a few late Holocene specimens, to large-scale studies of ancient populations, phenotypically important nuclear loci, and even whole mitochondrial genome sequences of extinct species. However, the field is still regularly marred by erroneous reports, which underestimate the extent of contamination within laboratories and samples themselves. An improved understanding of these processes and the effects of damage on ancient DNA templates has started to provide a more robust basis for research. Recent methodological advances have included the characterization of Pleistocene mammal populations and discoveries of DNA preserved in ancient sediments. Increasingly, ancient genetic information is providing a unique means to test assumptions used in evolutionary and population genetics studies to reconstruct the past. Initial results have revealed surprisingly complex population histories, and indicate that modern phylogeographic studies may give misleading impressions about even the recent evolutionary past. With the advent and uptake of appropriate methodologies, ancient DNA is now positioned to become a powerful tool in biological research and is also evolving new and unexpected uses, such as in the search for extinct or extant life in the deep biosphere and on other planets. PMID:15875564

  20. Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR.

    PubMed

    Longin, Cédric; Guilloux-Benatier, Michèle; Alexandre, Hervé

    2016-01-01

    Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage. PMID:27313572

  1. Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR

    PubMed Central

    Longin, Cédric; Guilloux-Benatier, Michèle; Alexandre, Hervé

    2016-01-01

    Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage. PMID:27313572

  2. Predicting adaptation to presymptomatic DNA testing for late onset disorders: who will experience distress? Rotterdam Leiden Genetics Workgroup.

    PubMed Central

    DudokdeWit, A C; Tibben, A; Duivenvoorden, H J; Niermeijer, M F; Passchier, J

    1998-01-01

    The first comparative study on predicting post-test distress (conceptualised by intrusion and avoidance, measured with the Impact of Event Scale) after presymptomatic genetic testing for Huntington's disease (HD, n=25), cancer syndromes (familial adenomatous polyposis (FAP, n=23)), and hereditary breast and ovarian cancer (HBOC, n=10) is reported. The variables with the highest predictive potential of post-test distress are presented. Participants who were depressed before the test were more distressed after testing, but we found that those who were anxious before the test were less distressed, that is, had less intrusive thoughts post-test. Other factors associated with a higher level of post-test intrusion were gender (being a woman), having children, and pre-test intrusion. Religion and being at risk for HBOC were associated with less post-test intrusion. Participants who showed avoidance behaviour before the test and those who had many people available for support showed more avoidance behaviour post-test. The test result did not additionally contribute to post-test distress. The prima facie simple notion that the test result, as such, determines the distress experienced seems to be a misrepresentation of the complex reality. PMID:9733033

  3. DNA-based diagnostic tests for Salmonella species targeting agfA, the structural gene for thin, aggregative fimbriae.

    PubMed Central

    Doran, J L; Collinson, S K; Burian, J; Sarlós, G; Todd, E C; Munro, C K; Kay, C M; Banser, P A; Peterkin, P I; Kay, W W

    1993-01-01

    Salmonella enteritidis 27655-3b and a few diarrheagenic Escherichia coli strains produce morphologically and antigenically related, thin, aggregative fimbriae, collectively named GVVPQ fimbriae (S. K. Collinson, L. Emödy, T. J. Trust, and W. W. Kay, J. Bacteriol. 174:4490-4495, 1992). To determine whether GVVPQ fimbriae are common to Salmonella spp. and other enteropathogenic members of the family Enterobacteriaceae, 113 isolates were phenotypically screened for Congo red binding and aggregative colony morphology. Presumptive positive and representative negative strains were examined by Western blotting (immunoblotting) by using antiserum to SEF 17, the native GVVPQ fimbria of S. enteritidis. Only four S. enteritidis strains and six E. coli isolates possessed substantial amounts of GVVPQ fimbriae after 24 h of incubation on T medium. Following 5 days of incubation, 56 of 93 Salmonella isolates (60%) and 1 of 7 additional E. coli clinical isolates possessed detectable levels of GVVPQ fimbriae. Since variable expression of GVVPQ fimbriae was observed among Salmonella isolates and some E. coli strains produced scant amounts, as revealed by immunoelectron microscopy, the ability to produce these fimbriae was evaluated by genotypic screening. The structural gene for the SEF 17 fimbrin, agfA, was amplified by the polymerase chain reaction, cloned, and sequenced to provide a characterized DNA probe. An agfA DNA fragment hybridized strongly to 603 of 604 (99.8%) Salmonella isolates but very weakly to 31 of 266 other members of the family Enterobacteriaceae including 26 of 137 E. coli strains, 3 of 14 Citrobacter spp., and single isolates of Shigella sonnei and Enterobacter cloacae. The agfA DNA probe proved to be a valuable diagnostic tool for Salmonella isolates arrayed on hydrophobic grid membrane filters. Unique agfA sequences were targeted in the development of a polymerase chain reaction assay specific for Salmonella spp. Images PMID:8104955

  4. A Transcontinental Challenge — A Test of DNA Barcode Performance for 1,541 Species of Canadian Noctuoidea (Lepidoptera)

    PubMed Central

    Zahiri, Reza; Lafontaine, J. Donald; Schmidt, B. Christian; deWaard, Jeremy R.; Zakharov, Evgeny V.; Hebert, Paul D. N.

    2014-01-01

    This study provides a first, comprehensive, diagnostic use of DNA barcodes for the Canadian fauna of noctuoids or “owlet” moths (Lepidoptera: Noctuoidea) based on vouchered records for 1,541 species (99.1% species coverage), and more than 30,000 sequences. When viewed from a Canada-wide perspective, DNA barcodes unambiguously discriminate 90% of the noctuoid species recognized through prior taxonomic study, and resolution reaches 95.6% when considered at a provincial scale. Barcode sharing is concentrated in certain lineages with 54% of the cases involving 1.8% of the genera. Deep intraspecific divergence exists in 7.7% of the species, but further studies are required to clarify whether these cases reflect an overlooked species complex or phylogeographic variation in a single species. Non-native species possess higher Nearest-Neighbour (NN) distances than native taxa, whereas generalist feeders have lower NN distances than those with more specialized feeding habits. We found high concordance between taxonomic names and sequence clusters delineated by the Barcode Index Number (BIN) system with 1,082 species (70%) assigned to a unique BIN. The cases of discordance involve both BIN mergers and BIN splits with 38 species falling into both categories, most likely reflecting bidirectional introgression. One fifth of the species are involved in a BIN merger reflecting the presence of 158 species sharing their barcode sequence with at least one other taxon, and 189 species with low, but diagnostic COI divergence. A very few cases (13) involved species whose members fell into both categories. Most of the remaining 140 species show a split into two or three BINs per species, while Virbia ferruginosa was divided into 16. The overall results confirm that DNA barcodes are effective for the identification of Canadian noctuoids. This study also affirms that BINs are a strong proxy for species, providing a pathway for a rapid, accurate estimation of animal diversity. PMID

  5. Testing.

    ERIC Educational Resources Information Center

    Killoran, James, Ed.

    1984-01-01

    This journal issue addresses the issue of testing in the social studies classroom. The first article, "The Role of Testing" (Bragaw), focuses on the need for tests to reflect the objectives of the study completed. The varying functions of pop quizzes, weekly tests, and unit tests are explored. "Testing Thinking Processes" (Killoran, Zimmer, and…

  6. Testing DNA Barcode Performance in 1000 Species of European Lepidoptera: Large Geographic Distances Have Small Genetic Impacts

    PubMed Central

    Huemer, Peter; Mutanen, Marko; Sefc, Kristina M.; Hebert, Paul D. N.

    2014-01-01

    This study examines the performance of DNA barcodes (mt cytochrome c oxidase 1 gene) in the identification of 1004 species of Lepidoptera shared by two localities (Finland, Austria) that are 1600 km apart. Maximum intraspecific distances for the pooled data were less than 2% for 880 species (87.6%), while deeper divergence was detected in 124 species. Despite such variation, the overall DNA barcode library possessed diagnostic COI sequences for 98.8% of the taxa. Because a reference library based on Finnish specimens was highly effective in identifying specimens from Austria, we conclude that barcode libraries based on regional sampling can often be effective for a much larger area. Moreover, dispersal ability (poor, good) and distribution patterns (disjunct, fragmented, continuous, migratory) had little impact on levels of intraspecific geographic divergence. Furthermore, the present study revealed that, despite the intensity of past taxonomic work on European Lepidoptera, nearly 20% of the species shared by Austria and Finland require further work to clarify their status. Particularly discordant BIN (Barcode Index Number) cases should be checked to ascertain possible explanatory factors such as incorrect taxonomy, hybridization, introgression, and Wolbachia infections. PMID:25541991

  7. Test of simultaneous synthetic DNA tracer injections for the estimation of the englacial and subglacial drainage system structure of Storglaciären, northern Sweden

    NASA Astrophysics Data System (ADS)

    Dahlke, H. E.; Leung, S.; Lyon, S. W.; Sharma, A. N.; Walter, M. T.; Williamson, A.

    2013-12-01

    Storglaciären glacier, located in the sub-arctic Tarfala catchment, in northern Sweden is one of the world's longest continuously monitored glaciers which provides a unique research platform for the long-term assessment of glacier and ice sheet processes. For example, small mountain glacier hydrological knowledge of the subglacial water distribution at the ice-bed interface has been applied to ice sheets to predict basal sliding processes. Basal sliding promoted by hydraulic jacking is an important glacial-velocity control that is dependent on the subglacial flow pathways' morphology. Thus, understanding subglacial water distribution and drainage system structure and morphology is crucial for modeling ice masses' flow. In order to estimate subglacial drainage system structure and morphology dye tracing experiments are widely employed. Tracer experiments provide quantitative parameters for any input location including tracer transit velocity, dispersivity, recovery and storage. However, spatial data coverage is limited by the finite number of tracers available for simultaneous tracing. In the presented study we test the use of synthetic DNA tracers for the assessment of the englacial and subglacial drainage system structure of Storglaciären. The synthetic DNA tracer is composed of polylactic acid (PLA) microspheres into which short strands of synthetic DNA and paramagnetic iron oxide nanoparticles are incorporated (Sharma et al., 2012, Environmental Science & Technology). Because the DNA sequences can be randomly combined the synthetic DNA tracer provides an enormous number of unique tracers (approximately 1.61 x 1060). Thus, these synthetic tracers have the advantage that multiple (>10) experiments can be conducted simultaneously, allowing a greater information gain within a shorter measurement period. Quantities of a certain DNA strand can be detected using biotechnology tools such as polymerase chain reaction (PCR) and quantitative PCR (qPCR). During the 2013

  8. Application of DNA Aptamers and Quantum Dots to Lateral Flow Test Strips for Detection of Foodborne Pathogens with Improved Sensitivity versus Colloidal Gold

    PubMed Central

    Bruno, John G.

    2014-01-01

    Preliminary studies aimed at improving the sensitivity of foodborne pathogen detection via lateral flow (LF) test strips by use of high affinity DNA aptamers for capture and reporter functions when coupled to red-emitting quantum dots (Qdot 655) are reported. A variety of DNA aptamers developed against Escherichia coli, Listeria monocytogenes, and Salmonella enterica were paired in capture and reporter combinations to determine which yielded the strongest detection of their cognate bacteria using a colloidal gold screening system. Several promising sandwich combinations were identified for each of the three bacterial LF strip systems. The best E. coli aptamer-LF system was further studied and yielded a visible limit of detection (LOD) of ~3,000 E. coli 8739 and ~6,000 E. coli O157:H7 in buffer. These LODs were reduced to ~300–600 bacterial cells per test respectively by switching to a Qdot 655 aptamer-LF system. Novel aspects of these assays such as the use of high levels of detergents to avoid quantum dot agglutination and enhance migration in analytical membranes, identification of optimal analytical membrane types, UV-immobilization of capture aptamers, and novel dual biotin/digoxigenin-end labeled aptamer streptavidin-colloidal gold or -Qdot 655 conjugates plus anti-digoxigenin antibody control lines are also discussed. In general, this work provides proof-of-principle for highly sensitive aptamer-Qdot LF strip assays for rapid foodborne pathogen detection. PMID:25437803

  9. Application of DNA Aptamers and Quantum Dots to Lateral Flow Test Strips for Detection of Foodborne Pathogens with Improved Sensitivity versus Colloidal Gold.

    PubMed

    Bruno, John G

    2014-01-01

    Preliminary studies aimed at improving the sensitivity of foodborne pathogen detection via lateral flow (LF) test strips by use of high affinity DNA aptamers for capture and reporter functions when coupled to red-emitting quantum dots (Qdot 655) are reported. A variety of DNA aptamers developed against Escherichia coli, Listeria monocytogenes, and Salmonella enterica were paired in capture and reporter combinations to determine which yielded the strongest detection of their cognate bacteria using a colloidal gold screening system. Several promising sandwich combinations were identified for each of the three bacterial LF strip systems. The best E. coli aptamer-LF system was further studied and yielded a visible limit of detection (LOD) of ~3,000 E. coli 8739 and ~6,000 E. coli O157:H7 in buffer. These LODs were reduced to ~300-600 bacterial cells per test respectively by switching to a Qdot 655 aptamer-LF system. Novel aspects of these assays such as the use of high levels of detergents to avoid quantum dot agglutination and enhance migration in analytical membranes, identification of optimal analytical membrane types, UV-immobilization of capture aptamers, and novel dual biotin/digoxigenin-end labeled aptamer streptavidin-colloidal gold or -Qdot 655 conjugates plus anti-digoxigenin antibody control lines are also discussed. In general, this work provides proof-of-principle for highly sensitive aptamer-Qdot LF strip assays for rapid foodborne pathogen detection. PMID:25437803

  10. Clinical Significance of an HPV DNA Chip Test with Emphasis on HPV-16 and/or HPV-18 Detection in Korean Gynecological Patients

    PubMed Central

    Yeo, Min-Kyung; Lee, Ahwon; Hur, Soo Young; Park, Jong Sup

    2016-01-01

    Background: Human papillomavirus (HPV) is a major risk factor for cervical cancer. Methods: We evaluated the clinical significance of the HPV DNA chip genotyping assay (MyHPV chip, Mygene Co.) compared with the Hybrid Capture 2 (HC2) chemiluminescent nucleic acid hybridization kit (Digene Corp.) in 867 patients. Results: The concordance rate between the MyHPV chip and HC2 was 79.4% (kappa coefficient, κ = 0.55). The sensitivity and specificity of both HPV tests were very similar (approximately 85% and 50%, respectively). The addition of HPV result (either MyHPV chip or HC2) to cytology improved the sensitivity (95%, each) but reduced the specificity (approximately 30%, each) compared with the HPV test or cytology alone. Based on the MyHPV chip results, the odds ratio (OR) for ≥ high-grade squamous intraepithelial lesions (HSILs) was 9.9 in the HPV-16/18 (+) group and 3.7 in the non-16/18 high-risk (HR)-HPV (+) group. Based on the HC2 results, the OR for ≥ HSILs was 5.9 in the HR-HPV (+) group. When considering only patients with cytological diagnoses of “negative for intraepithelial lesion or malignancy” and “atypical squamous cell or atypical glandular cell,” based on the MyHPV chip results, the ORs for ≥ HSILs were 6.8 and 11.7, respectively, in the HPV-16/18 (+) group. Conclusions: The sensitivity and specificity of the MyHPV chip test are similar to the HC2. Detecting HPV-16/18 with an HPV DNA chip test, which is commonly used in many Asian countries, is useful in assessing the risk of high-grade cervical lesions. PMID:27345180

  11. COLD-PCR enriches low-level variant DNA sequences and increases the sensitivity of genetic testing.

    PubMed

    Castellanos-Rizaldos, Elena; Milbury, Coren A; Guha, Minakshi; Makrigiorgos, G Mike

    2014-01-01

    Detection of low-level mutations is important for cancer biomarker and therapy targets discovery, but reliable detection remains a technical challenge. The newly developed method of CO-amplification at Lower Denaturation temperature PCR (COLD-PCR) helps to circumvent this issue. This PCR-based technology preferentially enriches minor known or unknown variants present in samples with a high background of wild type DNA which often hampers the accurate identification of these minority alleles. This is a simple process that consists of lowering the temperature at the denaturation step during the PCR-cycling protocol (critical denaturation temperature, T c) and inducing DNA heteroduplexing during an intermediate step. COLD-PCR in its simplest forms does not need additional reagents or specific instrumentation and thus, can easily replace conventional PCR and at the same time improve the mutation detection sensitivity limit of downstream technologies. COLD-PCR can be applied in two basic formats: fast-COLD-PCR that can enrich T m-reducing mutations and full-COLD-PCR that can enrich all mutations, though it requires an intermediate cross-hybridization step that lengthens the thermocycling program. An improved version of full-COLD-PCR (improved and complete enrichment, ice-COLD-PCR) has also been described. Finally, most recently, we developed yet another form of COLD-PCR, temperature-tolerant-COLD-PCR, which gradually increases the denaturation temperature during the COLD-PCR reaction, enriching diverse targets using a single cycling program. This report describes practical considerations for application of fast-, full-, ice-, and temperature-tolerant-COLD-PCR for enrichment of mutations prior to downstream screening. PMID:24259002

  12. Mitochondrial DNA variation in rainbow trout (Oncorhynchus mykiss) across its native range: testing biogeographical hypotheses and their relevance to conservation.

    PubMed

    McCusker, M R; Parkinson, E; Taylor, E B

    2000-12-01

    North-western North America has been repeatedly glaciated over most of the past two million years, with the most recent glaciation occurring between 60 000 and 10 000 years ago. Intraspecific genetic variation in many species has been shaped by where they survived glaciation and what postglacial recolonization routes were used. In this study, molecular techniques were used to investigate biogeographical, taxonomic and conservation issues in rainbow trout, Oncorhynchus mykiss. Mitochondrial DNA (mtDNA) variation was assessed using a restriction fragment length polymorphism (RFLP) analysis, focusing mainly on the previously understudied northern extent of the species' range. Two phylogenetically distinct mitochondrial lineages were found that differed from each other by up to 1.8% in sequence. Although the geographical distributions of the two clades overlap extensively, diversity and distributional analyses strongly suggest that trout survived glaciation in both coastal and inland refugia followed by postglacial gene flow and secondary contact. Postglacial dispersal into British Columbia most likely occurred from the Queen Charlotte Islands and the Columbia River. Although trout most likely also survived glaciation along the coast of Washington, Oregon and California, as well as near the Bering Strait, evidence suggests that dispersal into British Columbia from these areas was limited. Sequence analysis of mitochondrial haplotypes revealed higher diversity in California than in the northern part of the species' range, indicating an ancient presence of the species in the south. Phylogeographic divergence probably predates adaptive variation in the species as suggested by evidence for parallel evolution of life history types across the range of O. mykiss. PMID:11123621

  13. Cleaving DNA with DNA

    NASA Astrophysics Data System (ADS)

    Carmi, Nir; Balkhi, Shameelah R.; Breaker, Ronald R.

    1998-03-01

    A DNA structure is described that can cleave single-stranded DNA oligonucleotides in the presence of ionic copper. This ``deoxyribozyme'' can self-cleave or can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. Bimolecular deoxyribozyme-mediated strand scission proceeds with a kobs of 0.2 min-1, whereas the corresponding uncatalyzed reaction could not be detected. The duplex and triplex recognition domains can be altered, making possible the targeted cleavage of single-stranded DNAs with different nucleotide sequences. Several small synthetic DNAs were made to function as simple ``restriction enzymes'' for the site-specific cleavage of single-stranded DNA.

  14. BRCA1/2 testing in newly diagnosed breast and ovarian cancer patients without prior genetic counselling: the DNA-BONus study.

    PubMed

    Høberg-Vetti, Hildegunn; Bjorvatn, Cathrine; Fiane, Bent E; Aas, Turid; Woie, Kathrine; Espelid, Helge; Rusken, Tone; Eikesdal, Hans Petter; Listøl, Wenche; Haavind, Marianne T; Knappskog, Per M; Haukanes, Bjørn Ivar; Steen, Vidar M; Hoogerbrugge, Nicoline

    2016-06-01

    Germline BRCA1/2 testing of breast and ovarian cancer patients is growing rapidly as the result affects both treatment and cancer prevention in patients and relatives. Through the DNA-BONus study we offered BRCA1/2 testing and familial risk assessment to all new patients with breast (N=893) or ovarian (N=122) cancer diagnosed between September 2012 and April 2015, irrespective of family history or age, and without prior face-to-face genetic counselling. BRCA1/2 testing was accepted by 405 (45.4%) and 83 (68.0%) of the patients with breast or ovarian cancer, respectively. A pathogenic BRCA1/2 variant was found in 7 (1.7%) of the breast cancer patients and 19 (22.3%) of the ovarian cancer patients. In retrospect, all BRCA1/2 mutation carriers appeared to fulfill current criteria for BRCA1/2 testing. Hospital Anxiety and Depression Scale (HADS) scores showed that the mean levels of anxiety and depression were comparable to those reported for breast and gynecological cancer patients in general, with a significant drop in anxiety symptoms during a 6-month follow-up period, during which the test result was forwarded to the patients. These results show that BRCA1/2 testing is well accepted in newly diagnosed breast and ovarian cancer patients. Current test criteria based on age and family history are sufficient to identify most BRCA1/2 mutation carriers among breast cancer patients. We recommend germline BRCA1/2 testing in all patients with epithelial ovarian cancer because of the high prevalence of pathogenic BRCA1/2 variants. PMID:26350514

  15. Testing the validity of Northern European species in the Chrysis ignita species group (Hymenoptera: Chrysididae) with DNA barcoding.

    PubMed

    Soon, Villu; Budrys, Eduardas; Orlovskytė, Svetlana; Paukkunen, Juho; Odegaard, Frode; Ljubomirov, Toshko; Saarma, Urmas

    2014-01-01

    Containing more than a hundred species, the Chrysis ignita species group is the largest and one of the most taxonomically challenging groups in its genus. It has not been possible to resolve the taxonomy of the group using traditional methods due to the lack of robust diagnostic morphological characters. Here we present the results of a molecular analysis designed to delimit species in the Chrysis ignita group for the first time; using mitochondrial sequence data for 364 in-group specimens consisting of all 18 species known to occur in Northern Europe. Two mitochondrial loci were analysed: a COI gene fragment, and a continuous DNA sequence consisting of 16S rRNA, tRNAVal, 12S rRNA and ND4. Two approaches were employed for delimiting species: (1) genetic distance analysis based on the standard COI barcode sequences and; (2) phylogenetic analysis of the COI fragment together with rRNA genes. Both analyses yielded trees with similar topology, but support values for nodes were higher using the second approach. Fifteen species were distinguished in all analyses: Chrysis angustula Schenck, 1856, C. brevitarsis Thomson, 1870, C. clarinicollis Linsenmaier, 1951, C. corusca Valkeila, 1971, C. fulgida Linnaeus, 1761, C. ignita (Linnaeus, 1758), C. impressa Schenck, 1856, C. iris Christ, 1791, C. leptomandibularis Niehuis, 2000, C. longula Abeille de Perrin, 1879, C. ruddii Shuckard, 1837, C. schencki Linsenmaier, 1968, C. subcoriacea Linsenmaier, 1959, C. terminata Dahlbom, 1854 and C. vanlithi Linsenmaier, 1959. The specific status of C. mediata Linsenmaier, 1951 and C. solida Haupt, 1957 was not resolved. Included unidentified specimens grouped in three clusters, two of which are distinctly delimited and apparently represent cryptic species. The specific status of the unidentified samples in the third cluster remained unclear. Moreover, our data suggest the existence of additional cryptic species currently lumped under the names C. pseudobrevitarsis Linsenmaier

  16. Cross-Institute Evaluations of Inhibitor-Resistant PCR Reagents for Direct Testing of Aerosol and Blood Samples Containing Biological Warfare Agent DNA

    PubMed Central

    Minogue, Timothy D.; Rachwal, Phillip A.; Trombley Hall, Adrienne; Koehler, Jeffery W.

    2014-01-01

    Rapid pathogen detection is crucial for the timely introduction of therapeutics. Two groups (one in the United Kingdom and one in the United States) independently evaluated inhibitor-resistant PCR reagents for the direct testing of substrates. In the United Kingdom, a multiplexed Bacillus anthracis (target) and Bacillus subtilis (internal-control) PCR was used to evaluate 4 reagents against 5 PCR inhibitors and down-selected the TaqMan Fast Virus 1-Step master mix (Life Technologies Inc.). In the United States, four real-time PCR assays (targeting B. anthracis, Brucella melitensis, Venezuelan equine encephalitis virus [VEEV], and Orthopoxvirus spp.) were used to evaluate 5 reagents (plus the Fast Virus master mix) against buffer, blood, and soil samples and down-selected the KAPA Blood Direct master mix (KAPA Biosystems Inc.) with added Platinum Taq (Life Technologies). The down-selected reagents underwent further testing. In the United Kingdom experiments, both reagents were tested against seven contrived aerosol collector samples containing B. anthracis Ames DNA and B. subtilis spores from a commercial formulation (BioBall). In PCR assays with reaction mixtures containing 40% crude sample, an airfield-collected sample induced inhibition of the B. subtilis PCR with the KAPA reagent and complete failure of both PCRs with the Fast Virus reagent. However, both reagents allowed successful PCR for all other samples—which inhibited PCRs with a non-inhibitor-resistant reagent. In the United States, a cross-assay limit-of-detection (LoD) study in blood was conducted. The KAPA Blood Direct reagent allowed the detection of agent DNA (by four PCRs) at higher concentrations of blood in the reaction mixture (2.5%) than the Fast Virus reagent (0.5%), although LoDs differed between assays and reagent combinations. Across both groups, the KAPA Blood Direct reagent was determined to be the optimal reagent for inhibition relief in PCR. PMID:24334660

  17. Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol

    PubMed Central

    Elbrecht, Vasco; Leese, Florian

    2015-01-01

    Metabarcoding is an emerging genetic tool to rapidly assess biodiversity in ecosystems. It involves high-throughput sequencing of a standard gene from an environmental sample and comparison to a reference database. However, no consensus has emerged regarding laboratory pipelines to screen species diversity and infer species abundances from environmental samples. In particular, the effect of primer bias and the detection limit for specimens with a low biomass has not been systematically examined, when processing samples in bulk. We developed and tested a DNA metabarcoding protocol that utilises the standard cytochrome c oxidase subunit I (COI) barcoding fragment to detect freshwater macroinvertebrate taxa. DNA was extracted in bulk, amplified in a single PCR step, and purified, and the libraries were directly sequenced in two independent MiSeq runs (300-bp paired-end reads). Specifically, we assessed the influence of specimen biomass on sequence read abundance by sequencing 31 specimens of a stonefly species with known haplotypes spanning three orders of magnitude in biomass (experiment I). Then, we tested the recovery of 52 different freshwater invertebrate taxa of similar biomass using the same standard barcoding primers (experiment II). Each experiment was replicated ten times to maximise statistical power. The results of both experiments were consistent across replicates. We found a distinct positive correlation between species biomass and resulting numbers of MiSeq reads. Furthermore, we reliably recovered 83% of the 52 taxa used to test primer bias. However, sequence abundance varied by four orders of magnitudes between taxa despite the use of similar amounts of biomass. Our metabarcoding approach yielded reliable results for high-throughput assessments. However, the results indicated that primer efficiency is highly species-specific, which would prevent straightforward assessments of species abundance and biomass in a sample. Thus, PCR-based metabarcoding

  18. Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass--Sequence Relationships with an Innovative Metabarcoding Protocol.

    PubMed

    Elbrecht, Vasco; Leese, Florian

    2015-01-01

    Metabarcoding is an emerging genetic tool to rapidly assess biodiversity in ecosystems. It involves high-throughput sequencing of a standard gene from an environmental sample and comparison to a reference database. However, no consensus has emerged regarding laboratory pipelines to screen species diversity and infer species abundances from environmental samples. In particular, the effect of primer bias and the detection limit for specimens with a low biomass has not been systematically examined, when processing samples in bulk. We developed and tested a DNA metabarcoding protocol that utilises the standard cytochrome c oxidase subunit I (COI) barcoding fragment to detect freshwater macroinvertebrate taxa. DNA was extracted in bulk, amplified in a single PCR step, and purified, and the libraries were directly sequenced in two independent MiSeq runs (300-bp paired-end reads). Specifically, we assessed the influence of specimen biomass on sequence read abundance by sequencing 31 specimens of a stonefly species with known haplotypes spanning three orders of magnitude in biomass (experiment I). Then, we tested the recovery of 52 different freshwater invertebrate taxa of similar biomass using the same standard barcoding primers (experiment II). Each experiment was replicated ten times to maximise statistical power. The results of both experiments were consistent across replicates. We found a distinct positive correlation between species biomass and resulting numbers of MiSeq reads. Furthermore, we reliably recovered 83% of the 52 taxa used to test primer bias. However, sequence abundance varied by four orders of magnitudes between taxa despite the use of similar amounts of biomass. Our metabarcoding approach yielded reliable results for high-throughput assessments. However, the results indicated that primer efficiency is highly species-specific, which would prevent straightforward assessments of species abundance and biomass in a sample. Thus, PCR-based metabarcoding

  19. Preparing for presymptomatic DNA testing for early onset Alzheimer's disease/cerebral haemorrhage and hereditary Pick disease.

    PubMed Central

    Tibben, A; Stevens, M; de Wert, G M; Niermeijer, M F; van Duijn, C M; van Swieten, J C

    1997-01-01

    The acceptability of presymptomatic testing in 21 people at 50% risk for the APP-692 mutation causing presenile Alzheimer's disease or cerebral haemorrhage resulting from cerebral amyloid angiopathy (FAD-CH), and in 43 people at 50% risk for hereditary Pick disease (HPD) was assessed. Neither group differed in demographic variables. Thirty-nine people (64%) in the whole group would request presymptomatic testing if it were clinically available, although two-thirds did not yet feel ready to take it. The most important reasons in the HPD and FAD-CH group for taking the test were: to further basic research (42% and 47%, respectively), informing children (47% and 50%, respectively), future planning (29% and 47%, respectively), and relieving uncertainty (46% and 27%, respectively). The most commonly cited effect of an unfavourable test result concerned increasing problems for spouses (75% and 76%, respectively) and children (61% and 57%, respectively). Most respondents denied that an unfavourable result would have adverse effects on personal mood or relationship. One-third of all respondents favoured prenatal testing where one of the parents had an increased risk for HPD or FAD-CH. Participants would encourage their offspring to have the test before starting a relationship (35%) and before family planning (44%). Thirty-seven percent of the respondents would encourage their children to opt for prenatal diagnosis. People at risk for HPD were significantly more preoccupied with the occurrence of potential symptoms in themselves, compared with those at risk for FAD-CH, reflecting the devastating impact that disinhibition in the affected patient has on the family. Our findings underline the need for adequate counselling and the availability of professional and community resources to deal with the impact of test results in subjects and their relatives. PMID:9032652

  20. Design, characterization, teratogenicity testing, antibacterial, antifungal and DNA interaction of few high spin Fe(II) Schiff base amino acid complexes

    NASA Astrophysics Data System (ADS)

    Abdel-Rahman, Laila H.; El-Khatib, Rafat M.; Nassr, Lobna A. E.; Abu-Dief, Ahmed M.; Lashin, Fakhr El-Din

    2013-07-01

    In this study, new Fe(II) Schiff base amino acid chelates derived from the condensation of o-hydroxynaphthaldehyde with L-alanine, L-phenylalanine, L-aspartic acid, L-histidine and L-arginine were synthesized and characterized via elemental, thermogravimetric analysis, molar conductance, IR, electronic, mass spectra and magnetic moment measurements. The stoichiometry and the stability constants of the complexes were determined spectrophotometrically. Correlation of all spectroscopic data suggested that Schiff bases ligands exhibited tridentate with ONO sites coordinating to the metal ions via protonated phenolic-OH, azomethine-N and carboxylate-O with the general formulae [Fe(HL)2]·nH2O. But in case of L-histidine, the ligand acts as tetradentate via deprotonated phenolic-OH, azomethine-N, carboxylate-O and N-imidazole ring ([FeL(H2O)2]·2H2O), where HL = mono anion and L = dianion of the ligand. The structure of the prepared complexes is suggested to be octahedral. The prepared complexes were tested for their teratogenicity on chick embryos and found to be safe until a concentration of 100 μg/egg with full embryos formation. Moreover, the interaction between CT-DNA and the investigated complexes were followed by spectrophotometric and viscosity measurements. It was found that, the prepared complexes bind to DNA via classical intercalative mode and showed a different DNA activity with the sequence: nhi > nari > nali > nasi > nphali. Furthermore, the free ligands and their complexes are screened for their in vitro antibacterial and antifungal activity against three types of bacteria, Escherichia coli, Pseudomonas aeruginosa and Bacillus cereus and three types of anti fungal cultures, Penicillium purpurogenium, Aspergillus flavus and Trichotheium rosium in order to assess their antimicrobial potential. The results show that the metal complexes are more reactive with respect to their corresponding Schiff base amino acid ligands.

  1. Psychological distress in applicants for predictive DNA testing for autosomal dominant, heritable, late onset disorders. The Rotterdam/Leiden Genetics Workgroup.

    PubMed Central

    DudokdeWit, A C; Tibben, A; Duivenvoorden, H J; Frets, P G; Zoeteweij, M W; Losekoot, M; van Haeringen, A; Niermeijer, M F; Passchier, J

    1997-01-01

    In a comparative study on the effects of predictive DNA testing for late onset disorders, pre-test psychological distress was assessed in people at risk for Huntington's disease (HD, n = 41), cerebral haemorrhage (HCHWA-D, n = 9), breast and ovarian cancer (HBOC, n = 24), and polyposis coli (FAP, n = 45). Partners, if available, also participated in the study. Distress was measured with the subscales Intrusion and Avoidance of the Impact of Event Scale. People at risk for the neurodegenerative disorders reported more avoidance than those at risk for the cancer syndromes. People at risk for FAP and partners of those at risk for HBOC reported less intrusion than the others at risk and the other partners. Subjects who were more distressed reported more experiences with the disease in close relatives, the disease having a great impact on their lives, having considerations against predictive testing, expecting that being identified as a gene carrier would have adverse effects, and expecting relief after being identified as a non-carrier. Test candidates who expected an increase of personal problems showed higher avoidance, whereas those who could better anticipate future life as a carrier had higher intrusion levels. Generally, subjects with high distress levels are of more concern to the healthcare professional than those with low distress levels. However, high distress may reflect worrying as a mental preparation for the test result, whereas low distress may indicate denial-avoidance behaviour and poor anticipation of the test outcome. In pre-test counselling sessions, this should be acknowledged and addressed. PMID:9152835

  2. Measurement of circulating cell-free DNA levels by a new simple fluorescent test in patients with primary colorectal cancer.

    PubMed

    Czeiger, David; Shaked, Gad; Eini, Hadar; Vered, Ilan; Belochitski, Olga; Avriel, Avital; Ariad, Samuel; Douvdevani, Amos

    2011-02-01

    Elevated circulating cell-free DNA (CFD) levels were found in patients with cancer. The standard CFD assays are work-intensive and expensive. The aim was to evaluate in patients with cancer a new simple CFD assay. In mice inoculated with cancer cells, CFD levels correlated with tumor size. Compared with healthy subjects, 38 patients with colorectal cancer (CRC) had higher preoperative CFD levels (798 ± 409 vs 308 ± 256 ng/mL; P < .0001). Compared with patients free of disease at 1 year, CFD levels were elevated in patients who remained with disease or died (DD). CFD correlated with DD (P = .033), and a combined index of carcinoembryonic antigen × CFD exhibited a better correlation to DD than did pathologic staging (P = .0027 vs P = .0065). For patients with CRC, CFD levels were prognostic of death and disease. A large prospective study will need to be performed to truly evaluate the efficacy of this method for early detection, follow-up, and evaluation of patient response to treatment. PMID:21228367

  3. A possible screening test for inherited p53-related defects based on the apoptotic response of peripheral blood lymphocytes to DNA damage.

    PubMed Central

    Camplejohn, R. S.; Perry, P.; Hodgson, S. V.; Turner, G.; Williams, A.; Upton, C.; MacGeoch, C.; Mohammed, S.; Barnes, D. M.

    1995-01-01

    The cellular response, in terms of cell cycle arrest(s) and apoptosis, to radiation-induced DNA damage was studied. Experiments were performed on both mitogen-stimulated and resting peripheral blood lymphocytes (PBLs) from normal and cancer-prone (C-P) individuals. The C-P individuals comprised three patients carrying germline p53 mutations and three members of two families apparently without such mutations, but with an inherited defect which results in p53 deregulation as shown by high levels of stabilised p53 protein in normal tissues. Interestingly, mitogen-stimulated PBL, from both normal and C-P individuals failed to demonstrate a G1 arrest after gamma radiation. However, a clear difference was seen in the apoptotic response to DNA damage, of PBL from normal and C-P individuals; PBLs from C-P individuals with inherited p53-related defects had a reduced apoptotic response (P = 0.0003). There was a wide margin of separation, with no overlap between the two groups, supporting the possibility of using this altered apoptotic response as a screening test. This simple and rapid procedure could be used to identify those individuals in a C-P family who carry germline p53-related defects. The method appears to detect both individuals with p53 mutations and those apparently without mutations but with other p53-related defects. Images Figure 4 PMID:7669577

  4. DNA repair

    SciTech Connect

    Friedberg, E.C.; Hanawalt, P.C. )

    1988-01-01

    Topics covered in this book included: Eukaryote model systems for DNA repair study; Sensitive detection of DNA lesions and their repair; and Defined DNA sequence probes for analysis of mutagenesis and repair.

  5. Measurement of mRNA by solution hybridization with /sup 32/P-labelled single stranded cRNA probe (SP6 test). Comparison with a /sup 32/P-labelled single stranded cDNA as hybridization probe (S1 test) for measurement of AVP mRNA

    SciTech Connect

    Ludwig, G.; Haenze, J.L.; Lehmann, E.; Lang, R.E.; Burbach, J.H.; Ganten, D.

    1988-01-01

    Radioactively labelled cRNA for the rat AVP gene exon C was synthetized from a pSP64-vector and used for solution hybridization measurement of AVP mRNA (SP6 test). For comparison hybridization was carried out with a gel-purified radioactively labelled cDNA probe synthetized by primer extension of AVP gene exon C cloned into an M13mp9 phage vector DNA (S1 test). Both tests had a comparable sensitivity of up to 0.2 pg AVP mRNA. Under optimal hybridization conditions kinetics were similar in both tests. The fast and easy preparation of large amounts of labelled cDNA probe and simple determination of absolute amounts of mRNA by saturation kinetics without need of a mRNA standard makes the SP6 test an attractive alternative to the known S1 test. The SP6 test should be applicable for a wide variety of genes.

  6. Hepatitis B Test

    MedlinePlus

    ... IgM; anti-HBe; Hepatitis B e Antibody; HBV DNA Formal name: Hepatitis B Virus Testing Related tests: ... produced by the virus, and others detect viral DNA . The main uses for HBV tests include: To ...

  7. Accuracy of non-invasive prenatal testing using cell-free DNA for detection of Down, Edwards and Patau syndromes: a systematic review and meta-analysis

    PubMed Central

    Taylor-Phillips, Sian; Freeman, Karoline; Geppert, Julia; Agbebiyi, Adeola; Uthman, Olalekan A; Madan, Jason; Clarke, Angus; Quenby, Siobhan; Clarke, Aileen

    2016-01-01

    Objective To measure test accuracy of non-invasive prenatal testing (NIPT) for Down, Edwards and Patau syndromes using cell-free fetal DNA and identify factors affecting accuracy. Design Systematic review and meta-analysis of published studies. Data sources PubMed, Ovid Medline, Ovid Embase and the Cochrane Library published from 1997 to 9 February 2015, followed by weekly autoalerts until 1 April 2015. Eligibility criteria for selecting studies English language journal articles describing case–control studies with ≥15 trisomy cases or cohort studies with ≥50 pregnant women who had been given NIPT and a reference standard. Results 41, 37 and 30 studies of 2012 publications retrieved were included in the review for Down, Edwards and Patau syndromes. Quality appraisal identified high risk of bias in included studies, funnel plots showed evidence of publication bias. Pooled sensitivity was 99.3% (95% CI 98.9% to 99.6%) for Down, 97.4% (95.8% to 98.4%) for Edwards, and 97.4% (86.1% to 99.6%) for Patau syndrome. The pooled specificity was 99.9% (99.9% to 100%) for all three trisomies. In 100 000 pregnancies in the general obstetric population we would expect 417, 89 and 40 cases of Downs, Edwards and Patau syndromes to be detected by NIPT, with 94, 154 and 42 false positive results. Sensitivity was lower in twin than singleton pregnancies, reduced by 9% for Down, 28% for Edwards and 22% for Patau syndrome. Pooled sensitivity was also lower in the first trimester of pregnancy, in studies in the general obstetric population, and in cohort studies with consecutive enrolment. Conclusions NIPT using cell-free fetal DNA has very high sensitivity and specificity for Down syndrome, with slightly lower sensitivity for Edwards and Patau syndrome. However, it is not 100% accurate and should not be used as a final diagnosis for positive cases. Trial registration number CRD42014014947. PMID:26781507

  8. The utility of ancient human DNA for improving allele age estimates, with implications for demographic models and tests of natural selection

    PubMed Central

    Sams, Aaron J.; Hawks, John; Keinan, Alon

    2015-01-01

    The age of polymorphic alleles in humans is often estimated from population genetic patterns in extant human populations, such as allele frequencies, linkage disequilibrium, and rate of mutations. Ancient DNA can improve the accuracy of such estimates, as well as facilitate testing the validity of demographic models underlying many population genetic methods. Specifically, the presence of an allele in a genome derived from an ancient sample testifies that the allele is at least as old as that sample. In this study, we consider a common method for estimating allele age based on allele frequency as applied to variants from the US National Institutes of Health (NIH) Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project. We view these estimates in the context of the presence or absence of each allele in the genomes of the 5300 year old Tyrolean Iceman, Ötzi, and of the 50,000 year old Altai Neandertal. Our results illuminate the accuracy of these estimates and their sensitivity to demographic events that were not included in the model underlying age estimation. Specifically, allele presence in the Iceman genome provides a good fit of allele age estimates to the expectation based on the age of that specimen. The equivalent based on the Neandertal genome leads to a poorer fit. This is likely due in part to the older age of the Neandertal and the older time of the split between modern humans and Neandertals, but also due to gene flow from Neandertals to modern humans not being considered in the underlying demographic model. Thus, the incorporation of ancient DNA can improve allele age estimation, demographic modeling, and tests of natural selection. Our results also point to the importance of considering a more diverse set of ancient samples for understanding the geographic and temporal range of individual alleles. PMID:25467111

  9. Immunohistochemistry for hMLH1 and hMSH2: a practical test for DNA mismatch repair-deficient tumors.

    PubMed

    Marcus, V A; Madlensky, L; Gryfe, R; Kim, H; So, K; Millar, A; Temple, L K; Hsieh, E; Hiruki, T; Narod, S; Bapat, B V; Gallinger, S; Redston, M

    1999-10-01

    Inactivation of deoxyribonucleic acid (DNA) mismatch repair genes, most commonly human mutL homologue 1 (hMLH1) or human mutS homologue 2 (hMSH2), is a recently described alternate pathway in cancer development and progression. The resulting genetic instability is characterized by widespread somatic mutations in tumor DNA, and is termed high-frequency microsatellite instability (MSI-H). Although described in a variety of tumors, mismatch repair deficiency has been studied predominantly in colorectal carcinoma. Most MSI-H colorectal carcinomas are sporadic, but some occur in patients with hereditary nonpolyposis colorectal cancer (HNPCC), and are associated with germline mutations in mismatch repair genes. Until now, the identification of MSI-H cancers has required molecular testing. To evaluate the role of immunohistochemistry as a new screening tool for mismatch repair-deficient neoplasms, the authors studied the expression of hMLH1 and hMSH2, using commercially available monoclonal antibodies, in 72 formalin-fixed, paraffin-embedded tumors that had been tested previously for microsatellite instability. They compared immunohistochemical patterns of 38 MSI-H neoplasms, including 16 cases from HNPCC patients with known germline mutations in hMLH1 or hMSH2, with 34 neoplasms that did not show microsatellite instability. Thirty-seven of 38 MSI-H neoplasms were predicted to have a mismatch repair gene defect, as demonstrated by the absence of hMLH1 and/or hMSH2 expression. This included correspondence with all 16 cases with germline mutations. All 34 microsatellite-stable cancers had intact staining with both antibodies. These findings clearly demonstrate that immunohistochemistry can discriminate accurately between MSI-H and microsatellite-stable tumors, providing a practical new technique with important clinical and research applications. PMID:10524526

  10. The utility of ancient human DNA for improving allele age estimates, with implications for demographic models and tests of natural selection.

    PubMed

    Sams, Aaron J; Hawks, John; Keinan, Alon

    2015-02-01

    The age of polymorphic alleles in humans is often estimated from population genetic patterns in extant human populations, such as allele frequencies, linkage disequilibrium, and rate of mutations. Ancient DNA can improve the accuracy of such estimates, as well as facilitate testing the validity of demographic models underlying many population genetic methods. Specifically, the presence of an allele in a genome derived from an ancient sample testifies that the allele is at least as old as that sample. In this study, we consider a common method for estimating allele age based on allele frequency as applied to variants from the US National Institutes of Health (NIH) Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project. We view these estimates in the context of the presence or absence of each allele in the genomes of the 5300 year old Tyrolean Iceman, Ötzi, and of the 50,000 year old Altai Neandertal. Our results illuminate the accuracy of these estimates and their sensitivity to demographic events that were not included in the model underlying age estimation. Specifically, allele presence in the Iceman genome provides a good fit of allele age estimates to the expectation based on the age of that specimen. The equivalent based on the Neandertal genome leads to a poorer fit. This is likely due in part to the older age of the Neandertal and the older time of the split between modern humans and Neandertals, but also due to gene flow from Neandertals to modern humans not being considered in the underlying demographic model. Thus, the incorporation of ancient DNA can improve allele age estimation, demographic modeling, and tests of natural selection. Our results also point to the importance of considering a more diverse set of ancient samples for understanding the geographic and temporal range of individual alleles. PMID:25467111

  11. Development of a Rapid Point-of-Use DNA Test for the Screening of Genuity® Roundup Ready 2 Yield® Soybean in Seed Samples.

    PubMed

    Chandu, Dilip; Paul, Sudakshina; Parker, Mathew; Dudin, Yelena; King-Sitzes, Jennifer; Perez, Tim; Mittanck, Don W; Shah, Manali; Glenn, Kevin C; Piepenburg, Olaf

    2016-01-01

    Testing for the presence of genetically modified material in seed samples is of critical importance for all stakeholders in the agricultural industry, including growers, seed manufacturers, and regulatory bodies. While rapid antibody-based testing for the transgenic protein has fulfilled this need in the past, the introduction of new variants of a given transgene demands new diagnostic regimen that allows distinguishing different traits at the nucleic acid level. Although such molecular tests can be performed by PCR in the laboratory, their requirement for expensive equipment and sophisticated operation have prevented its uptake in point-of-use applications. A recently developed isothermal DNA amplification technique, recombinase polymerase amplification (RPA), combines simple sample preparation and amplification work-flow procedures with the use of minimal detection equipment in real time. Here, we report the development of a highly sensitive and specific RPA-based detection system for Genuity Roundup Ready 2 Yield (RR2Y) material in soybean (Glycine max) seed samples and present the results of studies applying the method in both laboratory and field-type settings. PMID:27314015

  12. Development of a Rapid Point-of-Use DNA Test for the Screening of Genuity® Roundup Ready 2 Yield® Soybean in Seed Samples

    PubMed Central

    Chandu, Dilip; Paul, Sudakshina; Parker, Mathew; Dudin, Yelena; King-Sitzes, Jennifer; Perez, Tim; Mittanck, Don W.; Shah, Manali; Glenn, Kevin C.; Piepenburg, Olaf

    2016-01-01

    Testing for the presence of genetically modified material in seed samples is of critical importance for all stakeholders in the agricultural industry, including growers, seed manufacturers, and regulatory bodies. While rapid antibody-based testing for the transgenic protein has fulfilled this need in the past, the introduction of new variants of a given transgene demands new diagnostic regimen that allows distinguishing different traits at the nucleic acid level. Although such molecular tests can be performed by PCR in the laboratory, their requirement for expensive equipment and sophisticated operation have prevented its uptake in point-of-use applications. A recently developed isothermal DNA amplification technique, recombinase polymerase amplification (RPA), combines simple sample preparation and amplification work-flow procedures with the use of minimal detection equipment in real time. Here, we report the development of a highly sensitive and specific RPA-based detection system for Genuity Roundup Ready 2 Yield (RR2Y) material in soybean (Glycine max) seed samples and present the results of studies applying the method in both laboratory and field-type settings. PMID:27314015

  13. Routine testing of fetal Rhesus D status in Rhesus D negative women using cell-free fetal DNA: an investigation into the preferences and information needs of women

    PubMed Central

    Oxenford, Kerry; Silcock, Caroline; Hill, Melissa; Chitty, Lyn

    2013-01-01

    Objective The goal of this study is to investigate women's preferences and information needs for routine implementation of fetal Rhesus D (RhD) typing using cell-free fetal DNA. Methods A questionnaire was developed following focus groups and interviews with both health professionals and RhD negative (RhD−) women offered fetal RhD genotyping within a research study and distributed to RhD− women attending routine antenatal appointments in four National Health Service hospitals. Current knowledge of blood types, anti-D administration, fetal RhD genotyping and future practices were explored. Results A total of 19 respondents participated in interviews and focus groups, and 270 respondents completed the questionnaires. Questionnaire respondents overwhelmingly felt that the test should be offered to all RhD− women (92.1%), and 75.9% said that they would accept this test. Most were happy to have the test even if it involved extra blood tests (89.3%) or appointments (79%). The knowledge of blood groups was poor. Although 90.7% knew that the baby could have a different blood group from themselves, only 34% knew that blood groups are inherited from both parents. More than 40% were not aware that anti-D would not be required if their baby was RhD−. Conclusions Women would welcome the introduction of routine fetal RhD genotyping. Information leaflets and training of midwives will be essential for implementation to ensure good understanding regarding testing. © 2013 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd. PMID:23625761

  14. The GeneInsight Suite: a platform to support laboratory and provider use of DNA-based genetic testing.

    PubMed

    Aronson, Samuel J; Clark, Eugene H; Babb, Lawrence J; Baxter, Samantha; Farwell, Lisa M; Funke, Birgit H; Hernandez, Amy Lovelette; Joshi, Victoria A; Lyon, Elaine; Parthum, Andrew R; Russell, Franklin J; Varugheese, Matthew; Venman, Thomas C; Rehm, Heidi L

    2011-05-01

    The future of personalized medicine will hinge on effective management of patient genetic profiles. Molecular diagnostic testing laboratories need to track knowledge surrounding an increasingly large number of genetic variants, incorporate this knowledge into interpretative reports, and keep ordering clinicians up to date as this knowledge evolves. Treating clinicians need to track which variants have been identified in each of their patients along with the significance of these variants. The GeneInsight(SM) Suite assists in these areas. The suite also provides a basis for interconnecting laboratories and clinicians in a manner that increases the scalability of personalized medicine processes. PMID:21432942

  15. Performance of Self-Collected Cervical Samples in Screening for Future Precancer Using Human Papillomavirus DNA Testing

    PubMed Central

    Hildesheim, Allan; González, Paula; Schiffman, Mark; Rodríguez, Ana Cecilia; Wacholder, Sholom; Jiménez, Silvia; Quint, Wim; Guillen, Diego; Kreimer, Aimée R.; Herrero, Rolando

    2015-01-01

    Background: Self-collected human papillomavirus (HPV) testing could reduce barriers to cervical cancer screening, with performance comparable to clinician-collected specimens. The ability of self-collected specimens to cross-sectionally and prospectively detect precursor lesions was investigated in an HPV vaccine randomized trial in Costa Rica. Methods: In the trial, 7466 women age 18 to 25 years received an HPV16/18 or control vaccine and were followed at least annually for four years. In this secondary analysis, we included all women who provided a self-collected cervicovaginal specimen six months after enrollment (5109 women = full analytical cohort). A subset (615 women = restricted cohort) also had clinician-collected specimens at the six-month postenrollment visit. High-grade squamous intraepithelial lesion or repeat low-grade squamous intraepithelial lesion prompted colposcopic referral throughout the study. HPV testing was performed with SPF10PCR/DEIA/LiPA25. Cross-sectional and prospective sensitivity, specificity, and predictive values were estimated. Results: In the full cohort, one-time HPV testing on self-collected samples detected prevalent CIN2+ with a sensitivity of 88.7% (95% confidence interval [CI] =77.0% to 95.7%) and a specificity of 68.9% (95% CI = 67.6% to 70.1%). For predicting incident CIN2+ in the subsequent four years, sensitivity was 73.9% (95% CI = 65.8% to 81.0%) and specificity 69.4% (95% CI = 68.1% to 70.7%). In the restricted cohort, for incident CIN2+, self-collected HPV was much more sensitive than cytology (80.0% vs 10.0%); relative sensitivity was 0.1 (95% CI = 0.03% to 0.5%). Furthermore, three times more women with normal baseline cytology developed incident CIN2+ than those with negative self-collected HPV. Self-collected and clinician-collected HPV testing had comparable performance. Agreement between self- and clinician-collected samples was 89.7% (kappa = 0.78, McNemar χ2 = 0.62) for carcinogenic HPV types. Conclusions

  16. Forensic DNA analysis.

    PubMed

    McDonald, Jessica; Lehman, Donald C

    2012-01-01

    Before the routine use of DNA profiling, blood typing was an important forensic tool. However, blood typing was not very discriminating. For example, roughly 30% of the United States population has type A-positive blood. Therefore, if A-positive blood were found at a crime scene, it could have come from 30% of the population. DNA profiling has a much better ability for discrimination. Forensic laboratories no longer routinely determine blood type. If blood is found at a crime scene, DNA profiling is performed. From Jeffrey's discovery of DNA fingerprinting to the development of PCR of STRs to the formation of DNA databases, our knowledge of DNA and DNA profiling have expanded greatly. Also, the applications for which we use DNA profiling have increased. DNA profiling is not just used for criminal case work, but it has expanded to encompass paternity testing, disaster victim identification, monitoring bone marrow transplants, detecting fetal cells in a mother's blood, tracing human history, and a multitude of other areas. The future of DNA profiling looks expansive with the development of newer instrumentation and techniques. PMID:22693781

  17. Method for assaying clustered DNA damages

    DOEpatents

    Sutherland, Betsy M.

    2004-09-07

    Disclosed is a method for detecting and quantifying clustered damages in DNA. In this method, a first aliquot of the DNA to be tested for clustered damages with one or more lesion-specific cleaving reagents under conditions appropriate for cleavage of the DNA to produce single-strand nicks in the DNA at sites of damage lesions. The number average molecular length (Ln) of double stranded DNA is then quantitatively determined for the treated DNA. The number average molecular length (Ln) of double stranded DNA is also quantitatively determined for a second, untreated aliquot of the DNA. The frequency of clustered damages (.PHI..sub.c) in the DNA is then calculated.

  18. Measurement of unscheduled DNA synthesis and S-phase synthesis in rodent hepatocytes following in vivo treatment: testing of 24 compounds.

    PubMed

    Mirsalis, J C; Tyson, C K; Steinmetz, K L; Loh, E K; Hamilton, C M; Bakke, J P; Spalding, J W

    1989-01-01

    The in vivo-in vitro hepatocyte DNA repair assay has been shown to be useful for studying genotoxic hepatocarcinogens. In addition, measurement of S-phase synthesis (SPS) provides an indirect indicator of hepatocellular proliferation, which may be an important mechanism in rodent carcinogenesis. This assay was used to examine 24 chemicals for their ability to induce unscheduled DNA synthesis (UDS) or SPS in Fischer-344 rats or B6C3F1 mice following in vivo treatment. Hepatocytes were isolated by liver perfusion and incubated with 3H-thymidine following in vivo treatment by gavage. UDS was measured by quantitative autoradiography as net grains/nucleus (NG). Controls from both sexes of both species yielded less than 0.0 NG. Chemicals chosen for testing were from the National Toxicology Program (NTP) genetic toxicology testing program and most were also evaluated in long-term animal studies conducted by the NTP. 11-Aminoundecanoic acid, benzyl acetate, bis(2-chloro-1-methylethyl)ether (BCMEE), C.I. Solvent Yellow 14, cinnamaldehyde, cinnamyl anthranilate, dichloromethane, dichlorvos, glutaraldehyde, 4,4'-methylenedianiline (MDA), 4-nitrotoluene, 4,4'-oxydianiline, a polybrominated biphenyl mixture (PBB), reserpine, 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane, trichloroethylene, and 2,6-xylidine all failed to induce UDS in rats and/or mice. Dinitrotoluene and Michler's Ketone induced positive UDS response in rat, while N-nitrosodiethanolamine and selenium sulfide induced equivocal UDS results in mouse and rat, respectively. BCMEE, bromoform, chloroform, PBB, 1,1,2-trichloroethane, and trichloroethylene were all potent inducers of SPS in mouse liver, while C.I. Solvent Yellow 14, and 1,1,2,2-tetrachloroethane yielded equivocal SPS results in rat and mouse, respectively. These results indicate that most of the test compounds do not induce UDS in the liver; however, the significant S-phase responses induced by many of these compounds, especially the halogenated

  19. Development of cost-effective real-time PCR test: to detect a wide range of HBV DNA concentrations in the western amazon region of Brazil

    PubMed Central

    2014-01-01

    Background Currently there is a significant risk of infection with hepatitis B virus (HBV) during blood transfusion in high epidemic area. This is due to the pre-seroconversion window period, immunovariant viral strains and the presence of occult HBV infection (OBI). The aim of this study was to develop an in-house real-time PCR-based method, which was both ultra-sensitive and efficient offering an alternative method for nucleic acid testing (NAT). Methods A precore fragment with 109 bp was cloned and serial diluted to standard curve construction. The calibration of the HBV - DNA values was performed against OptiQuant® HBV-DNA Quantification Panel, Acrometrix Europe B.V.). Results From our in-house plasmid we prepared serial dilutions ranging from 2 × 103 – 2 × 109 copies/ml. The threshold was adjusted automatically during analysis and the data collected were analyzed by linear regression (r2 = 0.99). The limit of detection for the assay with pHBVRO standards was 2000/ml in a total reaction volume of 30 μl. We found a strong correlation between the two methods (r2 = 0.9965 and p < 0.0001). The regression line give us the following equation: Log 10 (IU/mL) = 0.9038Log 10 (copies/mL) − 1.0643, suggesting that 1 IU/mL = 15 copies/mL. Conclusions Therefore, we can affirm that the qHBVRO PCR can detect HBV DNA in individuals with hepatitis B at any stage of the disease showing high capacity for NAT screening in hepatitis b donors. This results of sensitivity could provide an advance for automation in blood banks and increasing safety of patients who receive blood transfusions. PMID:24472141

  20. Amniote phylogenomics: testing evolutionary hypotheses with BAC library scanning and targeted clone analysis of large-scale DNA sequences from reptiles.

    PubMed

    Shedlock, Andrew M; Janes, Daniel E; Edwards, Scott V

    2008-01-01

    Phylogenomics research integrating established principles of systematic biology and taking advantage of the wealth of DNA sequences being generated by genome science holds promise for answering long-standing evolutionary questions with orders of magnitude more primary data than in the past. Although it is unrealistic to expect whole-genome initiatives to proceed rapidly for commercially unimportant species such as reptiles, practical approaches utilizing genomic libraries of large-insert clones pave the way for a phylogenomics of species that are nevertheless essential for testing evolutionary hypotheses within a phylogenetic framework. This chapter reviews the case for adopting genome-enabled approaches to evolutionary studies and outlines a program for using bacterial artificial chromosome (BAC) libraries or plasmid libraries as a basis for completing "genome scans" of reptiles. We have used BACs to close a critical gap in the genome database for Reptilia, the sister group of mammals, and present the methodological approaches taken to achieve this as a guideline for designing similar comparative studies. In addition, we provide a detailed step-by-step protocol for BAC-library screening and shotgun sequencing of specific clones containing target genes of evolutionary interest. Taken together, the genome scanning and shotgun sequencing techniques offer complementary diagnostic potential and can substantially increase the scale and power of analyses aimed at testing evolutionary hypotheses for nonmodel species. PMID:18629663

  1. DNA banking and DNA databanking by academic and commercial laboratories

    SciTech Connect

    McEwen, J.E. |; Reilly, P.R.

    1994-09-01

    The advent of DNA-based testing is giving rise to DNA banking (the long-term storage of cells, transformed cell lines, or extracted DNA for subsequent retrieval and analysis) and DNA data banking (the indefinite storage of information derived from DNA analysis). Large scale acquisition and storage of DNA and DNA data has important implications for the privacy rights of individuals. A survey of 148 academically based and commercial DNA diagnostic laboratories was conducted to determine: (1) the extent of their DNA banking activities; (2) their policies and experiences regarding access to DNA samples and data; (3) the quality assurance measures they employ; and (4) whether they have written policies and/or depositor`s agreements addressing specific issues. These issues include: (1) who may have access to DNA samples and data; (2) whether scientists may have access to anonymous samples or data for research use; (3) whether they have plans to contact depositors or retest samples if improved tests for a disorder become available; (4) disposition of samples at the end of the contract period if the laboratory ceases operations, if storage fees are unpaid, or after a death or divorce; (5) the consequence of unauthorized release, loss, or accidental destruction of samples; and (6) whether depositors may share in profits from the commercialization of tests or treatments developed in part from studies of stored DNA. The results suggest that many laboratories are banking DNA, that many have already amassed a large number of samples, and that a significant number plan to further develop DNA banking as a laboratory service over the next two years. Few laboratories have developed written policies governing DNA banking, and fewer still have drafted documents that define the rights and obligations of the parties. There may be a need for increased regulation of DNA banking and DNA data banking and for better defined policies with respect to protecting individual privacy.

  2. A capacitive DNA sensor-based test for simple and sensitive analysis of antibiotic resistance in field setting.

    PubMed

    Liu, Yanling; Hedström, Martin; Chen, Dongfeng; Fan, Xiaolong; Mattiasson, Bo

    2015-02-15

    To meet urgent needs for solving serious worldwide drug-resistance problems, a sensitive label-free capacitive sensor developed in our group was investigated as a tool to be applied in the field of antibiotic resistance genotyping, for instance the detection of ampicillin resistance gene (ampR). Proof-of-concept data demonstrated its detection sensitivity of pico-molar without any signal amplification step and a dynamic range of at least three orders of magnitude. The detection limits of less than 1 pM for the single-stranded ampR oligonucleotide and 4 pM for the double-stranded target can reliably be achieved after only 2.5 min sample reaction. Reusability of the probe-functionalized disposable electrode was investigated by comparing different regeneration solutions; mix of 25 mM NaOH/30% formamide was employed to regenerate the electrode for at least six cycles without significant loss of sensing ability. Assay is performed automatically and result is retrieved in 20 min. The developed sensitive genotyping tool is expected to provide simple, fast and affordable screening for monitoring spread of antibiotic resistances, which is suitable for testing in field setting. PMID:25238540

  3. Contribution to the diagnosis of Johne's disease in cattle. Comparative studies on the validity of Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test in detecting Mycobacterium paratuberculosis in faeces from cattle.

    PubMed

    Zimmer, K; Dräger, K G; Klawonn, W; Hess, R G

    1999-03-01

    In the present study, 132 selected faecal samples from clinically affected and subclinically infected cattle from dairy herds known to be affected by Johne's disease were investigated for the presence of Mycobacterium paratuberculosis using Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test. The sensitivity was 36.4% for Ziehl-Neelsen staining, 85.6% for faecal culture and 47.7% for the DNA-Probe test. Proving the presence of acid-fast bacteria in 49.3% of the samples from clinically affected cattle and 19.3% of those from subclinically infected cattle, Ziehl-Neelsen staining had the lowest detection rate of the three tests under investigation. Faecal culture showed the highest detection rate of M. paratuberculosis in samples from both clinically affected (84.0%) and subclinically infected (87.7%) animals. The DNA-Probe test showed a positive result in 68.0% of the samples from clinically affected cattle and 21.1% of those from subclinically infected cattle. Ziehl-Neelsen staining proved unreliable in diagnosing Johne's disease. Faecal culture was the most sensitive method for detecting M. paratuberculosis both in clinically affected and subclinically infected cattle. The sensitivity of a commercially available DNA-Probe test has to be enhanced to enable a quick and reliable diagnosis of Johne's disease. PMID:10216457

  4. HPV Test

    MedlinePlus

    ... test for wider range of HPV types. 2009 Mar 13. US Food and Drug Administration. Available online ... approves two DNA tests to detect HPV. 2009 Mar 17. Infectious Disease News. Available online at http:// ...

  5. POLG DNA testing as an emerging standard of care before instituting valproic acid therapy for pediatric seizure disorders

    PubMed Central

    Saneto, Russell P.; Lee, Inn-Chi; Koenig, Mary Kay; Bao, Xinhua; Weng, Shao-Wen; Naviaux, Robert K.; Wong, Lee-Jun C.

    2011-01-01

    Purpose To review our clinical experience and determine if there are appropriate signs and symptoms to consider POLG sequencing prior to valproic acid (VPA) dosing in patients with seizures. Methods Four patients who developed VPA-induced hepatotoxicity were examined for POLG sequence variations. A subsequent chart review was used to describe clinical course prior to and after VPA dosing. Results Four patients of multiple different ethnicities, age 3–18 years, developed VPA-induced hepatotoxicity. All were given VPA due to intrac partial seizures. Three of the patients had developed epilepsia partialis continua. The time from VPA exposure to liver failure was between 2 and 3 months. Liver failure was reversible in one patient. Molecular studies revealed homozygous p.R597W or p.A467T mutations in two patients. The other two patients showed compound heterozygous mutations, p.A467T/p.Q68X and p.L83P/p.G888S. Clinical findings and POLG mutations were diagnostic of Alpers–Huttenlocher syndrome. Conclusion Our cases underscore several important findings: POLG mutations have been observed in every ethnic group studied to date; early predominance of epileptiform discharges over the occipital region is common in POLG-induced epilepsy; the EEG and MRI findings varying between patients and stages of the disease; and VPA dosing at any stage of Alpers–Huttenlocher syndrome can precipitate liver failure. Our data support an emerging proposal that POLG gene testing should be considered in any child or adolescent who presents or develops intractable seizures with or without status epilepticus or epilepsia partialis continua, particularly when there is a history of psychomotor regression. PMID:20138553

  6. Tumor Mismatch Repair Immunohistochemistry and DNA MLH1 Methylation Testing of Patients With Endometrial Cancer Diagnosed at Age Younger Than 60 Years Optimizes Triage for Population-Level Germline Mismatch Repair Gene Mutation Testing

    PubMed Central

    Buchanan, Daniel D.; Tan, Yen Y.; Walsh, Michael D.; Clendenning, Mark; Metcalf, Alexander M.; Ferguson, Kaltin; Arnold, Sven T.; Thompson, Bryony A.; Lose, Felicity A.; Parsons, Michael T.; Walters, Rhiannon J.; Pearson, Sally-Ann; Cummings, Margaret; Oehler, Martin K.; Blomfield, Penelope B.; Quinn, Michael A.; Kirk, Judy A.; Stewart, Colin J.; Obermair, Andreas; Young, Joanne P.; Webb, Penelope M.; Spurdle, Amanda B.

    2014-01-01

    Purpose Clinicopathologic data from a population-based endometrial cancer cohort, unselected for age or family history, were analyzed to determine the optimal scheme for identification of patients with germline mismatch repair (MMR) gene mutations. Patients and Methods Endometrial cancers from 702 patients recruited into the Australian National Endometrial Cancer Study (ANECS) were tested for MMR protein expression using immunohistochemistry (IHC) and for MLH1 gene promoter methylation in MLH1-deficient cases. MMR mutation testing was performed on germline DNA of patients with MMR-protein deficient tumors. Prediction of germline mutation status was compared for combinations of tumor characteristics, age at diagnosis, and various clinical criteria (Amsterdam, Bethesda, Society of Gynecologic Oncology, ANECS). Results Tumor MMR-protein deficiency was detected in 170 (24%) of 702 cases. Germline testing of 158 MMR-deficient cases identified 22 truncating mutations (3% of all cases) and four unclassified variants. Tumor MLH1 methylation was detected in 99 (89%) of 111 cases demonstrating MLH1/PMS2 IHC loss; all were germline MLH1 mutation negative. A combination of MMR IHC plus MLH1 methylation testing in women younger than 60 years of age at diagnosis provided the highest positive predictive value for the identification of mutation carriers at 46% versus ≤ 41% for any other criteria considered. Conclusion Population-level identification of patients with MMR mutation-positive endometrial cancer is optimized by stepwise testing for tumor MMR IHC loss in patients younger than 60 years, tumor MLH1 methylation in individuals with MLH1 IHC loss, and germline mutations in patients exhibiting loss of MSH6, MSH2, or PMS2 or loss of MLH1/PMS2 with absence of MLH1 methylation. PMID:24323032

  7. Somatic Mutation Allelic Ratio Test Using ddPCR (SMART-ddPCR): An Accurate Method for Assessment of Preferential Allelic Imbalance in Tumor DNA.

    PubMed

    de Smith, Adam J; Walsh, Kyle M; Hansen, Helen M; Endicott, Alyson A; Wiencke, John K; Metayer, Catherine; Wiemels, Joseph L

    2015-01-01

    The extent to which heritable genetic variants can affect tumor development has yet to be fully elucidated. Tumor selection of single nucleotide polymorphism (SNP) risk alleles, a phenomenon called preferential allelic imbalance (PAI), has been demonstrated in some cancer types. We developed a novel application of digital PCR termed Somatic Mutation Allelic Ratio Test using Droplet Digital PCR (SMART-ddPCR) for accurate assessment of tumor PAI, and have applied this method to test the hypothesis that heritable SNPs associated with childhood acute lymphoblastic leukemia (ALL) may demonstrate tumor PAI. These SNPs are located at CDKN2A (rs3731217) and IKZF1 (rs4132601), genes frequently lost in ALL, and at CEBPE (rs2239633), ARID5B (rs7089424), PIP4K2A (rs10764338), and GATA3 (rs3824662), genes located on chromosomes gained in high-hyperdiploid ALL. We established thresholds of AI using constitutional DNA from SNP heterozygotes, and subsequently measured allelic copy number in tumor DNA from 19-142 heterozygote samples per SNP locus. We did not find significant tumor PAI at these loci, though CDKN2A and IKZF1 SNPs showed a trend towards preferential selection of the risk allele (p = 0.17 and p = 0.23, respectively). Using a genomic copy number control ddPCR assay, we investigated somatic copy number alterations (SCNA) underlying AI at CDKN2A and IKZF1, revealing a complex range of alterations including homozygous and hemizygous deletions and copy-neutral loss of heterozygosity, with varying degrees of clonality. Copy number estimates from ddPCR showed high agreement with those from multiplex ligation-dependent probe amplification (MLPA) assays. We demonstrate that SMART-ddPCR is a highly accurate method for investigation of tumor PAI and for assessment of the somatic alterations underlying AI. Furthermore, analysis of publicly available data from The Cancer Genome Atlas identified 16 recurrent SCNA loci that contain heritable cancer risk SNPs associated with a

  8. Somatic Mutation Allelic Ratio Test Using ddPCR (SMART-ddPCR): An Accurate Method for Assessment of Preferential Allelic Imbalance in Tumor DNA

    PubMed Central

    de Smith, Adam J.; Walsh, Kyle M.; Hansen, Helen M.; Endicott, Alyson A.; Wiencke, John K.; Metayer, Catherine; Wiemels, Joseph L.

    2015-01-01

    The extent to which heritable genetic variants can affect tumor development has yet to be fully elucidated. Tumor selection of single nucleotide polymorphism (SNP) risk alleles, a phenomenon called preferential allelic imbalance (PAI), has been demonstrated in some cancer types. We developed a novel application of digital PCR termed Somatic Mutation Allelic Ratio Test using Droplet Digital PCR (SMART-ddPCR) for accurate assessment of tumor PAI, and have applied this method to test the hypothesis that heritable SNPs associated with childhood acute lymphoblastic leukemia (ALL) may demonstrate tumor PAI. These SNPs are located at CDKN2A (rs3731217) and IKZF1 (rs4132601), genes frequently lost in ALL, and at CEBPE (rs2239633), ARID5B (rs7089424), PIP4K2A (rs10764338), and GATA3 (rs3824662), genes located on chromosomes gained in high-hyperdiploid ALL. We established thresholds of AI using constitutional DNA from SNP heterozygotes, and subsequently measured allelic copy number in tumor DNA from 19–142 heterozygote samples per SNP locus. We did not find significant tumor PAI at these loci, though CDKN2A and IKZF1 SNPs showed a trend towards preferential selection of the risk allele (p = 0.17 and p = 0.23, respectively). Using a genomic copy number control ddPCR assay, we investigated somatic copy number alterations (SCNA) underlying AI at CDKN2A and IKZF1, revealing a complex range of alterations including homozygous and hemizygous deletions and copy-neutral loss of heterozygosity, with varying degrees of clonality. Copy number estimates from ddPCR showed high agreement with those from multiplex ligation-dependent probe amplification (MLPA) assays. We demonstrate that SMART-ddPCR is a highly accurate method for investigation of tumor PAI and for assessment of the somatic alterations underlying AI. Furthermore, analysis of publicly available data from The Cancer Genome Atlas identified 16 recurrent SCNA loci that contain heritable cancer risk SNPs associated with a

  9. A non-organic and non-enzymatic extraction method gives higher yields of genomic DNA from whole-blood samples than do nine other methods tested.

    PubMed

    Lahiri, D K; Bye, S; Nurnberger, J I; Hodes, M E; Crisp, M

    1992-12-01

    We compared ten methods for extraction of DNA from whole blood. Nine methods require incubation with either enzymes or treatment of organic solvents or both. The 'Rapid Method' (RM) (Method 10) avoids the use of organic solvents (phenol/chloroform) and eliminates completely the use of proteinase K. Thus, the time and cost of DNA extraction are reduced significantly. This is accomplished by salting out and precipitation of the cellular proteins in saturated sodium chloride. This method takes less than an hour to completion, without compromising the yield or the quality of DNA. Using RM, we can make DNA from 0.1 ml of whole blood and as little as 0.5 ml of blood yields DNA sufficient to run a few Southern blots. The RM can also be applied to packed cells. The DNA is free of RNA, protein and degrading enzymes. The uncut DNA runs as a typical slow-migrating, high-molecular-weight and undegraded species in an agarose gel. The DNA is suitable for digestion by various restriction endonucleases. This procedure works equally well with fresh blood samples and with those that are stored at 4 degrees C and -70 degrees C. To our knowledge the RM reported here is the safest, fastest and most quantitative and economical method for preparation of DNA from whole blood and cells. PMID:1494032

  10. Mitochondrial DNA.

    ERIC Educational Resources Information Center

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  11. DNA Banking

    SciTech Connect

    Reilly, P.R. )

    1992-11-01

    The author is involved in the ethical, legal, and social issues of banking of DNA and data from DNA analysis. In his attempt to determine the extent of DNA banking in the U.S., the author surveyed some commercial companies performing DNA banking services. This article summarizes the results of that survey, with special emphasis on the procedures the companies use to protect the privacy of individuals. 4 refs.

  12. Quantification of human mitochondrial DNA using synthesized DNA standards.

    PubMed

    Kavlick, Mark F; Lawrence, Helen S; Merritt, R Travis; Fisher, Constance; Isenberg, Alice; Robertson, James M; Budowle, Bruce

    2011-11-01

    Successful mitochondrial DNA (mtDNA) forensic analysis depends on sufficient quantity and quality of mtDNA. A real-time quantitative PCR assay was developed to assess such characteristics in a DNA sample, which utilizes a duplex, synthetic DNA to ensure optimal quality assurance and quality control. The assay's 105-base pair target sequence facilitates amplification of degraded DNA and is minimally homologous to nonhuman mtDNA. The primers and probe hybridize to a region that has relatively few sequence polymorphisms. The assay can also identify the presence of PCR inhibitors and thus indicate the need for sample repurification. The results show that the assay provides information down to 10 copies and provides a dynamic range spanning seven orders of magnitude. Additional experiments demonstrated that as few as 300 mtDNA copies resulted in successful hypervariable region amplification, information that permits sample conservation and optimized downstream PCR testing. The assay described is rapid, reliable, and robust. PMID:21883207

  13. Sensitivity of APTIMA HPV E6/E7 mRNA test in comparison with hybrid capture 2 HPV DNA test for detection of high risk oncogenic human papillomavirus in 396 biopsy confirmed cervical cancers.

    PubMed

    Basu, Partha; Banerjee, Dipanwita; Mittal, Srabani; Dutta, Sankhadeep; Ghosh, Ishita; Chowdhury, Nilarun; Abraham, Priya; Chandna, Puneet; Ratnam, Sam

    2016-07-01

    The sensitivity of E6/E7 mRNA-based Aptima HPV test (AHPV; Hologic, Inc.) for detection of cervical cancer has been reported based on only a small number of cases. We determined the sensitivity of AHPV in comparison with the DNA-based Hybrid Capture 2 HPV test (HC2; Qiagen) for the detection of oncogenic HPV in a large number of cervical cancers at the time of diagnosis using cervical samples obtained in ThinPrep (Hologic). Samples yielding discordant results were genotyped using Linear Array assay (LA; Roche). Of 396 cases tested, AHPV detected 377 (sensitivity, 95.2%; 95%CI: 93.1-97.3), and HC2 376 (sensitivity, 94.9%; 95%CI: 92.7-97.1) with an agreement of 97.2% (kappa 0.7; 95%CI: 0.54-0.87). Among six AHPV+/HC2- cases, LA identified oncogenic HPV types in four including a type 73 and was negative in two. Among five AHPV-/HC2+ cases, LA detected oncogenic HPV types in two including a type 73 and was negative in three. Of 14 AHPV-/HC2- cases, 13 were genotyped. LA detected oncogenic HPV types in six, non-oncogenic types in three, and was negative in four. This is the largest study to demonstrate the sensitivity of AHPV for the detection of invasive cervical cancer and this assay showed equal sensitivity to HC2. J. Med. Virol. 88:1271-1278, 2016. © 2015 Wiley Periodicals, Inc. PMID:26693677

  14. Dna Sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  15. Phage Library Screening for the Rapid Identification and In Vivo Testing of Candidate Genes for a DNA Vaccine against Mycoplasma mycoides subsp. mycoides Small Colony Biotype

    PubMed Central

    March, John B.; Jepson, Catherine D.; Clark, Jason R.; Totsika, Makrina; Calcutt, Michael J.

    2006-01-01

    compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens. PMID:16368970

  16. The Red Queen Race between Parasitic Chytrids and Their Host, Planktothrix: A Test Using a Time Series Reconstructed from Sediment DNA

    PubMed Central

    Kyle, Marcia; Haande, Sigrid; Ostermaier, Veronika; Rohrlack, Thomas

    2015-01-01

    Parasitic chytrid fungi (phylum Chytridiomycota) are known to infect specific phytoplankton, including the filamentous cyanobacterium Planktothrix. Subspecies, or chemotypes of Planktothrix can be identified by the presence of characteristic oligopeptides. Some of these oligopeptides can be associated with important health concerns due to their potential for toxin production. However, the relationship between chytrid parasite and Planktothrix host is not clearly understood and more research is needed. To test the parasite - host relationship over time, we used a sediment core extracted from a Norwegian lake known to contain both multiple Planktothrix chemotype hosts and their parasitic chytrid. Sediment DNA of chytrids and Planktothrix was amplified and a 35-year coexistence was found. It is important to understand how these two antagonistic species can coexistence in a lake. Reconstruction of the time series showed that between 1979–1990 at least 2 strains of Planktothrix were present and parasitic pressure exerted by chytrids was low. After this period one chemotype became dominant and yet showed continued low susceptibility to chytrid parasitism. Either environmental conditions or intrinsic characteristics of Planktothrix could have been responsible for this continued dominance. One possible explanation could be found in the shift of Planktothrix to the metalimnion, an environment that typically consists of low light and decreased temperatures. Planktothrix are capable of growth under these conditions while the chytrid parasites are constrained. Another potential explanation could be due to the differences between cellular oligopeptide variations found between Planktothrix chemotypes. These oligopeptides can function as defense systems against chytrids. Our findings suggest that chytrid driven diversity was not maintained over time, but that the combination of environmental constraints and multiple oligopeptide production to combat chytrids could have allowed

  17. The ERCC2/XPD Lys751Gln polymorphism affects DNA repair of benzo[a]pyrene induced damage, tested in an in vitro model.

    PubMed

    Xiao, Sha; Cui, Su; Lu, Xiaobo; Guan, Yangyang; Li, Dandan; Liu, Qiufang; Cai, Yuan; Jin, Cuihong; Yang, Jinghua; Wu, Shengwen; van der Straaten, Tahar

    2016-08-01

    Nucleotide excision repair (NER) is an important defense mechanism of the body to exogenous carcinogens and mutagens, such as benzo[a]pyrene (B[a]P). Genetic polymorphisms in ERCC2/XPD, a critical element in NER, are thought to be associated with individual's cancer susceptibility. Although ERCC2/XPD Lys751Gln (rs13181) is the most studied polymorphism, the impact of this polymorphism on DNA repair capacity to carcinogen remains unclear. In the present study, cDNA clones carrying different genotypes of ERCC2/XPD (Lys751Gln) were introduced into an ERCC2/XPD deficient cell line (UV5) in a well-controlled biological system. After B[a]P treatment, cell growth inhibition rates and DNA damage levels in all cells were detected respectively. As expected, we found that the DNA repair capacity in UV5 cells was restored to levels similar to wildtype parent AA8 cells upon introduction of the cDNA clone of ERCC2/XPD (Lys751). Interestingly, after B[a]P treatment, transfected cells expressing variant ERCC2/XPD (751Gln) showed an enhanced cellular sensitivity and a diminished DNA repair capacity. The wildtype genotype AA (Lys) was found to be associated with a higher DNA repair capacity as compared to its polymorphic genotype CC (Gln). These data indicate that ERCC2/XPD Lys751Gln polymorphism affects DNA repair capacity after exposure to environmental carcinogens such as B[a]P in this well-controlled in vitro system and could act as a biomarker to increase the predictive value to develop cancer. PMID:27139774

  18. DNA Immunization

    PubMed Central

    Wang, Shixia; Lu, Shan

    2013-01-01

    DNA immunization was discovered in early 1990s and its use has been expanded from vaccine studies to a broader range of biomedical research, such as the generation of high quality polyclonal and monoclonal antibodies as research reagents. In this unit, three common DNA immunization methods are described: needle injection, electroporation and gene gun. In addition, several common considerations related to DNA immunization are discussed. PMID:24510291

  19. Serological tests for syphilis in Saudi Arabia.

    PubMed Central

    Hossain, A

    1986-01-01

    A total of 6684 sera were initially screened for syphilis by the Venereal Disease Research Laboratory (VDRL) test and the Treponema pallidum haemagglutination assay (TPHA). Reactive sera from either or both these tests were tested for confirmation by the fluorescent treponemal antibody-absorbed (FTA-ABS) test. VDRL biological false positive reactors were detected in 0.5% of the total sera examined, with 0.4% and 0.8%, respectively, obtained in pregnant women and blood donors. Eight sera (0.1%) were found to be positive in the TPHA test alone. An overall positivity of 2.7% for syphilis was detected, with a 0.85% positivity in antenatal patients. Infection with T pallidum seemed to be more common in men than in women (1.6:1) and predominated in the age group 20-39 years. Serological testing of sera from 26 mother and infant pairs allowed one case of congenital syphilis to be detected by FTA-ABS (IgM) and identified VDRL biological false positivity in seven infants. PMID:3770753

  20. DNA adducts-chemical addons.

    PubMed

    Rajalakshmi, T R; AravindhaBabu, N; Shanmugam, K T; Masthan, K M K

    2015-04-01

    DNA adduct is a piece of DNA covalently bond to a chemical (safrole, benzopyrenediol epoxide, acetaldehyde). This process could be the start of a cancerous cell. When a chemical binds to DNA, it gets damaged resulting in abnormal replication. This could be the start of a mutation and without proper DNA repair, this can lead to cancer. It is this chemical that binds with the DNA is our prime area of concern. Instead of performing the whole body analysis for diagnosing cancer, this test could be carried out for early detection of cancer. When scanning tunneling microscope is used, the DNA results can be obtained earlier. DNA adducts in scientific experiments are used as biomarkers. PMID:26015708

  1. DNA adducts-chemical addons

    PubMed Central

    Rajalakshmi, T. R.; AravindhaBabu, N.; Shanmugam, K. T.; Masthan, K. M. K.

    2015-01-01

    DNA adduct is a piece of DNA covalently bond to a chemical (safrole, benzopyrenediol epoxide, acetaldehyde). This process could be the start of a cancerous cell. When a chemical binds to DNA, it gets damaged resulting in abnormal replication. This could be the start of a mutation and without proper DNA repair, this can lead to cancer. It is this chemical that binds with the DNA is our prime area of concern. Instead of performing the whole body analysis for diagnosing cancer, this test could be carried out for early detection of cancer. When scanning tunneling microscope is used, the DNA results can be obtained earlier. DNA adducts in scientific experiments are used as biomarkers. PMID:26015708

  2. FBI's DNA analysis program

    NASA Astrophysics Data System (ADS)

    Brown, John R.

    1994-03-01

    Forensic DNA profiling technology is a significant law enforcement tool due to its superior discriminating power. Applying the principles of population genetics to the DNA profile obtained in violent crime investigations results in low frequency of occurrence estimates for the DNA profile. These estimates often range from a frequency of occurrence of 1 in 50 unrelated individuals to 1 in a million unrelated individuals or even smaller. It is this power to discriminate among individuals in the population that has propelled forensic DNA technology to the forefront of forensic testing in violent crime cases. Not only is the technology extremely powerful in including or excluding a criminal suspect as the perpetrator, but it also gives rise to the potential of identifying criminal suspects in cases where the investigators of unknown suspect cases have exhausted all other available leads.

  3. DNA ALTERATIONS

    EPA Science Inventory

    The exposure of an organism to genotoxic chemicals may induce a cascade of genetic events. nitially, structural alterations to DNA are formed. ext, the DNA damage is processed and subsequently expressed in mutant gene products. inally, diseases result from the genetic damage. he ...

  4. [Sensitivity and specificity of new commercial tests for the detection of specific Echinococcus antibodies].

    PubMed

    Auer, Herbert; Stöckl, Cornelia; Suhendra, Susanne; Schneider, Renate

    2009-10-01

    Cystic echinococcosis (CE), caused by Echinococcus granulosus, and alveolar echinococcosis (AE), caused by E. multilocularis belong to the most serious parasitic diseases. Both forms of echinococcosis occur in Austria; in addition, imported cases are diagnosed and treated regularly in Austria. Diagnosis of echinococcosis is based on clinical symptoms, imaging techniques and particularly on the detection of specific antibodies in serum specimens of patients. For decades several companies have been providing commercial Echinococcus antigens and echinococcosis tests based on different methods, i.e. complement fixation test (CFT) and electrophoretic methods (CIEP, IEP) in the past and enzyme-linked immunosorbent assays (ELISA), western blot assays (WB) and indirect haemagglutination assays (IHA) in recent years. During the last years two studies have been carried out in our laboratory in order to evaluate the sensitivity and specificity of two commercial E. granulosus antigens (the synthetic p176 antigen, arc 5 antigen) and three commercial testkits (IHA from Dade Behring, IHA from Fumouze, ELISA from Novagnost-Dade Behring). Sera of patients with histologically and/or molecular biologically confirmed cystic or alveolar echinococcosis, of patients with other parasitic infections and of healthy people were tested comparatively for specific Echinococcus antibodies. The synthetic p176 antigen proved to be a highly specific but a insensitive antigen, whereas both the indirect haemagglutination assay as well as the Novagnost-ELISA showed a much higher sensitivity but only moderate specificity. Our studies demonstrated that neither the commercial antigens nor the test kits tested should be used as a primary test in a routine laboratory for the diagnosis of cystic echinococcosis or of alveolar echinococcosis. PMID:19915815

  5. In vitro studies of the genotoxic effects of bitumen and coal-tar fume condensates: comparison of data obtained by mutagenicity testing and DNA adduct analysis by 32P-postlabelling.

    PubMed

    De Méo, M; Genevois, C; Brandt, H; Laget, M; Bartsch, H; Castegnaro, M

    1996-08-14

    Bitumens contain traces of polycyclic aromatic compounds (PACs), a part of which will end up in the fumes emitted during hot handling of bitumen-containing products, e.g. during roadpaving. Although exposure of workers to these fumes is low, it might lead to health problems. Studies on bitumen fume condensates (BFCs) showed weak to moderate mutagenic activities, but studies on DNA adduct formation have not been reported. Therefore, a study was initiated in which fumes were generated from two road grade bitumens, in such a way that they were representative of the fumes produced in the field. The combined vapour/particulates were tested in vitro for their ability to produce DNA adducts and in modified Ames mutation assays, using a number of different strains. An attempt was made to relate the results to chemical data, such as the content of a number of individual polycyclic aromatic hydrocarbons (PAHs) and with a measure for the total PAC content. As a reference material fume condensate from coal-tar (coal-tar pitch volatiles; CTPV) were subjected to the same tests. All fume condensates tested were mutagenic to all strains and induced the formation of DNA adducts. The patterns of DNA adducts, obtained by 32P-postlabelling, arising from the BFCs were qualitatively different from the patterns of adducts obtained from the CTPVs, implying qualitative differences in the nature of the compounds responsible for the formation of these adducts. This is corroborated by the observation that for BFCs quantitative adduct levels are higher than would be expected based on the PAH content. These data thus indicate that the PAHs analysed are not the sole components responsible for adduct formation from BFCs, but that an important contribution comes from other (hetero- and/or substituted-) PACs. PMID:8760390

  6. An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

    PubMed Central

    Santos, Ana Paula de Torres; Levi, José Eduardo; Lemos, Marcilio Figueiredo; Calux, Samira Julien; Oba, Isabel Takano; Moreira, Regina Célia

    2016-01-01

    This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy. PMID:26872342

  7. [DNA computing].

    PubMed

    Błasiak, Janusz; Krasiński, Tadeusz; Popławski, Tomasz; Sakowski, Sebastian

    2011-01-01

    Biocomputers can be an alternative for traditional "silicon-based" computers, which continuous development may be limited due to further miniaturization (imposed by the Heisenberg Uncertainty Principle) and increasing the amount of information between the central processing unit and the main memory (von Neuman bottleneck). The idea of DNA computing came true for the first time in 1994, when Adleman solved the Hamiltonian Path Problem using short DNA oligomers and DNA ligase. In the early 2000s a series of biocomputer models was presented with a seminal work of Shapiro and his colleguas who presented molecular 2 state finite automaton, in which the restriction enzyme, FokI, constituted hardware and short DNA oligomers were software as well as input/output signals. DNA molecules provided also energy for this machine. DNA computing can be exploited in many applications, from study on the gene expression pattern to diagnosis and therapy of cancer. The idea of DNA computing is still in progress in research both in vitro and in vivo and at least promising results of these research allow to have a hope for a breakthrough in the computer science. PMID:21735816

  8. Using mitochondrial DNA to test the hypothesis of a European post-glacial human recolonization from the Franco-Cantabrian refuge

    PubMed Central

    García, O; Fregel, R; Larruga, J M; Álvarez, V; Yurrebaso, I; Cabrera, V M; González, A M

    2011-01-01

    It has been proposed that the distribution patterns and coalescence ages found in Europeans for mitochondrial DNA (mtDNA) haplogroups V, H1 and H3 are the result of a post-glacial expansion from a Franco-Cantabrian refuge that recolonized central and northern areas. In contrast, in this refined mtDNA study of the Cantabrian Cornice that contributes 413 partial and 9 complete new mtDNA sequences, including a large Basque sample and a sample of Asturians, no experimental evidence was found to support the human refuge-expansion theory. In fact, all measures of gene diversity point to the Cantabrian Cornice in general and the Basques in particular, as less polymorphic for V, H1 and H3 than other southern regions in Iberia or in Central Europe. Genetic distances show the Cantabrian Cornice is a very heterogeneous region with significant local differences. The analysis of several minor subhaplogroups, based on complete sequences, also suggests different focal expansions over a local and peninsular range that did not affect continental Europe. Furthermore, all detected clinal trends show stronger longitudinal than latitudinal profiles. In Northern Iberia, it seems that the highest diversity values for some haplogroups with Mesolithic coalescence ages are centred on the Mediterranean side, including Catalonia and South-eastern France. PMID:20407470

  9. Parvovirus B19 Test

    MedlinePlus

    ... Viral detection involves finding parvovirus B19 genetic material ( DNA ) in a blood sample or, less commonly, in ... fetal cord blood, or amniotic fluid . Parvovirus B19 DNA testing is performed primarily to detect active parvovirus ...

  10. Dancing DNA.

    ERIC Educational Resources Information Center

    Pennisi, Elizabeth

    1991-01-01

    An imaging technique that uses fluorescent dyes and allows scientists to track DNA as it moves through gels or in solution is described. The importance, opportunities, and implications of this technique are discussed. (KR)

  11. Identification of Lactobacillus UFV H2B20 (probiotic strain) using DNA-DNA hybridization

    PubMed Central

    de Magalhães, J.T.; Uetanabaro, A.P. T.; de Moraes, C.A.

    2008-01-01

    Sequence analyses of the 16S rDNA gene and DNA-DNA hybridization tests were performed for identification of the species of the probiotic Lactobacillus UFV H2b20 strain. Using these two tests, we concluded that this strain, originally considered Lact. acidophilus, should be classified as Lact. delbrueckii. PMID:24031263

  12. Unravelling DNA

    NASA Astrophysics Data System (ADS)

    Conroy, Rs; Danilowicz, C.

    2004-04-01

    The forces involved in the biology of life are carefully balanced between stopping thermal fluctuations ripping our DNA apart and having bonds weak enough to allow enzymes to function. The application of recently developed techniques for measuring piconewton forces and imaging at the nanometre scale on a molecule-by-molecule basis has dramatically increased the impact of single-molecule biophysics. This article describes the most commonly used techniques for imaging and manipulating single biomolecules. Using these techniques, the mechanical properties of DNA can be investigated, for example through measurements of the forces required to stretch and unzip the DNA double helix. These properties determine the ease with which DNA can be folded into the cell nucleus and the size and complexity of the accompanying cellular machinery. Part of this cellular machinery is enzymes, which manipulate, repair and transcribe the DNA helix. Enzymatic function is increasingly being investigated at the single molecule level to give better understanding of the forces and processes involved in the genetic cycle. One of the challenges is to transfer this understanding of single molecules into living systems. Already there have been some notable successes, such as the development of techniques for gene expression through the application of mechanical forces to cells, and the imaging and control of viral infection of a cell. This understanding and control of DNA has also been used to design molecules, which can self-assemble into a range of structures.

  13. Testing the Potential of Proposed DNA Barcoding Markers in Nezara virudula and Nezara antennata When Geographic Variation and Closely Related Species Were Considered

    PubMed Central

    Li, Min; Liu, Qiang; Xi, Li; Liu, Yang; Zhu, Gengping; Zhao, Yanni; Bu, Wenjun

    2014-01-01

    The COI gene as the core of the DNA barcoding system for animals has received significant attention. The observed wide overlap between intra- and interspecific sequence variability has led researchers to envisage the primary COI-based method. The sequences of 16S rDNA, COI, and Cyt b genes of Nezara virudula (L.) (Hemiptera: Pentatomidae) from 13 countries and the same sequences of N. antennata Scott were chosen as molecular markers to analyze the intra- and interspecific relationships between the closely related species in this study. The results support that Cyt b gene may be a good candidate alongside COI, when the combined factors of geographic variation and closely related species are taken into account. PMID:25373226

  14. What Is Mitochondrial DNA?

    MedlinePlus

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  15. Dynamics of interaction between complement-fixing antibody/dsDNA immune complexes and erythrocytes. In vitro studies and potential general applications to clinical immune complex testing

    SciTech Connect

    Taylor, R.P.; Horgan, C.; Hooper, M.; Burge, J.

    1985-01-01

    Soluble antibody//sub 3/H-double-stranded PM2 DNA (dsDNA) immune complexes were briefly opsonized with complement and then allowed to bind to human erythrocytes (via complement receptors). The cells were washed and subsequently a volume of autologous blood in a variety of media was added, and the release of the bound immune complexes from the erythrocytes was studied as a function of temperature and time. After 1-2 h, the majority of the bound immune complexes were not released into the serum during blood clotting at either 37 degrees C or room temperature, but there was a considerably greater release of the immune complexes into the plasma of blood that was anticoagulated with EDTA. Similar results were obtained using various conditions of opsonization and also using complexes that contained lower molecular weight dsDNA. Thus, the kinetics of release of these antibody/dsDNA immune complexes differed substantially from the kinetics of release of antibody/bovine serum albumin complexes that was reported by others. Studies using the solution phase C1q immune complex binding assay confirmed that in approximately half of the SLE samples that were positive for immune complexes, there was a significantly higher level of detectable immune complexes in plasma vs. serum. Freshly drawn erythrocytes from some SLE patients exhibiting this plasma/serum discrepancy had IgG antigen on their surface that was released by incubation in EDTA plasma. Thus, the higher levels of immune complexes observed in EDTA plasma vs. serum using the C1q assay may often reflect the existence of immune complexes circulating in vivo bound to erythrocytes.

  16. What Is Genetic Ancestry Testing?

    MedlinePlus

    ... from relatives or from historical documentation. Examination of DNA variations can provide clues about where a person's ... families with the same surname are related. Mitochondrial DNA testing: This type of testing identifies genetic variations ...

  17. Diagnostic Accuracy of PIK3CA Mutation Detection by Circulating Free DNA in Breast Cancer: A Meta-Analysis of Diagnostic Test Accuracy

    PubMed Central

    Zhu, Hanjiang; Lin, Yan; Pan, Bo; Zhang, Xiaohui; Huang, Xin; Xu, Qianqian; Xu, Yali; Sun, Qiang

    2016-01-01

    Mutation of p110 alpha-catalytic subunit of phosphatidylinositol 3-kinase (PIK3CA) has high predictive and prognostic values for breast cancer. Hence, there has been a marked interest in detecting and monitoring PIK3CA genotype with non-invasive technique, such as circulating free DNA (cfDNA). However, the diagnostic accuracy of PIK3CA genotyping by cfDNA is still a problem of controversy. Here, we conducted the first meta-analysis to evaluate overall diagnostic performance of cfDNA for PIK3CA mutation detection. Literature search was performed in Pubmed, Embase and Cochrane Central Register of Controlled Trials databases. Seven cohorts from five studies with 247 patients were included. The pooled sensitivity, specificity, positive and negative likelihood ratio, diagnostic odds ratio and area under summary receiver operating characteristic curve were calculated for accuracy evaluation. The pooled sensitivity and specificity were 0.86 (95% confidence interval [CI] 0.32–0.99) and 0.98 (95% CI 0.86–1.00), respectively; the pooled positive and negative likelihood ratio were 42.8 (95% CI 5.1–356.9) and 0.14 (95% CI 0.02–1.34), respectively; diagnostic odds ratio for evaluating the overall diagnostic performance was 300 (95% CI 8–11867); area under summary receiver operating characteristic curve reached 0.99 (95% CI 0.97–0.99). Subgroup analysis with metastatic breast cancer revealed remarkable improvement in diagnostic performance (sensitivity: 0.86–0.91; specificity: 0.98; diagnostic odds ratio: 300–428). This meta-analysis proved that detecting PIK3CA gene mutation by cfDNA has high diagnostic accuracy in breast cancer, especially for metastatic breast cancer. It may serve as a reliable non-invasive assay for detecting and monitoring PIK3CA mutation status in order to deliver personalized and precise treatment. PMID:27336598

  18. DNA vaccines

    NASA Astrophysics Data System (ADS)

    Gregersen, Jens-Peter

    2001-12-01

    Immunization by genes encoding immunogens, rather than with the immunogen itself, has opened up new possibilities for vaccine research and development and offers chances for new applications and indications for future vaccines. The underlying mechanisms of antigen processing, immune presentation and regulation of immune responses raise high expectations for new and more effective prophylactic or therapeutic vaccines, particularly for vaccines against chronic or persistent infectious diseases and tumors. Our current knowledge and experience of DNA vaccination is summarized and critically reviewed with particular attention to basic immunological mechanisms, the construction of plasmids, screening for protective immunogens to be encoded by these plasmids, modes of application, pharmacokinetics, safety and immunotoxicological aspects. DNA vaccines have the potential to accelerate the research phase of new vaccines and to improve the chances of success, since finding new immunogens with the desired properties is at least technically less demanding than for conventional vaccines. However, on the way to innovative vaccine products, several hurdles have to be overcome. The efficacy of DNA vaccines in humans appears to be much less than indicated by early studies in mice. Open questions remain concerning the persistence and distribution of inoculated plasmid DNA in vivo, its potential to express antigens inappropriately, or the potentially deleterious ability to insert genes into the host cell's genome. Furthermore, the possibility of inducing immunotolerance or autoimmune diseases also needs to be investigated more thoroughly, in order to arrive at a well-founded consensus, which justifies the widespread application of DNA vaccines in a healthy population.

  19. The evolutionary conservation of DNA polymerase. alpha

    SciTech Connect

    Miller, M.A.; Korn, D.; Wang, T.S.F. )

    1988-08-25

    The evolutionary conservation of DNA polymerase {alpha} was assessed by immunological and molecular genetic approaches. Four anti-human KB cell DNA polymerase {alpha} monoclonal antibodies were tested for their ability to recognize a phylogenetically broad array of eukaryotic DNA polymerases. While the single non-neutralizing antibody used in this study recognizes higher mammalian (human, simian, canine, and bovine) polymerases only, three neutralizing antibodies exhibit greater, but variable, extents of cross-reactivity among vertebrate species. Genomic Southern hybridization studies with the cDNA of the human DNA polymerase {alpha} catalytic polypeptide identify the existence of many consensus DNA sequences within the DNA polymerase genes of vertebrate, invertebrate, plant and unicellular organisms. These findings illustrate the differential evolutionary conservation of four unique epitopes on DNA sequences, presumably reflective of critical functional domains, in the DNA polymerase genes from a broad diversity of living forms.

  20. Alpha-1 Antitrypsin Test

    MedlinePlus

    ... measures the level of the protein AAT in blood. Alpha-1 antitrypsin phenotype testing evaluates the amount and type of AAT being produced and compares it to normal patterns. Alpha-1 antitrypsin genotype testing ( DNA testing) can ...

  1. Performance of an Early Infant Diagnostic Test, AmpliSens DNA-HIV-FRT, Using Dried Blood Spots Collected from Children Born to Human Immunodeficiency Virus-Infected Mothers in Ukraine.

    PubMed

    Chang, Joy; Tarasova, Tetyana; Shanmugam, Vedapuri; Azarskova, Marianna; Nguyen, Shon; Hurlston, Mackenzie; Sabatier, Jennifer; Zhang, Guoqing; Osmanov, Saladin; Ellenberger, Dennis; Yang, Chunfu; Vitek, Charles; Liulchuk, Maria; Nizova, Natalya

    2015-12-01

    An accurate accessible test for early infant diagnosis (EID) is crucial for identifying HIV-infected infants and linking them to treatment. To improve EID services in Ukraine, dried blood spot (DBS) samples obtained from 237 HIV-exposed children (≤18 months of age) in six regions in Ukraine in 2012 to 2013 were tested with the AmpliSens DNA-HIV-FRT assay, the Roche COBAS AmpliPrep/COBAS TaqMan (CAP/CTM) HIV-1 Qual test, and the Abbott RealTime HIV-1 Qualitative assay. In comparison with the paired whole-blood results generated from AmpliSens testing at the oblast HIV reference laboratories in Ukraine, the sensitivity was 0.99 (95% confidence interval [CI], 0.95 to 1.00) for the AmpliSens and Roche CAP/CTM Qual assays and 0.96 (95% CI, 0.90 to 0.98) for the Abbott Qualitative assay. The specificity was 1.00 (95% CI, 0.97 to 1.00) for the AmpliSens and Abbott Qualitative assays and 0.99 (95% CI, 0.96 to 1.00) for the Roche CAP/CTM Qual assay. McNemar analysis indicated that the proportions of positive results for the tests were not significantly different (P > 0.05). Cohen's kappa (0.97 to 0.99) indicated almost perfect agreement among the three tests. These results indicated that the AmpliSens DBS and whole-blood tests performed equally well and were comparable to the two commercially available EID tests. More importantly, the performance characteristics of the AmpliSens DBS test meets the World Health Organization EID test requirements; implementing AmpliSens DBS testing might improve EID services in resource-limited settings. PMID:26447114

  2. DNA codes

    SciTech Connect

    Torney, D. C.

    2001-01-01

    We have begun to characterize a variety of codes, motivated by potential implementation as (quaternary) DNA n-sequences, with letters denoted A, C The first codes we studied are the most reminiscent of conventional group codes. For these codes, Hamming similarity was generalized so that the score for matched letters takes more than one value, depending upon which letters are matched [2]. These codes consist of n-sequences satisfying an upper bound on the similarities, summed over the letter positions, of distinct codewords. We chose similarity 2 for matches of letters A and T and 3 for matches of the letters C and G, providing a rough approximation to double-strand bond energies in DNA. An inherent novelty of DNA codes is 'reverse complementation'. The latter may be defined, as follows, not only for alphabets of size four, but, more generally, for any even-size alphabet. All that is required is a matching of the letters of the alphabet: a partition into pairs. Then, the reverse complement of a codeword is obtained by reversing the order of its letters and replacing each letter by its match. For DNA, the matching is AT/CG because these are the Watson-Crick bonding pairs. Reversal arises because two DNA sequences form a double strand with opposite relative orientations. Thus, as will be described in detail, because in vitro decoding involves the formation of double-stranded DNA from two codewords, it is reasonable to assume - for universal applicability - that the reverse complement of any codeword is also a codeword. In particular, self-reverse complementary codewords are expressly forbidden in reverse-complement codes. Thus, an appropriate distance between all pairs of codewords must, when large, effectively prohibit binding between the respective codewords: to form a double strand. Only reverse-complement pairs of codewords should be able to bind. For most applications, a DNA code is to be bi-partitioned, such that the reverse-complementary pairs are separated

  3. Performance testing of a semi-automatic card punch system, using direct STR profiling of DNA from blood samples on FTA™ cards.

    PubMed

    Ogden, Samantha J; Horton, Jeffrey K; Stubbs, Simon L; Tatnell, Peter J

    2015-01-01

    The 1.2 mm Electric Coring Tool (e-Core™) was developed to increase the throughput of FTA(™) sample collection cards used during forensic workflows and is similar to a 1.2 mm Harris manual micro-punch for sampling dried blood spots. Direct short tandem repeat (STR) DNA profiling was used to compare samples taken by the e-Core tool with those taken by the manual micro-punch. The performance of the e-Core device was evaluated using a commercially available PowerPlex™ 18D STR System. In addition, an analysis was performed that investigated the potential carryover of DNA via the e-Core punch from one FTA disc to another. This contamination study was carried out using Applied Biosystems AmpflSTR™ Identifiler™ Direct PCR Amplification kits. The e-Core instrument does not contaminate FTA discs when a cleaning punch is used following excision of discs containing samples and generates STR profiles that are comparable to those generated by the manual micro-punch. PMID:25407399

  4. Fundamental research on scalable DNA molecular computation

    NASA Astrophysics Data System (ADS)

    Wang, Sixue

    Beginning with the ground-breaking work on DNA computation by Adleman in 1994 [2], the idea of using DNA molecules to perform computations has been explored extensively. In this thesis, a computation based on a scalable DNA neural network was discussed and a neuron model was partially implemented using DNA molecules. In order to understand the behavior of short DNA strands in a polyacrylamide gel, we have measured the mobilities of various short single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) shorter than 100 bases. We found that sufficiently short lengths of ssDNA had a higher mobility than same lengths of dsDNA, with a crossover length Lx at which the mobilities are equal. The crossover length decreases approximately linearly with polyacrylamide gel acrylamide concentration. At the same time, the influence of DNA structure on its mobility was studied and the effect of single-stranded overhangs on dsDNA was discussed. The idea to make a scalable DNA neural network was discussed. To prepare our basis vector DNA oligomers, a 90 base DNA template with 50 base random strand in the middle and two 20 base primers on the ends was designed and purchased. By a series of dilutions, we obtained several aliquots, containing only 30 random sequence molecules each. These were amplified to roughly 5 pico mole quantities by 38 cycles of PCR with hot start DNA polymerase. We then used asymmetric PCR followed by polyacrylamide gel purification to get the necessary single-stranded basis vectors (ssDNA) and their complements. We tested the suitability of this scheme by adding two vectors formed from different linear of the basis vectors. The full scheme for DNA neural network computation was tested using two determinate ssDNA strands. We successfully transformed an input DNA oligomer to a different output oligomer using the polymerase reaction required by the proposed DNA neural network algorithm. Isothermal linear amplification was used to obtain a sufficient quantity of

  5. DNA computing.

    PubMed

    Gibbons, A; Amos, M; Hodgson, D

    1997-02-01

    DNA computation is a novel and exciting recent development at the interface of computer science and molecular biology. We describe the current activity in this field following the seminal work of Adleman, who recently showed how techniques of molecular biology may be applied to the solution of a computationally intractable problem. PMID:9013647

  6. DNA Music.

    ERIC Educational Resources Information Center

    Miner, Carol; della Villa, Paula

    1997-01-01

    Describes an activity in which students reverse-translate proteins from their amino acid sequences back to their DNA sequences then assign musical notes to represent the adenine, guanine, cytosine, and thymine bases. Data is obtained from the National Institutes of Health (NIH) on the Internet. (DDR)

  7. DNA Investigations.

    ERIC Educational Resources Information Center

    Mayo, Ellen S.; Bertino, Anthony J.

    1991-01-01

    Presents a simulation activity that allow students to work through the exercise of DNA profiling and to grapple with some analytical and ethical questions involving a couple arranging with a surrogate mother to have a baby. Can be used to teach the principles of restriction enzyme digestion, gel electrophoresis, and probe hybridization. (MDH)

  8. DNA Methylation

    PubMed Central

    Marinus, M.G.; Løbner-Olesen, A.

    2014-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential and in C. crescentus, it is important for temporal gene expression which, in turn, is required for coordinating chromosome initiation, replication and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage; decrease transformation frequency in certain bacteria; decrease the stability of short direct repeats; are necessary for site-directed mutagenesis; and to probe eukaryotic structure and function. PMID:26442938

  9. Comparison of the benzoyl-DL-arginine-naphthylamide (BANA) test, DNA probes, and immunological reagents for ability to detect anaerobic periodontal infections due to Porphyromonas gingivalis, Treponema denticola, and Bacteroides forsythus.

    PubMed Central

    Loesche, W J; Lopatin, D E; Giordano, J; Alcoforado, G; Hujoel, P

    1992-01-01

    Most forms of periodontal disease are associated with the presence or overgrowth of anaerobic species that could include Treponema denticola, Porphyromonas gingivalis, and Bacteroides forsythus among others. These three organisms are among the few cultivable plaque species that can hydrolyze the synthetic trypsin substrate benzoyl-DL-arginine-naphthylamide (BANA). In turn, BANA hydrolysis by the plaque can be associated with periodontal morbidity and with the presence of these three BANA-positive organisms in the plaque. In this investigation, the results of the BANA test, which simultaneously detects one or more of these organisms, were compared with the detection of these organisms by (i) highly specific antibodies to P. gingivalis, T. denticola, and B. forsythus; (ii) whole genomic DNA probes to P. gingivalis and T. denticola; and (iii) culturing or microscopic procedures. The BANA test, the DNA probes, and an enzyme-linked immunosorbent assay or an indirect immunofluorescence assay procedure exhibited high sensitivities, i.e., 90 ot 96%, and high accuracies, i.e., 83 to 92%, in their ability to detect combinations of these organisms in over 200 subgingival plaque samples taken from the most periodontally diseased sites in 67 patients. This indicated that if P. gingivalis, T. denticola, and B. forsythus are appropriate marker organisms for an anaerobic periodontal infection, then the three detection methods are equally accurate in their ability to diagnose this infection. The same statement could not be made for the culturing approach, where accuracies of 50 to 62% were observed. PMID:1311335

  10. DNA Fingerprinting To Improve Data Collection Efficiency and Yield in a Host-Specificity Test of a Weed Biological Control Candidate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An open-field test was conducted in southern France to assess the host-specificity of Ceratapion basicorne, a candidate for biological control of yellow starthistle (Centaurea solstitialis; YST). Test plants were infested by naturally occurring populations of C. basicorne but were also exposed to s...

  11. DNA: The Strand that Connects Us All

    SciTech Connect

    Kaplan, Matt

    2011-03-29

    Learn how the methods and discoveries of human population genetics are applied for personal genealogical reconstruction and anthropological testing. Dr. Kaplan starts with a short general review of human genetics and the biology behind this form of DNA testing. He looks at how DNA testing is performed and how samples are processed in the University of Arizona laboratory. He also examines examples of personal genealogical results from Family Tree DNA and personal anthropological results from the Genographic Project. Finally, he describes the newest project in the UA laboratory, the DNA Shoah Project.

  12. An improved DNA-based test for detection of the codon 616 mutation in the alpha cyclic GMP phosphodiesterase gene that causes progressive retinal atrophy in the Cardigan Welsh Corgi.

    PubMed

    Petersen-Jones, Simon M; Entz, David D

    2002-06-01

    The aim of the study was to develop an improved test to detect the codon 616 gene mutation in the alpha cyclic GMP phosphodiesterase gene that causes progressive retinal atrophy in the Cardigan Welsh Corgi. We studied 10 control dogs of known genotype at codon 616 of the alpha cyclic GMP phosphodiesterase gene and 80 Cardigan Welsh Corgis of unknown genotype. A polymerase chain reaction (PCR) utilizing a mismatched primer was designed so that it introduced a HinfI restriction enzyme digestion site into the PCR product only if the normal gene sequence was present, the restriction site was not introduced if the codon 616 mutation was present. An additional HinfI site present in the amplified section from both normal and mutant alleles acted as a positive control for restriction enzyme digestion. The PCR reliably amplified a portion of the alpha cyclic GMP phosphodiesterase gene spanning the codon 616 mutation site. Restriction enzyme digestion with HinfI and analysis on a suitable agarose gel reliably ascertained the genotype of the control dogs and was used to identify the genotype of a further 80 test dogs. An improved DNA-based test for detection of the codon 616 mutation in the alpha cyclic GMP phosphodiesterase gene that causes progressive retinal atrophy in the Cardigan Welsh Corgi has been designed. This overcomes potential problems that could be associated with allele-specific PCR tests such as that used previously in a diagnostic test for this gene mutation. PMID:12071867

  13. Authentication of forensic DNA samples.

    PubMed

    Frumkin, Dan; Wasserstrom, Adam; Davidson, Ariane; Grafit, Arnon

    2010-02-01

    Over the past twenty years, DNA analysis has revolutionized forensic science, and has become a dominant tool in law enforcement. Today, DNA evidence is key to the conviction or exoneration of suspects of various types of crime, from theft to rape and murder. However, the disturbing possibility that DNA evidence can be faked has been overlooked. It turns out that standard molecular biology techniques such as PCR, molecular cloning, and recently developed whole genome amplification (WGA), enable anyone with basic equipment and know-how to produce practically unlimited amounts of in vitro synthesized (artificial) DNA with any desired genetic profile. This artificial DNA can then be applied to surfaces of objects or incorporated into genuine human tissues and planted in crime scenes. Here we show that the current forensic procedure fails to distinguish between such samples of blood, saliva, and touched surfaces with artificial DNA, and corresponding samples with in vivo generated (natural) DNA. Furthermore, genotyping of both artificial and natural samples with Profiler Plus((R)) yielded full profiles with no anomalies. In order to effectively deal with this problem, we developed an authentication assay, which distinguishes between natural and artificial DNA based on methylation analysis of a set of genomic loci: in natural DNA, some loci are methylated and others are unmethylated, while in artificial DNA all loci are unmethylated. The assay was tested on natural and artificial samples of blood, saliva, and touched surfaces, with complete success. Adopting an authentication assay for casework samples as part of the forensic procedure is necessary for maintaining the high credibility of DNA evidence in the judiciary system. PMID:20129467

  14. The future of forensic DNA analysis

    PubMed Central

    Butler, John M.

    2015-01-01

    The author's thoughts and opinions on where the field of forensic DNA testing is headed for the next decade are provided in the context of where the field has come over the past 30 years. Similar to the Olympic motto of ‘faster, higher, stronger’, forensic DNA protocols can be expected to become more rapid and sensitive and provide stronger investigative potential. New short tandem repeat (STR) loci have expanded the core set of genetic markers used for human identification in Europe and the USA. Rapid DNA testing is on the verge of enabling new applications. Next-generation sequencing has the potential to provide greater depth of coverage for information on STR alleles. Familial DNA searching has expanded capabilities of DNA databases in parts of the world where it is allowed. Challenges and opportunities that will impact the future of forensic DNA are explored including the need for education and training to improve interpretation of complex DNA profiles. PMID:26101278

  15. Ligation errors in DNA computing.

    PubMed

    Aoi, Y; Yoshinobu, T; Tanizawa, K; Kinoshita, K; Iwasaki, H

    1999-10-01

    DNA computing is a novel method of computing proposed by Adleman (1994), in which the data is encoded in the sequences of oligonucleotides. Massively parallel reactions between oligonucleotides are expected to make it possible to solve huge problems. In this study, reliability of the ligation process employed in the DNA computing is tested by estimating the error rate at which wrong oligonucleotides are ligated. Ligation of wrong oligonucleotides would result in a wrong answer in the DNA computing. The dependence of the error rate on the number of mismatches between oligonucleotides and on the combination of bases is investigated. PMID:10636043

  16. A DNA extraction protocol for improved DNA yield from individual mosquitoes.

    PubMed

    Nieman, Catelyn C; Yamasaki, Youki; Collier, Travis C; Lee, Yoosook

    2015-01-01

    Typical DNA extraction protocols from commercially available kits provide an adequate amount of DNA from a single individual mosquito sufficient for PCR-based assays. However, next-generation sequencing applications and high-throughput SNP genotyping assays exposed the limitation of DNA quantity one usually gets from a single individual mosquito. Whole genome amplification could alleviate the issue but it also creates bias in genome representation. While trying to find alternative DNA extraction protocols for improved DNA yield, we found that a combination of the tissue lysis protocol from Life Technologies and the DNA extraction protocol from Qiagen yielded a higher DNA amount than the protocol using the Qiagen or Life Technologies kit only. We have not rigorously tested all the possible combinations of extraction protocols; we also only tested this on mosquito samples. Therefore, our finding should be noted as a suggestion for improving people's own DNA extraction protocols and not as an advertisement of a commercially available product. PMID:26937269

  17. Testing Potential Effects of Maize Expressing the Bacillus thuringiensis Cry1Ab Endotoxin (Bt Maize) on Mycorrhizal Fungal Communities via DNA- and RNA-Based Pyrosequencing and Molecular Fingerprinting

    PubMed Central

    Kuramae, Eiko E.; Hillekens, Remy; de Hollander, Mattias; Kiers, E. Toby; Röling, Wilfred F. M.; Kowalchuk, George A.; van der Heijden, Marcel G. A.

    2012-01-01

    The cultivation of genetically modified (GM) crops has increased significantly over the last decades. However, concerns have been raised that some GM traits may negatively affect beneficial soil biota, such as arbuscular mycorrhizal fungi (AMF), potentially leading to alterations in soil functioning. Here, we test two maize varieties expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt maize) for their effects on soil AM fungal communities. We target both fungal DNA and RNA, which is new for AM fungi, and we use two strategies as an inclusive and robust way of detecting community differences: (i) 454 pyrosequencing using general fungal rRNA gene-directed primers and (ii) terminal restriction fragment length polymorphism (T-RFLP) profiling using AM fungus-specific markers. Potential GM-induced effects were compared to the normal natural variation of AM fungal communities across 15 different agricultural fields. AM fungi were found to be abundant in the experiment, accounting for 8% and 21% of total recovered DNA- and RNA-derived fungal sequences, respectively, after 104 days of plant growth. RNA- and DNA-based sequence analyses yielded most of the same AM fungal lineages. Our research yielded three major conclusions. First, no consistent differences were detected between AM fungal communities associated with GM plants and non-GM plants. Second, temporal variation in AMF community composition (between two measured time points) was bigger than GM trait-induced variation. Third, natural variation of AMF communities across 15 agricultural fields in The Netherlands, as well as within-field temporal variation, was much higher than GM-induced variation. In conclusion, we found no indication that Bt maize cultivation poses a risk for AMF. PMID:22885748

  18. Testing potential effects of maize expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt maize) on mycorrhizal fungal communities via DNA- and RNA-based pyrosequencing and molecular fingerprinting.

    PubMed

    Verbruggen, Erik; Kuramae, Eiko E; Hillekens, Remy; de Hollander, Mattias; Kiers, E Toby; Röling, Wilfred F M; Kowalchuk, George A; van der Heijden, Marcel G A

    2012-10-01

    The cultivation of genetically modified (GM) crops has increased significantly over the last decades. However, concerns have been raised that some GM traits may negatively affect beneficial soil biota, such as arbuscular mycorrhizal fungi (AMF), potentially leading to alterations in soil functioning. Here, we test two maize varieties expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt maize) for their effects on soil AM fungal communities. We target both fungal DNA and RNA, which is new for AM fungi, and we use two strategies as an inclusive and robust way of detecting community differences: (i) 454 pyrosequencing using general fungal rRNA gene-directed primers and (ii) terminal restriction fragment length polymorphism (T-RFLP) profiling using AM fungus-specific markers. Potential GM-induced effects were compared to the normal natural variation of AM fungal communities across 15 different agricultural fields. AM fungi were found to be abundant in the experiment, accounting for 8% and 21% of total recovered DNA- and RNA-derived fungal sequences, respectively, after 104 days of plant growth. RNA- and DNA-based sequence analyses yielded most of the same AM fungal lineages. Our research yielded three major conclusions. First, no consistent differences were detected between AM fungal communities associated with GM plants and non-GM plants. Second, temporal variation in AMF community composition (between two measured time points) was bigger than GM trait-induced variation. Third, natural variation of AMF communities across 15 agricultural fields in The Netherlands, as well as within-field temporal variation, was much higher than GM-induced variation. In conclusion, we found no indication that Bt maize cultivation poses a risk for AMF. PMID:22885748

  19. Post-hybridization recovery of membrane filter-bound DNA for enzymatic DNA amplification.

    PubMed

    Chong, K Y; Chen, C M; Choo, K B

    1993-04-01

    We describe here a simple and rapid method for enzymatic DNA amplification using DNA template recovered from membrane filters previously used in hybridization analysis. This is done by first solubilizing membrane pieces carrying DNA of interest in dimethyl sulfoxide, followed by isopropanol precipitation and polymerase chain reaction amplification. The source of membrane-bound DNA successfully tested includes plasmid and human leukocyte DNA and DNA immobilized on bacterial colony filters and plaque lifts. The sensitivity of the procedure is such that DNA recovered from 0.5 microgram of filter-bound total human DNA was enough for PCR amplification of a 0.3-kb fragment. Our protocol will be useful for recycling of scarce DNA samples for cloning and sequencing purposes. PMID:8476600

  20. DNA Vaccines: Experiences in the Swine Model.

    PubMed

    Accensi, Francesc; Rodríguez, Fernando; Monteagudo, Paula L

    2016-01-01

    DNA vaccination is one of the most fascinating vaccine-strategies currently in development. Two of the main advantages of DNA immunization rely on its simplicity and flexibility, being ideal to dissect both the immune mechanisms and the antigens involved in protection against a given pathogen. Here, we describe several strategies used to enhance the immune responses induced and the protection afforded by experimental DNA vaccines tested in swine and provide with very basic protocol describing the generation and in vivo application of a prototypic DNA vaccine. Only time will tell the last word regarding the definitive implementation of DNA vaccination in the field. PMID:26458829

  1. Enzyme immunoassay for anti-treponemal IgG: screening or confirmatory test?

    PubMed Central

    Young, H; Moyes, A; McMillan, A; Patterson, J

    1992-01-01

    AIMS: To review the performance of the Venereal Diseases Research Laboratory (VDRL) test and the Treponema pallidum haemagglutination assay (TPHA) as a combined screen for syphilis to provide a baseline for assessing screening by anti-treponemal IGG EIA. METHODS: Between 1980 and 1987 all serum samples were screened by both VDRL and TPHA tests. The FTA-ABS test was also used in suspected early primary syphilis, or when one of the other tests was positive. A positive result in a screening test was confirmed by quantitative testing. From 1988 all specimens were screened with an enzyme immunoassay (Captia Syph G) as a single screening test. RESULTS: Of the 44 primary, 47 secondary, and 38 early latent cases of syphilis, the VDRL and TPHA detected 32 (73%) and 31 (71%) of the primary cases; the combination detected 37 (84%). All 85 cases of cases of secondary and early latent infection were reactive in the TPHA test, whereas the VDRL was reactive in only 68 (80%). EIA had a reported sensitivity of 82% for primary infection. CONCLUSIONS: EIA can be used as a single screening test for detecting early syphilis because its results are comparable with those of the combined VDRL and TPHA tests. The conventional VDRL test should not be used as a single screening test. PMID:1740512

  2. DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  3. Cell-free DNA testing in a trisomy 21 pregnancy with confined placental mosaicism for a cell line with trisomy for both chromosomes 18 and 21.

    PubMed

    Crooks, Kristy; Edwardsen, Ginger; O'Connor, Siobhan; Powell, Cynthia; Vargo, Diane; Vora, Neeta; Kaiser-Rogers, Kathleen

    2016-01-01

    NIPT (noninvasive prenatal testing) detected trisomy for two chromosomes. One trisomy reflected the fetal karyotype, and the other resulted from CPM (confined placental mosaicism). This case illustrates that extensive cytogenetic analysis can be required to identify CPM, and that patients should be counseled regarding the possibility of discordant NIPT results. PMID:26783428

  4. Testing the use of ITS rDNA and protein-coding genes in the generic and species delimitation of the lichen genus Usnea (Parmeliaceae, Ascomycota).

    PubMed

    Truong, Camille; Divakar, Pradeep K; Yahr, Rebecca; Crespo, Ana; Clerc, Philippe

    2013-08-01

    In lichen-forming fungi, traditional taxonomical concepts are frequently in conflict with molecular data, and identifying appropriate taxonomic characters to describe phylogenetic clades remains challenging in many groups. The selection of suitable markers for the reconstruction of solid phylogenetic hypotheses is therefore fundamental. The lichen genus Usnea is highly diverse, with more than 350 estimated species, distributed in polar, temperate and tropical regions. The phylogeny and classification of Usnea have been a matter of debate, given the lack of phenotypic characters to describe phylogenetic clades and the low degree of resolution of phylogenetic trees. In this study, we investigated the phylogenetic relationships of 52 Usnea species from across the genus, based on ITS rDNA, nuLSU, and two protein-coding genes RPB1 and MCM7. ITS comprised several highly variable regions, containing substantial genetic signal, but also susceptible to causing bias in the generation of the alignment. We compared several methods of alignment of ITS and found that a simultaneous optimization of alignment and phylogeny (using BAli-phy) improved significantly both the topology and the resolution of the phylogenetic tree. However the resolution was even better when using protein-coding genes, especially RPB1 although it is less variable. The phylogeny based on the concatenated dataset revealed that the genus Usnea is subdivided into four highly-supported clades, corresponding to the traditionally circumscribed subgenera Eumitria, Dolichousnea, Neuropogon and Usnea. However, characters that have been used to describe these clades are often homoplasious within the phylogeny and their parallel evolution is suggested. On the other hand, most of the species were reconstructed as monophyletic, indicating that combinations of phenotypic characters are suitable discriminators for delimitating species, but are inadequate to describe generic subdivisions. PMID:23603312

  5. Comparison of Target-Capture and Restriction-Site Associated DNA Sequencing for Phylogenomics: A Test in Cardinalid Tanagers (Aves, Genus: Piranga).

    PubMed

    Manthey, Joseph D; Campillo, Luke C; Burns, Kevin J; Moyle, Robert G

    2016-07-01

    Restriction-site associated DNA sequencing (RAD-seq) and target capture of specific genomic regions, such as ultraconserved elements (UCEs), are emerging as two of the most popular methods for phylogenomics using reduced-representation genomic data sets. These two methods were designed to target different evolutionary timescales: RAD-seq was designed for population-genomic level questions and UCEs for deeper phylogenetics. The utility of both data sets to infer phylogenies across a variety of taxonomic levels has not been adequately compared within the same taxonomic system. Additionally, the effects of uninformative gene trees on species tree analyses (for target capture data) have not been explored. Here, we utilize RAD-seq and UCE data to infer a phylogeny of the bird genus Piranga The group has a range of divergence dates (0.5-6 myr), contains 11 recognized species, and lacks a resolved phylogeny. We compared two species tree methods for the RAD-seq data and six species tree methods for the UCE data. Additionally, in the UCE data, we analyzed a complete matrix as well as data sets with only highly informative loci. A complete matrix of 189 UCE loci with 10 or more parsimony informative (PI) sites, and an approximately 80% complete matrix of 1128 PI single-nucleotide polymorphisms (SNPs) (from RAD-seq) yield the same fully resolved phylogeny of Piranga We inferred non-monophyletic relationships of Piranga lutea individuals, with all other a priori species identified as monophyletic. Finally, we found that species tree analyses that included predominantly uninformative gene trees provided strong support for different topologies, with consistent phylogenetic results when limiting species tree analyses to highly informative loci or only using less informative loci with concatenation or methods meant for SNPs alone. PMID:26821912

  6. Paternity testing.

    PubMed

    Onoja, A M

    2011-01-01

    Molecular diagnostic techniques have found application in virtually all areas of medicine, including criminal investigations and forensic analysis. The techniques have become so precise that it is now possible to conclusively determine paternity using DNA from grand parents, cousins, or even saliva left on a discarded cigarette butt. This is a broad overview of paternity testing. PMID:22288312

  7. Wrinkled DNA.

    PubMed Central

    Arnott, S; Chandrasekaran, R; Puigjaner, L C; Walker, J K; Hall, I H; Birdsall, D L; Ratliff, R L

    1983-01-01

    The B form of poly d(GC):poly d(GC) in orthorhombic microcrystallites in oriented fibers has a secondary structure in which a dinucleotide is the repeated motif rather than a mononucleotide as in standard, smooth B DNA. One set of nucleotides (probably GpC) has the same conformations as the smooth form but the alternate (CpG) nucleotides have a different conformation at C3'-O3'. This leads to a distinctive change in the orientation of the phosphate groups. Similar perturbations can be detected in other poly d(PuPy):poly d(PuPy) DNAs such as poly d(IC):poly d(IC) and poly d(AT):poly d(AT) in their D forms which have tetragonal crystal environments. This suggests that such perturbations are intrinsic to all stretches of duplex DNA where purines and pyrimidines alternate and may play a role in the detection and exploitation of such sequences by regulatory proteins. Images PMID:6572358

  8. Optical DNA

    NASA Astrophysics Data System (ADS)

    Vijaywargi, Deepak; Lewis, Dave; Kirovski, Darko

    A certificate of authenticity (COA) is an inexpensive physical object with a random and unique structure S which is hard to near-exactly replicate. An inexpensive device should be able to scan object’s physical “fingerprint,” a set of features that represents S. In this paper, we explore one set of requirements that optical media such as DVDs should satisfy, to be considered as COAs. As manufacturing of such media produces inevitable errors, we use the locations and count of these errors as a “fingerprint” for each optical disc: its optical DNA. The “fingerprint” is signed using publisher’s private-key and the resulting signature is stored onto the optical medium using a post-production process. Standard DVD players with altered firmware that includes publisher’s public-key, should be able to verify the authenticity of DVDs protected with optical DNA. Our key finding is that for the proposed protocol, only DVDs with exceptional wear-and-tear characteristics would result in an inexpensive and viable anti-counterfeiting technology.

  9. Laser mass spectrometry for DNA sequencing, disease diagnosis, and fingerprinting

    NASA Astrophysics Data System (ADS)

    Chen, C. H. Winston; Taranenko, N. I.; Zhu, Y. F.; Chung, C. N.; Allman, S. L.

    1997-05-01

    Since laser mass spectrometry has the potential for achieving very fast DNA analysis, we recently applied it to DNA sequencing, DNA typing for fingerprinting, and DNA screening for disease diagnosis. Two different approaches for sequencing DNA have been successfully demonstrated. One is to sequence DNA with DNA ladders produced from Sanger's enzymatic method. The other is to do direct sequencing without DNA ladders. The need for quick DNA typing for identification purposes is critical for forensic application. Our preliminary results indicate laser mass spectrometry can possible be used for rapid DNA fingerprinting applications at a much lower cost than gel electrophoresis. Population screening for certain genetic disease can be a very efficient step to reducing medical costs through prevention. Since laser mass spectrometry can provide very fast DNA analysis, we applied laser mass spectrometry to disease diagnosis. Clinical samples with both base deletion and point mutation have been tested with complete success.

  10. Laser mass spectrometry for DNA sequencing, disease diagnosis, and fingerprinting

    SciTech Connect

    Winston Chen, C.H.; Taranenko, N.I.; Zhu, Y.F.; Chung, C.N.; Allman, S.L.

    1997-03-01

    Since laser mass spectrometry has the potential for achieving very fast DNA analysis, the authors recently applied it to DNA sequencing, DNA typing for fingerprinting, and DNA screening for disease diagnosis. Two different approaches for sequencing DNA have been successfully demonstrated. One is to sequence DNA with DNA ladders produced from Snager`s enzymatic method. The other is to do direct sequencing without DNA ladders. The need for quick DNA typing for identification purposes is critical for forensic application. The preliminary results indicate laser mass spectrometry can possibly be used for rapid DNA fingerprinting applications at a much lower cost than gel electrophoresis. Population screening for certain genetic disease can be a very efficient step to reducing medical costs through prevention. Since laser mass spectrometry can provide very fast DNA analysis, the authors applied laser mass spectrometry to disease diagnosis. Clinical samples with both base deletion and point mutation have been tested with complete success.

  11. Implication of complex vertebral malformation and bovine leukocyte adhesion deficiency DNA-based testing on disease frequency in the Holstein population.

    PubMed

    Schütz, E; Scharfenstein, M; Brenig, B

    2008-12-01

    Two inherited lethal disorders, bovine leukocyte adhesion deficiency (BLAD) and complex vertebral malformation (CVM), play a major role in breeding of Holstein cattle. Both inherited diseases are based on single nucleotide polymorphisms that have been known for 12 and 7 yr, respectively. A total of 25,753 cattle were genotyped for BLAD (18,200 tests) and CVM (14,493 tests) in our laboratory since the beginning of the genotyping programs for these diseases. Based on founder effects, the CVM mutation is thought to be linked to milk production. The BLAD was genotyped using RFLP until 2001; then a fluorescence resonance energy transfer assay on a LightCycler was used, as for CVM genotyping. By using single nucleotide polymorphism-aided breeding, the allelic frequency of the BLAD and CVM mutations in the active sire population was reduced from 9.4% in 1997 to 0.3% in 2007 (BLAD) and from 8.3% in 2002 to 2.3% in 2007 (CVM), with calculated half-life of the mutant allele of 2.1 yr for BLAD and 3.6 yr for CVM. An observed increase of BLAD frequency in 1999 could be attributed to the massive use of a BLAD-positive sire tested falsely negative in another laboratory. These data show that marker-assisted selection is capable of substantially reducing the frequency of a mutation within a period of not more than 5 yr. The different selection strategies against the lethal recessive allele in CVM and BLAD are reflected in the different reduction rates of the specific allele frequencies. PMID:19038961

  12. DNA mimicry by proteins.

    PubMed

    Dryden, D T F; Tock, M R

    2006-04-01

    It has been discovered recently, via structural and biophysical analyses, that proteins can mimic DNA structures in order to inhibit proteins that would normally bind to DNA. Mimicry of the phosphate backbone of DNA, the hydrogen-bonding properties of the nucleotide bases and the bending and twisting of the DNA double helix are all present in the mimics discovered to date. These mimics target a range of proteins and enzymes such as DNA restriction enzymes, DNA repair enzymes, DNA gyrase and nucleosomal and nucleoid-associated proteins. The unusual properties of these protein DNA mimics may provide a foundation for the design of targeted inhibitors of DNA-binding proteins. PMID:16545103

  13. Recombinant DNA Paper Model Simulation: The Genetic Engineer.

    ERIC Educational Resources Information Center

    Wagner, Joan

    1998-01-01

    Describes a course for talented high school students that focuses on DNA science and technology. Employs Cold Spring Harbor's DNA Science laboratory manual. Engages students in performing sickle-cell anemia and thalassemia tests in rabbits. (DDR)

  14. Evaluation of primary HPV-DNA testing in relation to visual inspection methods for cervical cancer screening in rural China: an epidemiologic and cost-effectiveness modelling study

    PubMed Central

    2011-01-01

    Background A new lower-cost rapid-throughput human papillomavirus (HPV) test (careHPV, Qiagen, Gaithersburg, USA) has been shown to have high sensitivity for the detection of high grade cervical intraepithelial neoplasia. Methods We assessed the outcomes and cost-effectiveness of careHPV screening in rural China, compared to visual inspection with acetic acid, when used alone (VIA) or in combination with Lugol's iodine (VIA/VILI). Using data on sexual behaviour, test accuracy, diagnostic practices and costs from studies performed in rural China, we estimated the cost-effectiveness ratio (CER) and associated lifetime outcomes for once-lifetime and twice-lifetime screening strategies, and for routine screening at 5-yearly, 10-yearly and IARC-recommended intervals. The optimal age range for once-lifetime screening was also assessed. Results For all strategies, the relative ordering of test technologies in reducing cervical cancer incidence and mortality was VIA (least effective); VIA/VILI; careHPV@1.0 pg/ml and careHPV@0.5 pg/ml (most effective). For once-lifetime strategies, maximum effectiveness was achieved if screening occurred between 35-50 years. Assuming a participation rate of ~70%, once-lifetime screening at age 35 years would reduce cancer mortality by 8% (for VIA) to 12% (for careHPV@0.5) over the long term, with a CER of US$557 (for VIA) to $959 (for careHPV@1.0) per life year saved (LYS) compared to no intervention; referenced to a 2008 GDP per capita in Shanxi Province of $2,975. Correspondingly, regular screening with an age-standardised participation rate of 62% (which has been shown to be achievable in this setting) would reduce cervical cancer mortality by 19-28% (for 10-yearly screening) to 43-54% (using IARC-recommended intervals), with corresponding CERs ranging from $665 (for 10-yearly VIA) to $2,269 (for IARC-recommended intervals using careHPV@1.0) per LYS. Conclusions This modelled analysis suggests that primary careHPV screening compares

  15. Short Tandem Repeat DNA Internet Database

    National Institute of Standards and Technology Data Gateway

    SRD 130 Short Tandem Repeat DNA Internet Database (Web, free access)   Short Tandem Repeat DNA Internet Database is intended to benefit research and application of short tandem repeat DNA markers for human identity testing. Facts and sequence information on each STR system, population data, commonly used multiplex STR systems, PCR primers and conditions, and a review of various technologies for analysis of STR alleles have been included.

  16. Compressive Sensing DNA Microarrays

    PubMed Central

    2009-01-01

    Compressive sensing microarrays (CSMs) are DNA-based sensors that operate using group testing and compressive sensing (CS) principles. In contrast to conventional DNA microarrays, in which each genetic sensor is designed to respond to a single target, in a CSM, each sensor responds to a set of targets. We study the problem of designing CSMs that simultaneously account for both the constraints from CS theory and the biochemistry of probe-target DNA hybridization. An appropriate cross-hybridization model is proposed for CSMs, and several methods are developed for probe design and CS signal recovery based on the new model. Lab experiments suggest that in order to achieve accurate hybridization profiling, consensus probe sequences are required to have sequence homology of at least 80% with all targets to be detected. Furthermore, out-of-equilibrium datasets are usually as accurate as those obtained from equilibrium conditions. Consequently, one can use CSMs in applications in which only short hybridization times are allowed. PMID:19158952

  17. The DNA Files

    SciTech Connect

    1998-06-09

    The DNA Files is a radio documentary which disseminates genetics information over public radio. The documentaries explore subjects which include the following: How genetics affects society. How human life began and how it evolved. Could new prenatal genetic tests hold the key to disease prevention later in life? Would a national genetic data base sacrifice individual privacy? and Should genes that may lead to the cure for cancer be privately owned? This report serves as a project update for the second quarter of 1998. It includes the spring/summer 1998 newsletter, the winter 1998 newsletter, the program clock, and the latest flyer.

  18. Mitochondrial DNA replacement versus nuclear DNA persistence

    NASA Astrophysics Data System (ADS)

    Serva, Maurizio

    2006-10-01

    In this paper we consider two populations whose generations are not overlapping and whose size is large. The number of males and females in both populations is constant. Any generation is replaced by a new one and any individual has two parents concerning nuclear DNA and a single one (the mother) concerning mtDNA. Moreover, at any generation some individuals migrate from the first population to the second. In a finite random time T, the mtDNA of the second population is completely replaced by the mtDNA of the first. In the same time, the nuclear DNA is not completely replaced and a fraction F of the ancient nuclear DNA persists. We compute both T and F. Since this study shows that complete replacement of mtDNA in a population is compatible with the persistence of a large fraction of nuclear DNA, it may have some relevance for the 'out of Africa'/multiregional debate in palaeoanthropology.

  19. DNA modifications: Another stable base in DNA

    NASA Astrophysics Data System (ADS)

    Brazauskas, Pijus; Kriaucionis, Skirmantas

    2014-12-01

    Oxidation of 5-methylcytosine has been proposed to mediate active and passive DNA demethylation. Tracking the history of DNA modifications has now provided the first solid evidence that 5-hydroxymethylcytosine is a stable epigenetic modification.

  20. Development of an Optimized Protocol for NMR Metabolomics Studies of Human Colon Cancer Cell Lines and First Insight from Testing of the Protocol Using DNA G-Quadruplex Ligands as Novel Anti-Cancer Drugs

    PubMed Central

    Lauri, Ilaria; Savorani, Francesco; Iaccarino, Nunzia; Zizza, Pasquale; Pavone, Luigi Michele; Novellino, Ettore; Engelsen, Søren Balling; Randazzo, Antonio

    2016-01-01

    The study of cell lines by nuclear magnetic resonance (NMR) spectroscopy metabolomics represents a powerful tool to understand how the local metabolism and biochemical pathways are influenced by external or internal stimuli. In particular, the use of adherent mammalian cells is emerging in the metabolomics field in order to understand the molecular mechanism of disease progression or, for example, the cellular response to drug treatments. Hereto metabolomics investigations for this kind of cells have generally been limited to mass spectrometry studies. This study proposes an optimized protocol for the analysis of the endo-metabolome of human colon cancer cells (HCT116) by NMR. The protocol includes experimental conditions such as washing, quenching and extraction. In order to test the proposed protocol, it was applied to an exploratory study of cancer cells with and without treatment by anti-cancer drugs, such as DNA G-quadruplex binders and Adriamycin (a traditional anti-cancer drug). The exploratory NMR metabolomics analysis resulted in NMR assignment of all endo-metabolites that could be detected and provided preliminary insights about the biological behavior of the drugs tested. PMID:26784246

  1. The characterization of four gene expression analysis in circulating tumor cells made by Multiplex-PCR from the AdnaTest kit on the lab-on-a-chip Agilent DNA 1000 platform

    PubMed Central

    Škereňová, Markéta; Mikulová, Veronika; Čapoun, Otakar; Zima, Tomáš

    2016-01-01

    Introduction Nowadays, on-a-chip capillary electrophoresis is a routine method for the detection of PCR fragments. The Agilent 2100 Bioanalyzer was one of the first commercial devices in this field. Our project was designed to study the characteristics of Agilent DNA 1000 kit in PCR fragment analysis as a part of circulating tumour cell (CTC) detection technique. Despite the common use of this kit a complex analysis of the results from a long-term project is still missing. Materials and methods A commercially available Agilent DNA 1000 kit was used as a final step in the CTC detection (AdnaTest) for the determination of the presence of PCR fragments generated by Multiplex PCR. Data from 30 prostate cancer patients obtained during two years of research were analyzed to determine the trueness and precision of the PCR fragment size determination. Additional experiments were performed to demonstrate the precision (repeatability, reproducibility) and robustness of PCR fragment concentration determination. Results The trueness and precision of the size determination was below 3% and 2% respectively. The repeatability of the concentration determination was below 15%. The difference in concentration determination increases when Multiplex-PCR/storage step is added between the two measurements of one sample. Conclusions The characteristics established in our study are in concordance with the manufacturer’s specifications established for a ladder as a sample. However, the concentration determination may vary depending on chip preparation, sample storage and concentration. The 15% variation of concentration determination repeatability was shown to be partly proportional and can be suppressed by proper normalization. PMID:26981024

  2. What is a "DNA-Compatible" Reaction?

    PubMed

    Malone, Marie L; Paegel, Brian M

    2016-04-11

    DNA-encoded synthesis can generate vastly diverse screening libraries of arbitrarily complex molecules as long as chemical reaction conditions do not compromise DNA's informational integrity, a fundamental constraint that "DNA-compatible" reaction development does not presently address. We devised DNA-encoded reaction rehearsal, an integrated analysis of reaction yield and impact on DNA, to acquire these key missing data. Magnetic DNA-functionalized sensor beads quantitatively report the % DNA template molecules remaining viable for PCR amplification after exposure to test reaction conditions. Analysis of solid-phase bond forming (e.g., Suzuki-Miyaura cross-coupling, reductive amination) and deprotection reactions (e.g., allyl esters, silyl ethers) guided the definition and optimization of DNA-compatible reaction conditions (>90% yield, >30% viable DNA molecules), most notably in cases that involved known (H(+), Pd) and more obscure (Δ, DMF) hazards to DNA integrity. The data provide an empirical yet mechanistically consistent and predictive framework for designing successful DNA-encoded reaction sequences for combinatorial library synthesis. PMID:26971959

  3. DNA fingerprinting and the substantive law.

    PubMed

    Singh, D

    1994-01-01

    The ability to analyse human DNA has the potential to revolutionize legal proceedings--both civil and criminal--especially those which turn on biological or genetic evidence. Examining the positive and negative aspects of DNA fingerprinting, it would appear that the benefits certainly outweigh any disadvantages. In paternity determinations, DNA fingerprinting renders otiose many of the controversial concerns, particularly regarding the determination of legal probability, providing as it does unequivocal evidence of blood relationship. Furthermore, in criminal law, its value is well-documented. The relevant legislation is already in place, therefore no further legislation is required to enable South African law enforcement agencies to make use of the DNA test. The apparently high cost may, however, frustrate its general application to the civil law but with the assistance of the state and law societies, the envisaged problem could be alleviated. To date no other forensic tests rival the accuracy of the DNA test. PMID:7968306

  4. Construction of DNA Hemicatenanes from Two Small Circular DNA Molecules

    PubMed Central

    Gaillard, Claire; Strauss, François

    2015-01-01

    DNA hemicatenanes, one of the simplest possible junctions between two double stranded DNA molecules, have frequently been mentioned in the literature for their possible function in DNA replication, recombination, repair, and organization in chromosomes. They have been little studied experimentally, however, due to the lack of an appropriate method for their preparation. Here we have designed a method to build hemicatenanes from two small circular DNA molecules. The method involves, first, the assembly of two linear single strands and their circularization to form a catenane of two single stranded circles, and, second, the addition and base-pairing of the two single stranded circles complementary to the first ones, followed by their annealing using DNA topoisomerase I. The product was purified by gel electrophoresis and characterized. The arrangement of strands was as expected for a hemicatenane and clearly distinct from a full catenane. In addition, each circle was unwound by an average of half a double helical turn, also in excellent agreement with the structure of a hemicatenane. It was also observed that hemicatenanes are quickly destabilized by a single cut on either of the two strands passing inside the junction, strongly suggesting that DNA strands are able to slide easily inside the hemicatenane. This method should make it possible to study the biochemical properties of hemicatenanes and to test some of the hypotheses that have been proposed about their function, including a possible role for this structure in the organization of complex genomes in loops and chromosomal domains. PMID:25799010

  5. Synthesis of DNA

    DOEpatents

    Mariella, Jr., Raymond P.

    2008-11-18

    A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

  6. Sperm DNA oxidative damage and DNA adducts.

    PubMed

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-12-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps=0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps=0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps=0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on sperm

  7. DNA encoding a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-08-15

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  8. DNA polymerases and cancer

    PubMed Central

    Lange, Sabine S.; Takata, Kei-ichi; Wood, Richard D.

    2013-01-01

    There are fifteen different DNA polymerases encoded in mammalian genomes, which are specialized for replication, repair or the tolerance of DNA damage. New evidence is emerging for lesion-specific and tissue-specific functions of DNA polymerases. Many point mutations that occur in cancer cells arise from the error-generating activities of DNA polymerases. However, the ability of some of these enzymes to bypass DNA damage may actually defend against chromosome instability in cells and at least one DNA polymerase, POLζ, is a suppressor of spontaneous tumorigenesis. Because DNA polymerases can help cancer cells tolerate DNA damage, some of these enzymes may be viable targets for therapeutic strategies. PMID:21258395

  9. Salmon redd identification using environmental DNA (eDNA)

    USGS Publications Warehouse

    Pilliod, David S.; Laramie, Matthew B.

    2016-01-01

    IntroductionThe purpose of this project was to develop a technique to use environmental DNA (eDNA) to distinguish between redds made by Chinook salmon (Oncorhynchus tshawytscha) and redds made by Coho salmon (O. kisutch) and to distinguish utilized redds from test/abandoned redds or scours that have the appearance of redds. The project had two phases:Phase 1. Develop, test, and optimize a molecular assay for detecting and identifying Coho salmon DNA and differentiating it from Chinook salmon DNA.Phase 2. Demonstrate the efficacy of the technique.Collect and preserve water samples from the interstitial spaces of 10 known redds (as identified by expert observers) of each species and 10 gravel patches that do not include a redd of either species.Collect control samples from the water column adjacent to each redd to establish background eDNA levels.Analyze the samples using the developed molecular assays for Coho salmon (phase I) and Chinook salmon (Laramie and others, 2015).Evaluate whether samples collected from Chinook and Coho redds have significantly higher levels of eDNA of the respective species than background levels (that is, from gravel, water column).Evaluate whether samples collected from the interstitial spaces of gravel patches that are not redds are similar to background eDNA levels.The Sandy River is a large tributary of the Columbia River. The Sandy River meets the Columbia River approximately 23 km upstream of Portland, Oregon. The Sandy River Basin provides overlapping spawning habitat for both Chinook and Coho salmon.Samples provided by Portland Water Bureau for analysis were collected from the Bull Run River, Sixes Creek, Still Creek, Arrah Wanna Side Channel, and Side Channel 18.

  10. Method of quantitating dsDNA

    DOEpatents

    Stark, Peter C.; Kuske, Cheryl R.; Mullen, Kenneth I.

    2002-01-01

    A method for quantitating dsDNA in an aqueous sample solution containing an unknown amount of dsDNA. A first aqueous test solution containing a known amount of a fluorescent dye-dsDNA complex and at least one fluorescence-attenutating contaminant is prepared. The fluorescence intensity of the test solution is measured. The first test solution is diluted by a known amount to provide a second test solution having a known concentration of dsDNA. The fluorescence intensity of the second test solution is measured. Additional diluted test solutions are similarly prepared until a sufficiently dilute test solution having a known amount of dsDNA is prepared that has a fluorescence intensity that is not attenuated upon further dilution. The value of the maximum absorbance of this solution between 200-900 nanometers (nm), referred to herein as the threshold absorbance, is measured. A sample solution having an unknown amount of dsDNA and an absorbance identical to that of the sufficiently dilute test solution at the same chosen wavelength is prepared. Dye is then added to the sample solution to form the fluorescent dye-dsDNA-complex, after which the fluorescence intensity of the sample solution is measured and the quantity of dsDNA in the sample solution is determined. Once the threshold absorbance of a sample solution obtained from a particular environment has been determined, any similarly prepared sample solution taken from a similar environment and having the same value for the threshold absorbance can be quantified for dsDNA by adding a large excess of dye to the sample solution and measuring its fluorescence intensity.

  11. Changes in chromosome structure, mitotic activity and nuclear DNA content from cells of Allium Test induced by bark water extract of Uncaria tomentosa (Willd.) DC.

    PubMed

    Kuraś, Mieczysław; Nowakowska, Julita; Sliwińska, Elwira; Pilarski, Radosław; Ilasz, Renata; Tykarska, Teresa; Zobel, Alicja; Gulewicz, Krzysztof

    2006-09-19

    The influence of water extract of Uncaria tomentosa (Willd.) DC bark on the meristematic cells of the root tips of Allium cepa L., e.g. cells of Allium Test, was investigated. The experiment was carried out in two variants: (1) continuous incubation at different concentrations (2, 4, 8 and 16 mg/ml) of the extract for 3, 6, 12, 24, 48 and 72h; and (2) 24-h incubation in three concentrations of the extract (4, 8 or 16 mg/ml), followed by post-incubation in distilled water for 3, 6, 12, 24 and 48h. During the continuous incubation, the mitotic activity was reduced (2 and 4 mg/ml) or totally inhibited (8 and 16 mg/ml), depending on the concentration of the extract. All the concentrations resulted in gradual reduction of the mitotic activity. In the concentration of 2 mg/ml, the mitotic activity reached its lowest value after 12h (2 mg/ml) and after 24h in 4 mg/ml, followed by spontaneous intensification of divisions during further incubation. Instead, in higher concentrations of the extracts (8 and 16 mg/ml), the mitotic activity was totally inhibited within 24h and did not resume even after 72h. Incubation caused changes in the phase index, mainly as an increase in the number of prophases. After 24h of incubation, in all phases, condensation and contraction of chromosomes were observed. During post-incubation, divisions resumed in all concentrations, reaching even higher values than the control. Cytometric analysis showed that the extract caused inhibition of the cell cycle at the border between gap(2) and beginning of mitosis (G(2)/M). PMID:16793229

  12. Combining Push Pull Tracer Tests and Microbial DNA and mRNA Analysis to Assess In-Situ Groundwater Nitrate Transformations

    NASA Astrophysics Data System (ADS)

    Henson, W.; Graham, W. D.; Huang, L.; Ogram, A.

    2015-12-01

    Nitrogen transformation mechanisms in the Upper Floridan Aquifer (UFA) are still poorly understood because of karst aquifer complexity and spatiotemporal variability in nitrate and carbon loading. Transformation rates have not been directly measured in the aquifer. This study quantifies nitrate-nitrogen transformation potential in the UFA using single well push-pull tracer injection (PPT) experiments combined with microbial characterization of extracted water via qPCR and RT-qPCR of selected nitrate reduction genes. Tracer tests with chloride and nitrate ± carbon were executed in two wells representing anoxic and oxic geochemical end members in a spring groundwater contributing area. A significant increase in number of microbes with carbon addition suggests stimulated growth. Increases in the activities of denitrification genes (nirK and nirS) as measured by RT-qPCR were not observed. However, only microbes suspended in the tracer were obtained, ignoring effects of aquifer material biofilms. Increases in nrfA mRNA and ammonia concentrations were observed, supporting Dissimilatory Reduction of Nitrate to Ammonia (DNRA) as a reduction mechanism. In the oxic aquifer, zero order nitrate loss rates ranged from 32 to 89 nmol /L*hr with no added carbon and 90 to 240 nmol /L*hr with carbon. In the anoxic aquifer, rates ranged from 18 to 95 nmol /L*hr with no added carbon and 34 to 207 nmol /L*hr with carbon. These loss rates are low; 13 orders of magnitude less than the loads applied in the contributing area each year, however they do indicate that losses can occur in oxic and anoxic aquifers with and without carbon. These rates may include, ammonia adsorption, uptake, or denitrification in aquifer material biofilms. Rates with and without carbon addition for both aquifers were similar, suggesting aquifer redox state and carbon availability alone are insufficient to predict response to nutrient additions without characterization of microbial response. Surprisingly, these

  13. DNA Nanotechnology-- Architectures Designed with DNA

    NASA Astrophysics Data System (ADS)

    Han, Dongran

    As the genetic information storage vehicle, deoxyribonucleic acid (DNA) molecules are essential to all known living organisms and many viruses. It is amazing that such a large amount of information about how life develops can be stored in these tiny molecules. Countless scientists, especially some biologists, are trying to decipher the genetic information stored in these captivating molecules. Meanwhile, another group of researchers, nanotechnologists in particular, have discovered that the unique and concise structural features of DNA together with its information coding ability can be utilized for nano-construction efforts. This idea culminated in the birth of the field of DNA nanotechnology which is the main topic of this dissertation. The ability of rationally designed DNA strands to self-assemble into arbitrary nanostructures without external direction is the basis of this field. A series of novel design principles for DNA nanotechnology are presented here, from topological DNA nanostructures to complex and curved DNA nanostructures, from pure DNA nanostructures to hybrid RNA/DNA nanostructures. As one of the most important and pioneering fields in controlling the assembly of materials (both DNA and other materials) at the nanoscale, DNA nanotechnology is developing at a dramatic speed and as more and more construction approaches are invented, exciting advances will emerge in ways that we may or may not predict.

  14. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma

    PubMed Central

    Burnham, Philip; Kim, Min Seong; Agbor-Enoh, Sean; Luikart, Helen; Valantine, Hannah A.; Khush, Kiran K.; De Vlaminck, Iwijn

    2016-01-01

    Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that ssDNA library preparation yields a greater portion of sub-100 bp nuclear genomic cfDNA (p 10−5, Mann-Whitney U Test), and an increased relative abundance of mitochondrial (10.7x, p 10−5) and microbial cfDNA (71.3x, p 10−5). The higher yield of microbial sequences from this method increases the sensitivity of cfDNA-based monitoring for infections following transplantation. We detail the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We report the observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. A ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods. PMID:27297799

  15. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma.

    PubMed

    Burnham, Philip; Kim, Min Seong; Agbor-Enoh, Sean; Luikart, Helen; Valantine, Hannah A; Khush, Kiran K; De Vlaminck, Iwijn

    2016-01-01

    Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that ssDNA library preparation yields a greater portion of sub-100 bp nuclear genomic cfDNA (p 10(-5), Mann-Whitney U Test), and an increased relative abundance of mitochondrial (10.7x, p 10(-5)) and microbial cfDNA (71.3x, p 10(-5)). The higher yield of microbial sequences from this method increases the sensitivity of cfDNA-based monitoring for infections following transplantation. We detail the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We report the observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. A ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods. PMID:27297799

  16. Priming DNA replication from triple helix oligonucleotides: possible threestranded DNA in DNA polymerases.

    PubMed

    Lestienne, Patrick P

    2011-01-01

    Triplex associate with a duplex DNA presenting the same polypurine or polypyrimidine-rich sequence in an antiparallel orientation. So far, triplex forming oligonucleotides (TFOs) are known to inhibit transcription, replication, and to induce mutations. A new property of TFO is reviewed here upon analysis of DNA breakpoint yielding DNA rearrangements; the synthesized sequence of the first direct repeat displays a skewed polypurine- rich sequence. This synthesized sequence can bind the second homologous duplex sequence through the formation of a triple helix, which is able to prime further DNA replication. In these case, the d(G)-rich Triple Helix Primers (THP) bind the homologous strand in a parallel manner, possibly via a RecA-like mechanism. This novel property is shared by all tested DNA polymerases: phage, retrovirus, bacteria, and human. These features may account for illegitimate initiation of replication upon single-strand breakage and annealing to a homologous sequence where priming may occur. Our experiments suggest that DNA polymerases can bind three instead of two polynucleotide strands in their catalytic centre. PMID:22229092

  17. Priming DNA Replication from Triple Helix Oligonucleotides: Possible Threestranded DNA in DNA Polymerases

    PubMed Central

    Lestienne, Patrick P.

    2011-01-01

    Triplex associate with a duplex DNA presenting the same polypurine or polypyrimidine-rich sequence in an antiparallel orientation. So far, triplex forming oligonucleotides (TFOs) are known to inhibit transcription, replication, and to induce mutations. A new property of TFO is reviewed here upon analysis of DNA breakpoint yielding DNA rearrangements; the synthesized sequence of the first direct repeat displays a skewed polypurine- rich sequence. This synthesized sequence can bind the second homologous duplex sequence through the formation of a triple helix, which is able to prime further DNA replication. In these case, the d(G)-rich Triple Helix Primers (THP) bind the homologous strand in a parallel manner, possibly via a RecA-like mechanism. This novel property is shared by all tested DNA polymerases: phage, retrovirus, bacteria, and human. These features may account for illegitimate initiation of replication upon single-strand breakage and annealing to a homologous sequence where priming may occur. Our experiments suggest that DNA polymerases can bind three instead of two polynucleotide strands in their catalytic centre. PMID:22229092

  18. Technologies for enhanced efficacy of DNA vaccines

    PubMed Central

    Saade, Fadi; Petrovsky, Nikolai

    2012-01-01

    Despite many years of research, human DNA vaccines have yet to fulfill their early promise. Over the past 15 years, multiple generations of DNA vaccines have been developed and tested in preclinical models for prophylactic and therapeutic applications in the areas of infectious disease and cancer, but have failed in the clinic. Thus, while DNA vaccines have achieved successful licensure for veterinary applications, their poor immunogenicity in humans when compared with traditional protein-based vaccines has hindered their progress. Many strategies have been attempted to improve DNA vaccine potency including use of more efficient promoters and codon optimization, addition of traditional or genetic adjuvants, electroporation, intradermal delivery and various prime–boost strategies. This review summarizes these advances in DNA vaccine technologies and attempts to answer the question of when DNA vaccines might eventually be licensed for human use. PMID:22309668

  19. DNA detection using origami paper analytical devices

    PubMed Central

    Ellington, Andrew D.; Crooks, Richard M.

    2013-01-01

    We demonstrate the hybridization-induced fluorescence detection of DNA on an origami-based paper analytical device (oPAD). The paper substrate was patterned by wax printing and controlled heating to construct hydrophilic channels and hydrophobic barriers in a three-dimensional fashion. A competitive assay was developed where the analyte, a single-stranded DNA (ssDNA), and a quencher-labeled ssDNA competed for hybridization with a fluorophore-labeled ssDNA probe. Upon hybridization of the analyte with the fluorophore-labeled ssDNA, a linear response of fluorescence vs. analyte concentration was observed with an extrapolated limit of detection < 5 nM and a sensitivity relative standard deviation as low as 3%. The oPAD setup was also tested against OR/AND logic gates, proving to be successful in both detection systems. PMID:24070108

  20. DNA detection using origami paper analytical devices.

    PubMed

    Scida, Karen; Li, Bingling; Ellington, Andrew D; Crooks, Richard M

    2013-10-15

    We demonstrate the hybridization-induced fluorescence detection of DNA on an origami-based paper analytical device (oPAD). The paper substrate was patterned by wax printing and controlled heating to construct hydrophilic channels and hydrophobic barriers in a three-dimensional fashion. A competitive assay was developed where the analyte, a single-stranded DNA (ssDNA), and a quencher-labeled ssDNA competed for hybridization with a fluorophore-labeled ssDNA probe. Upon hybridization of the analyte with the fluorophore-labeled ssDNA, a linear response of fluorescence vs analyte concentration was observed with an extrapolated limit of detection <5 nM and a sensitivity relative standard deviation as low as 3%. The oPAD setup was also tested against OR/AND logic gates, proving to be successful in both detection systems. PMID:24070108

  1. Quantitative DNA fiber mapping

    DOEpatents

    Gray, Joe W.; Weier, Heinz-Ulrich G.

    1998-01-01

    The present invention relates generally to the DNA mapping and sequencing technologies. In particular, the present invention provides enhanced methods and compositions for the physical mapping and positional cloning of genomic DNA. The present invention also provides a useful analytical technique to directly map cloned DNA sequences onto individual stretched DNA molecules.

  2. Mitochondrial DNA haplotype predicts deafness risk

    SciTech Connect

    Hutchin, T.; Cortopassi, G.

    1995-12-18

    Since mitochondrial DNA (mtDNA) does not recombine in humans, once deleterious variation arises within a particular mtDNA clone it remains linked to that clonal type. An A to G mutation at mtDNA position 1555 confers matrilineal deafness among Asians and others. Two major mtDNA types (I and II) have been defined in Asians by D-loop sequencing. We have determined the D-loop sequence of 8 unrelated deaf Asians bearing the 1555G mutation, and find that 7 are of type II, whereas only one is of type I. Thus the frequency of the 1555G mutation is higher in type II mtDNA than type I (P = 0.035, binomial test), and persons with type II mtDNA are more likely to become deaf. Type II mtDNAs are rare in the Caucasian population, which may explain the rarity of this form of deafness in the United States. Negative Darwinian selection is expected to rapidly eliminate mtDNAs bearing severely deleterious mutations; but mildly deleterious mutations whose phenotype is expressed after reproduction should persist on the mtDNA background in which they arose. Thus determination of mtDNA clonal type has the potential to predict human risk for diseases that are the result of mildly deleterious mtDNA mutations which confer a post-reproductive phenotype. 4 refs., 1 fig.

  3. Engineered DNA polymerase improves PCR results for plastid DNA1

    PubMed Central

    Schori, Melanie; Appel, Maryke; Kitko, AlexaRae; Showalter, Allan M.

    2013-01-01

    • Premise of the study: Secondary metabolites often inhibit PCR and sequencing reactions in extractions from plant material, especially from silica-dried and herbarium material. A DNA polymerase that is tolerant to inhibitors improves PCR results. • Methods and Results: A novel DNA amplification system, including a DNA polymerase engineered via directed evolution for improved tolerance to common plant-derived PCR inhibitors, was evaluated and PCR parameters optimized for three species. An additional 31 species were then tested with the engineered enzyme and optimized protocol, as well as with regular Taq polymerase. • Conclusions: PCR products and high-quality sequence data were obtained for 96% of samples for rbcL and 79% for matK, compared to 29% and 21% with regular Taq polymerase. PMID:25202519

  4. Accelerating peroxidase mimicking nanozymes using DNA

    NASA Astrophysics Data System (ADS)

    Liu, Biwu; Liu, Juewen

    2015-08-01

    DNA-capped iron oxide nanoparticles are nearly 10-fold more active as a peroxidase mimic for TMB oxidation than naked nanoparticles. To understand the mechanism, the effect of DNA length and sequence is systematically studied, and other types of polymers are also compared. This rate enhancement is more obvious with longer DNA and, in particular, poly-cytosine. Among the various polymer coatings tested, DNA offers the highest rate enhancement. A similar acceleration is also observed for nanoceria. On the other hand, when the positively charged TMB substrate is replaced by the negatively charged ABTS, DNA inhibits oxidation. Therefore, the negatively charged phosphate backbone and bases of DNA can increase TMB binding by the iron oxide nanoparticles, thus facilitating the oxidation reaction in the presence of hydrogen peroxide.DNA-capped iron oxide nanoparticles are nearly 10-fold more active as a peroxidase mimic for TMB oxidation than naked nanoparticles. To understand the mechanism, the effect of DNA length and sequence is systematically studied, and other types of polymers are also compared. This rate enhancement is more obvious with longer DNA and, in particular, poly-cytosine. Among the various polymer coatings tested, DNA offers the highest rate enhancement. A similar acceleration is also observed for nanoceria. On the other hand, when the positively charged TMB substrate is replaced by the negatively charged ABTS, DNA inhibits oxidation. Therefore, the negatively charged phosphate backbone and bases of DNA can increase TMB binding by the iron oxide nanoparticles, thus facilitating the oxidation reaction in the presence of hydrogen peroxide. Electronic supplementary information (ESI) available: Methods, TEM, UV-vis and DLS data. See DOI: 10.1039/c5nr04176g

  5. Assessment and mitigation of DNA loss utilizing centrifugal filtration devices.

    PubMed

    Doran, Ashley E; Foran, David R

    2014-11-01

    Maximizing DNA recovery during its isolation can be vital in forensic casework, particularly when DNA yields are expected to be low, such as from touch samples. Many forensic laboratories utilize centrifugal filtration devices to purify and concentrate the DNA; however, DNA loss has been reported when using them. In this study, all centrifugal filtration devices tested caused substantial DNA loss, affecting low molecular weight DNA (PCR product) somewhat more than high molecular weight DNA. Strategies for mitigating DNA loss were then examined, including pre-treatment with glucose, glycogen, silicone (RainX(®)), bovine serum albumin, yeast RNA, or high molecular weight DNA. The length of pre-treatment and UV irradiation of pre-treatment reagents were also investigated. Pre-treatments with glucose and glycogen resulted in little or no improvement in DNA recovery, and most or all DNA was lost after silicone pre-treatment. Devices pre-treated with BSA produced irregular and uninterpretable quantitative PCR amplification curves for the DNA and internal PCR control. On the other hand, nucleic acid pre-treatments greatly improved recovery of all DNAs. Pre-treatment time and its UV irradiation did not influence DNA recovery. Overall, the results show that centrifugal filtration devices trap DNA, yet their proper pre-treatment can circumvent that loss, which is critical in the case of low copy forensic DNA samples. PMID:25173492

  6. DNA Microarrays for Identifying Fishes

    PubMed Central

    Nölte, M.; Weber, H.; Silkenbeumer, N.; Hjörleifsdottir, S.; Hreggvidsson, G. O.; Marteinsson, V.; Kappel, K.; Planes, S.; Tinti, F.; Magoulas, A.; Garcia Vazquez, E.; Turan, C.; Hervet, C.; Campo Falgueras, D.; Antoniou, A.; Landi, M.; Blohm, D.

    2008-01-01

    In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commercially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a “Fish Chip” for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products. PMID:18270778

  7. Pax-3-DNA interaction: flexibility in the DNA binding and induction of DNA conformational changes by paired domains.

    PubMed Central

    Chalepakis, G; Wijnholds, J; Gruss, P

    1994-01-01

    The mouse Pax-3 gene encodes a protein that is a member of the Pax family of DNA binding proteins. Pax-3 contains two DNA binding domains: a paired domain (PD) and a paired type homeodomain (HD). Both domains are separated by 53 amino acids and interact synergistically with a sequence harboring an ATTA motif (binding to the HD) and a GTTCC site (binding to the PD) separated by 5 base pairs. Here we show that the interaction of Pax-3 with these two binding sites is independent of their angular orientation. In addition, the protein spacer region between the HD and the PD can be shortened without changing the spatial flexibility of the two DNA binding domains which interact with DNA. Furthermore, by using circular permutation analysis we determined that binding of Pax-3 to a DNA fragment containing a specific binding site causes conformational changes in the DNA, as indicated by the different mobilities of the Pax-3-DNA complexes. The ability to change the conformation of the DNA was found to be an intrinsic property of the Pax-3 PD and of all Pax proteins that we tested so far. These in vitro studies suggest that interaction of Pax proteins with their specific sequences in vivo may result in an altered DNA conformation. Images PMID:8065927

  8. Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity

    PubMed Central

    Levikova, Maryna; Klaue, Daniel; Seidel, Ralf; Cejka, Petr

    2013-01-01

    Dna2 is a nuclease-helicase involved in several key pathways of eukaryotic DNA metabolism. The potent nuclease activity of Saccharomyces cerevisiae Dna2 was reported to be required for all its in vivo functions tested to date. In contrast, its helicase activity was shown to be weak, and its inactivation affected only a subset of Dna2 functions. We describe here a complex interplay of the two enzymatic activities. We show that the nuclease of Dna2 inhibits its helicase by cleaving 5′ flaps that are required by the helicase domain for loading onto its substrate. Mutational inactivation of Dna2 nuclease unleashes unexpectedly vigorous DNA unwinding activity, comparable with that of the most potent eukaryotic helicases. Thus, the ssDNA-specific nuclease activity of Dna2 limits and controls the enzyme's capacity to unwind dsDNA. We postulate that regulation of this interplay could modulate the biochemical properties of Dna2 and thus license it to carry out its distinct cellular functions. PMID:23671118

  9. Abstractions for DNA circuit design

    PubMed Central

    Lakin, Matthew R.; Youssef, Simon; Cardelli, Luca; Phillips, Andrew

    2012-01-01

    DNA strand displacement techniques have been used to implement a broad range of information processing devices, from logic gates, to chemical reaction networks, to architectures for universal computation. Strand displacement techniques enable computational devices to be implemented in DNA without the need for additional components, allowing computation to be programmed solely in terms of nucleotide sequences. A major challenge in the design of strand displacement devices has been to enable rapid analysis of high-level designs while also supporting detailed simulations that include known forms of interference. Another challenge has been to design devices capable of sustaining precise reaction kinetics over long periods, without relying on complex experimental equipment to continually replenish depleted species over time. In this paper, we present a programming language for designing DNA strand displacement devices, which supports progressively increasing levels of molecular detail. The language allows device designs to be programmed using a common syntax and then analysed at varying levels of detail, with or without interference, without needing to modify the program. This allows a trade-off to be made between the level of molecular detail and the computational cost of analysis. We use the language to design a buffered architecture for DNA devices, capable of maintaining precise reaction kinetics for a potentially unbounded period. We test the effectiveness of buffered gates to support long-running computation by designing a DNA strand displacement system capable of sustained oscillations. PMID:21775321

  10. Requirement for the Kinase Activity of Human DNA-Dependent Protein Kinase Catalytic Subunit in DNA Strand Break Rejoining

    PubMed Central

    Kurimasa, Akihiro; Kumano, Satoshi; Boubnov, Nikolai V.; Story, Michael D.; Tung, Chang-Shung; Peterson, Scott R.; Chen, David J.

    1999-01-01

    The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enormous, 470-kDa protein serine/threonine kinase that has homology with members of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors Ku70 and Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in DNA-PKcs (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the DNA-PKcs gene to test the importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These results indicate that the protein kinase activity of DNA-PKcs is essential for the rejoining of DNA DSBs in mammalian cells. We have also determined a model structure for the DNA-PKcs kinase domain based on comparisons to the crystallographic structure of a cyclic AMP-dependent protein kinase. This structure gives some insight into which amino acid residues are crucial for the kinase activity in DNA-PKcs. PMID:10207111

  11. A review of testing used in seroprevalence studies on measles and rubella.

    PubMed

    Dimech, Wayne; Mulders, Mick N

    2016-07-29

    Seroprevalence studies are an essential tool to monitor the efficacy of vaccination programmes, to understand population immunity and to identify populations at higher risk of infection. An overarching review of all aspects of seroprevalence studies for measles and rubella published between 1998 and June 2014 was undertaken and the findings reported elsewhere. This paper details the considerable variation in the testing formats identified in the review. Apart from serum/plasma samples, testing of oral fluid, breast milk, dry blood spots and capillary whole blood were reported. Numerous different commercial assays were employed, including microtitre plate assays, automated immunoassays and classical haemagglutination inhibition and neutralisation assays. A total of 29 of the 68 (43%) measles and 14 of the 58 (24%) rubella studies reported qualitative test results. Very little information on the testing environment, including quality assurance mechanisms used, was provided. Due to the large numbers of testing systems, the diversity of sample types used and the difficulties in accurate quantification of antibody levels, the results reported in individual studies were not necessarily comparable. Further efforts to standardise seroprevalence studies may overcome this deficiency. PMID:27340096

  12. LCAT DNA shearing.

    PubMed

    Okabe, Yuka; Lee, Abraham P

    2014-04-01

    We present a novel method to fragment DNA by using lateral cavity acoustic transducers (LCATs). DNA solution is placed within a microfluidic device containing LCATs. The LCATs cause microstreaming, which fragments DNA within the solution without any need for purification or downstream processing. The LCAT-based DNA fragmentation method offers an easy-to-use, low-cost, low-energy way to fragment DNA that is amenable to integration on microfluidic platforms to further automate DNA processing. Furthermore, the LCAT microdevice requires less than 10 µL of sample, and no external equipment is needed besides a piezoelectric transducer. PMID:23850863

  13. Detection and quantitation of HBV DNA in miniaturized samples: multi centre study to evaluate the performance of the COBAS ® AmpliPrep/COBAS ® TaqMan ® hepatitis B virus (HBV) test v2.0 by the use of plasma or serum specimens.

    PubMed

    Berger, Annemarie; Gohl, Peter; Stürmer, Martin; Rabenau, Holger Felix; Nauck, Markus; Doerr, Hans Wilhelm

    2010-11-01

    Laboratory analysis of blood specimens is an increasingly important tool for rapid diagnosis and control of therapy. So, miniaturization of test systems is needed, but reduced specimens might impair test quality. For rapid detection and quantitation of HBV DNA, the COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HBV test has proved a robust instrument in routine diagnostic services. The test system has been modified recently for application of reduced samples of blood plasma and for blood serum, too. The performance of this modified COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HBV v2.0 (HBV v2.0 (this test is currently not available in the USA)) test was evaluated by comparison with the former COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HBV v1.0 (HBV v1.0) test. In this study a platform correlation of both assay versions was done including 275 HBV DNA positive EDTA plasma samples. Comparable results were obtained (R(2)=0.97, mean difference -0.03 log(10)IU/ml). The verification of equivalency of the sample matrix (plasma vs. serum samples tested in HBV v2.0 in the same run) showed comparable results for all 278 samples with a R(2)=0.99 and a mean difference of 0.06 log(10)IU/ml. In conclusion, the new test version HBV v2.0 is highly specific and reproducible and quantifies accurately HBV DNA in EDTA plasma and serum samples from patients with chronic HBV infection. PMID:20728470

  14. Mouse zygotes respond to severe sperm DNA damage by delaying paternal DNA replication and embryonic development.

    PubMed

    Gawecka, Joanna E; Marh, Joel; Ortega, Michael; Yamauchi, Yasuhiro; Ward, Monika A; Ward, W Steven

    2013-01-01

    Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF). SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B) followed by irreversible DNA degradation by a nuclease(s). Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes) and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development. PMID:23431372

  15. Increase in the frequency of hepadnavirus DNA integrations by oxidative DNA damage and inhibition of DNA repair.

    PubMed Central

    Petersen, J; Dandri, M; Bürkle, A; Zhang, L; Rogler, C E

    1997-01-01

    Persistent hepadnavirus infection leads to oxidative stress and DNA damage through increased production of toxic oxygen radicals. In addition, hepadnaviral DNA integrations into chromosomal DNA can promote the process of hepatocarcinogenesis (M. Feitelson, Clin. Microbiol. Rev. 5:275-301, 1992). While previous studies have identified preferred integration sites in hepadnaviral genomes and suggested integration mechanisms (M. A. Buendia, Adv. Cancer Res. 59:167-226, 1992; C. E. Rogler, Curr. Top. Microbiol. Immunol. 168:103-141, 1991; C. Shih et al., J. Virol. 61:3491-3498, 1987), very little is known about the effects of agents which damage chromosomal DNA on the frequency of hepadnaviral DNA integrations. Using a recently developed subcloning approach to detect stable new integrations of duck hepatitis B virus (DHBV) (S. S. Gong, A. D. Jensen, and C. E. Rogler, J. Virol. 70:2000-2007, 1996), we tested the effects of increased chromosomal DNA damage induced by H2O2, or of the disturbance in DNA repair due to the inhibition of poly(ADP-ribose) polymerase (PARP), on the frequency of DHBV DNA integrations. Subclones of LMH-D21-6 cells, which replicate DHBV, were grown in the presence of various H2O2 concentrations and exhibited up to a threefold increase in viral DNA integration frequency in a dose-dependent manner. Moreover, inhibition of PARP, which plays a role in cellular responses to DNA breakage, by 3-aminobenzamide (3-AB) resulted in a sevenfold increase in the total number of new DHBV DNA integrations into host chromosomal DNA. Removal of either H2O2 or 3-AB from the culture medium in a subsequent cycle of subcloning was accompanied by a reversion back towards the original lower frequency of stable DHBV DNA integrations for LMH-D21-6 cells. These data support the hypothesis that DNA damage sites can serve as sites for hepadnaviral DNA integration, and that increasing the number of DNA damage sites dramatically increases viral integration frequency. PMID

  16. Comparison of the diagnostic accuracy of a rapid immunochromatographic test and the rapid plasma reagin test for antenatal syphilis screening in Mozambique.

    PubMed Central

    Montoya, Pablo J.; Lukehart, Sheila A.; Brentlinger, Paula E.; Blanco, Ana J.; Floriano, Florencia; Sairosse, Josefa; Gloyd, Stephen

    2006-01-01

    OBJECTIVE: Programmes to control syphilis in developing countries are hampered by a lack of laboratory services, delayed diagnosis, and doubts about current screening methods. We aimed to compare the diagnostic accuracy of an immunochromatographic strip (ICS) test and the rapid plasma reagin (RPR) test with the combined gold standard (RPR, Treponema pallidum haemagglutination assay and direct immunofluorescence stain done at a reference laboratory) for the detection of syphilis in pregnancy. METHODS: We included test results from 4789 women attending their first antenatal visit at one of six health facilities in Sofala Province, central Mozambique. We compared diagnostic accuracy (sensitivity, specificity, and positive and negative predictive values) of ICS and RPR done at the health facilities and ICS performed at the reference laboratory. We also made subgroup comparisons by human immunodeficiency virus (HIV) and malaria status. FINDINGS: For active syphilis, the sensitivity of the ICS was 95.3% at the reference laboratory, and 84.1% at the health facility. The sensitivity of the RPR at the health facility was 70.7%. Specificity and positive and negative predictive values showed a similar pattern. The ICS outperformed RPR in all comparisons (P<0.001). CONCLUSION: The diagnostic accuracy of the ICS compared favourably with that of the gold standard. The use of the ICS in Mozambique and similar settings may improve the diagnosis of syphilis in health facilities, both with and without laboratories. PMID:16501726

  17. Structural Organization of DNA.

    ERIC Educational Resources Information Center

    Banfalvi, Gaspar

    1986-01-01

    Explains the structural organization of DNA by providing information on the primary, secondary, tertiary, and higher organization levels of the molecule. Also includes illustrations and descriptions of sign-inversion and rotating models for supercoiling of DNA. (ML)

  18. Modeling DNA Replication.

    ERIC Educational Resources Information Center

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  19. DNA tagged microparticles

    DOEpatents

    Farquar, George Roy; Leif, Roald N; Wheeler, Elizabeth

    2015-05-05

    A simulant that includes a carrier and DNA encapsulated in the carrier. Also a method of making a simulant including the steps of providing a carrier and encapsulating DNA in the carrier to produce the simulant.

  20. Developmental validation of the ParaDNA(®) Intelligence System-A novel approach to DNA profiling.

    PubMed

    Blackman, Stephen; Dawnay, Nick; Ball, Glyn; Stafford-Allen, Beccy; Tribble, Nicholas; Rendell, Paul; Neary, Kelsey; Hanson, Erin K; Ballantyne, Jack; Kallifatidis, Beatrice; Mendel, Julian; Mills, DeEtta K; Wells, Simon

    2015-07-01

    DNA profiling through the analysis of STRs remains one of the most widely used tools in human identification across the world. Current laboratory STR analysis is slow, costly and requires expert users and interpretation which can lead to instances of delayed investigations or non-testing of evidence on budget grounds. The ParaDNA(®) Intelligence System has been designed to provide a simple, fast and robust way to profile DNA samples in a lab or field-deployable manner. The system analyses 5-STRs plus amelogenin to deliver a DNA profile that enables users to gain rapid investigative leads and intelligent prioritisation of samples in human identity testing applications. Utilising an innovative sample collector, minimal training is required to enable both DNA analysts and nonspecialist personnel to analyse biological samples directly, without prior processing, in approximately 75min. The test uses direct PCR with fluorescent HyBeacon(®) detection of STR allele lengths to provide a DNA profile. The developmental validation study described here followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines and tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Intelligence System on a range of mock evidence items. The data collected demonstrate that the ParaDNA Intelligence System displays useful DNA profiles when sampling a variety of evidence items including blood, saliva, semen and touch DNA items indicating the potential to benefit a number of applications in fields such as forensic, military and disaster victim identification (DVI). PMID:25980941

  1. Is DNA a language?

    PubMed

    Tsonis, A A; Elsner, J B; Tsonis, P A

    1997-01-01

    DNA sequences usually involve local construction rules that affect different scales. As such their "dictionary" may not follow Zipf's law (a power law) which is followed in every natural language. Indeed, analysis of many DNA sequences suggests that no linguistics connections to DNA exist and that even though it has structure DNA is not a language. Computer simulations and a biological approach to this problem further support these results. PMID:9039397

  2. Comparison of DNA-lipid complexes and DNA alone for gene transfer to cystic fibrosis airway epithelia in vivo.

    PubMed Central

    Zabner, J; Cheng, S H; Meeker, D; Launspach, J; Balfour, R; Perricone, M A; Morris, J E; Marshall, J; Fasbender, A; Smith, A E; Welsh, M J

    1997-01-01

    Cationic lipids show promise as vectors for transfer of CFTR cDNA to airway epithelia of patients with cystic fibrosis (CF). However, previous studies have not compared the effect of DNA-lipid to DNA alone. Recently, we developed a formulation of plasmid encoding CFTR (pCF1-CFTR) and cationic lipid (GL-67:DOPE) that generated greater gene transfer in mouse lung than previously described DNA-lipid vectors. Therefore, we tested the hypothesis that DNA-lipid complexes were more effective than DNA alone at transferring CFTR cDNA to airway epithelia in vivo. We administered complexes of DNA-lipid to one nostril and DNA alone to the other nostril in a randomized, double-blind study. Electrophysiologic measurements showed that DNA-lipid complexes partially corrected the Cl- transport defect. Importantly, the pCF1-CFTR plasmid alone was at least as effective as complexes of DNA with lipid. Measurements of vector-specific CFTR transcripts also showed gene transfer with both DNA-lipid and DNA alone. These results indicate that nonviral vectors can transfer CFTR cDNA to airway epithelia and at least partially restore the Cl- transport defect characteristic of CF. However, improvements in the overall efficacy of gene transfer are required to develop a treatment for CF. PMID:9294121

  3. Efficient DNA sequencing on microtiter plates using dried reagents and Bst DNA polymerase.

    PubMed

    Earley, J J; Kuivaniemi, H; Prockop, D J; Tromp, G

    1993-01-01

    Sequenase, Taq DNA polymerase and Bst DNA polymerase were tested for sequencing of DNA on microtiter plates using dried down reagents. Several parameters were investigated to expedite the drying process while minimizing damage to the enzyme. Sequenase did not tolerate drying very well, and frequently generated sequences with weak signals and many sites of premature termination. With Taq DNA polymerase it was possible to obtain sequences of good quality. However, there was considerable variation of results between experiments and between batches of microtiter plates. Bst DNA polymerase generated sequences of excellent quality. It was stable for more than a week in dried-down state at -20 degrees C and at least overnight at room temperature. The method described here using Bst DNA polymerase is well suited for laboratory robots and workstations that typically employ 96-well microtiter plates. PMID:8173079

  4. DNA barcode goes two-dimensions: DNA QR code web server.

    PubMed

    Liu, Chang; Shi, Linchun; Xu, Xiaolan; Li, Huan; Xing, Hang; Liang, Dong; Jiang, Kun; Pang, Xiaohui; Song, Jingyuan; Chen, Shilin

    2012-01-01

    The DNA barcoding technology uses a standard region of DNA sequence for species identification and discovery. At present, "DNA barcode" actually refers to DNA sequences, which are not amenable to information storage, recognition, and retrieval. Our aim is to identify the best symbology that can represent DNA barcode sequences in practical applications. A comprehensive set of sequences for five DNA barcode markers ITS2, rbcL, matK, psbA-trnH, and CO1 was used as the test data. Fifty-three different types of one-dimensional and ten two-dimensional barcode symbologies were compared based on different criteria, such as coding capacity, compression efficiency, and error detection ability. The quick response (QR) code was found to have the largest coding capacity and relatively high compression ratio. To facilitate the further usage of QR code-based DNA barcodes, a web server was developed and is accessible at http://qrfordna.dnsalias.org. The web server allows users to retrieve the QR code for a species of interests, convert a DNA sequence to and from a QR code, and perform species identification based on local and global sequence similarities. In summary, the first comprehensive evaluation of various barcode symbologies has been carried out. The QR code has been found to be the most appropriate symbology for DNA barcode sequences. A web server has also been constructed to allow biologists to utilize QR codes in practical DNA barcoding applications. PMID:22574113

  5. DNA polymerases drive DNA sequencing-by-synthesis technologies: both past and present

    PubMed Central

    Chen, Cheng-Yao

    2014-01-01

    Next-generation sequencing (NGS) technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. Escherichia coli DNA polymerase I proteolytic (Klenow) fragment was originally utilized in Sanger’s dideoxy chain-terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today’s standard capillary electrophoresis (CE) and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ϕ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ϕ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies. PMID:25009536

  6. Effect of DNA type on response of DNA biosensor for carcinogens

    NASA Astrophysics Data System (ADS)

    Sani, Nor Diyana bt. Md.; Heng, Lee Yook; Surif, Salmijah; Lazim, Azwani Mat

    2013-11-01

    Carcinogens are cancer causing chemicals that can bind to DNA and cause damage to the DNA. These chemicals are available everywhere including in water, air, soil and food. Therefore, a sensor that can detect the presence of these chemicals will be a very useful tool. Since carcinogens bind to DNA, DNA can be used as the biological element in a biosensor. This study has utilized different types of DNA in a biosensor for carcinogen detection. The DNAs include double stranded calf thymus DNA, single stranded calf thymus DNA and guanine rich single stranded DNA. The modified SPE was exposed to a carcinogen followed by interaction with methylene blue which acts as the electroactive indicator. The SPE was then analysed using differential pulse voltammetry (DPV). Optimization studies were conducted for MB concentration and accumulation time, DNA concentration, as well as effect of buffer concentration, buffer pH and ionic strength. The performance of the biosensor was tested on a group 1 carcinogen, formaldehyde. The results indicated that the usage of guanine rich single stranded DNA also gives higher response as carcinogens prefer to bind with guanine compared to other bases.

  7. DNAzymes in DNA Nanomachines and DNA Analysis

    NASA Astrophysics Data System (ADS)

    He, Yu; Tian, Ye; Chen, Yi; Mao, Chengde

    This chapter discusses our efforts in using DNAzymes in DNA nano-machines and DNA analysis systems. 10-23 DNAzymes can cleave specific phos-phodiester bonds in RNA. We use them to construct an autonomous DNA-RNA chimera nanomotor, which constantly extracts chemical energy from RNA substrates and transduces the energy into a mechanical motion: cycles of contraction and extension. The motor's motion can be reversibly turned on and off by a DNA analogue (brake) of the RNA substrate. Addition and removal of the brake stops and restarts, respectively, the motor's motion. Furthermore, when the RNA substrates are preorganized into a one-dimensional track, a DNAzyme can continuously move along the track so long as there are substrates available ahead. Based on a similar mechanism, a novel DNA detection system has been developed. A target DNA activates a DNAzyme to cleave RNA-containing molecular beacons (MB), which generates an enhanced fluorescence signal. A following work integrates two steps of signal amplifications: a rolling-circle amplification (RCA) to synthesize multiple copies of DNAzymes, and the DNAzymes catalyze a chemical reaction to generate a colorimetric signal. This method allows detection of DNA analytes whose concentration is as low as 1 pM.

  8. The Many Sides of DNA.

    ERIC Educational Resources Information Center

    Flannery, Maura C.

    1997-01-01

    Explores the meaning of DNA. Discusses histories of DNA, literature on DNA, the contributions of Max Delbruck and Barbara McClintock, life, views of control, current research, and the language of DNA. Contains 24 references. (JRH)

  9. An evaluation of the performance of five extraction methods: Chelex® 100, QIAamp® DNA Blood Mini Kit, QIAamp® DNA Investigator Kit, QIAsymphony® DNA Investigator® Kit and DNA IQ™.

    PubMed

    Ip, Stephen C Y; Lin, Sze-Wah; Lai, Kam-Ming

    2015-05-01

    DNA left at a crime scene was often limited in amount and far from pristine. To maximize the chance of recovering as much information as possible from such compromised samples, an appropriate extraction method using the available technologies needs to be devised. In this study, we used human blood, buffy coat and a total of 76 simulated touch DNA samples to test the effectiveness of the following five common DNA extraction methods, namely, Chelex® 100, QIAamp® DNA Blood Mini Kit, QIAamp® DNA Investigator Kit, QIAsymphony® DNA Investigator® Kit and DNA IQ™ system, in the recovery of such DNA. We demonstrated that the QIAamp® and QIAsymphony® DNA Investigator® Kits, and the DNA IQ™ system, exhibited a better effectiveness in DNA recovery amongst these methods and yielded extracts with higher success rate in subsequent DNA profiling. These extracts also generated profiles with better intra-colour signal balance. The findings in this work allowed us to propose an extraction approach as follows: 1) casework samples shall be extracted with the QIAamp®/QIAsymphony® DNA Investigator® Kits or the DNA IQ™ system, viz., QIAsymphony® DNA Investigator® Kit and DNA IQ™, due to their higher throughput, are for the touched DNA evidence from the volume crime, while QIAamp® DNA Investigator Kit is preferable for challenging bloodstain samples; and 2) control samples, such as buccal swab, with known identity can be extracted with the Chelex, due to their cheaper cost per sample. PMID:25934373

  10. DNA probes for two Microsporidia, Nosema bombycis and Nosema costelytrae.

    PubMed

    Malone, L A; McIvor, C A

    1995-05-01

    Two DNA fragments which hybridize specifically with DNA of Nosema bombycis and Nosema costelytrae, respectively, were obtained from genomic DNA of each microsporidian species and sequenced. Neither fragment hybridized with genomic DNA from four other microsporidian isolates tested: Nosema apis, Vairimorpha sp. from cabbage white butterfly (Pieris rapae), and two isolates of Vavraia oncoperae, one from New Zealand grass grubs, Costelytrae zealandica, and another from porina caterpillars, Wiseana spp. The probe for N. bombycis did not hybridize with genomic DNA from N. costelytrae or with DNA from silkworms (Bombyx mori), the primary insect host of this microsporidium. Likewise, the probe for N. costelytrae did not hybridize with genomic DNA from N. bombycis or with DNA from grass grubs (C. zealandica). Both fragments were AT-rich (59 and 79% of total bases, respectively), had G+C/A+T ratios of 0.70 and 0.25, respectively, and represented repeated sequences dispersed throughout the genome. PMID:7745281

  11. The interaction of radio frequency and lambda DNA

    NASA Astrophysics Data System (ADS)

    Pearson, Mary Elizabeth

    By exposing an aqueous DNA solution to a spectrum of radio frequency (RF) energy this research identifies frequencies, if any, where DNA interacts with RF energy. Interaction is determined by the amount of RF energy either absorbed or reflected by the DNA solution. Previous studies have shown that RF energy at high power levels causes destruction of DNA. The method outlined in this thesis will radiate a DNA solution at a low power level of non-ionizing RF energy. This will determine if DNA behavioral changes can be induced without heating the DNA solution. Any frequencies interacting with DNA within the frequency band areas will be identified as potential frequencies to induce change in genetic function. This thesis sets a foundational experimental protocol to test RF energy interaction with a variety of biological molecules.

  12. DNA Sequencing apparatus

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1992-01-01

    An automated DNA sequencing apparatus having a reactor for providing at least two series of DNA products formed from a single primer and a DNA strand, each DNA product of a series differing in molecular weight and having a chain terminating agent at one end; separating means for separating the DNA products to form a series bands, the intensity of substantially all nearby bands in a different series being different, band reading means for determining the position an This invention was made with government support including a grant from the U.S. Public Health Service, contract number AI-06045. The U.S. government has certain rights in the invention.

  13. DNA evidence: current perspective and future challenges in India.

    PubMed

    Verma, Sunil K; Goswami, Gajendra K

    2014-08-01

    Since the discovery of DNA fingerprinting technology in 1985 it has been used extensively as evidence in the court of law world-wide to establish the individual identity both in civil and criminal matters. In India, the first case of parentage dispute solved by the use of DNA fingerprinting technology was in 1989. Since then till date, the DNA technology has been used not only to resolve the cases of paternity and maternity disputes, but also for the establishment of individual identity in various criminal cases and for wildlife forensic identification. Since last half a decade, India is exercising to enact legislation on the use of DNA in the judicial realm and the draft 'Human DNA Bill-2012' is pending in the parliament. Largely, the promoters of forensic DNA testing have anticipated that DNA tests are nearly infallible and DNA technology could be the greatest single advance step in search for truth, conviction of the perpetrator, and acquittal of the innocent. The current article provides a comprehensive review on the status of DNA testing in India and elucidates the consequences of the admissibility of DNA as 'evidence' in the judicial dominion. In this backdrop of civil and criminal laws and changing ethical and societal attitudes, it is concluded that the DNA legislation in India and world-wide needs to be designed with utmost care. PMID:24967868

  14. DNA probe specific for Legionella pneumophila.

    PubMed Central

    Grimont, P A; Grimont, F; Desplaces, N; Tchen, P

    1985-01-01

    A procedure for preparing a DNA probe to be used in the specific detection of Legionella pneumophila by dot or colony hybridization has been devised. When total DNA from L. pneumophila was used as a radioactive probe, cross-hybridization occurred with DNA from many other species belonging to various families (including Legionellaceae, Enterobacteriaceae, Pseudomonadaceae, and Vibrionaceae). Cross-hybridizing restriction fragments in L. pneumophila ATCC 33152 DNA were identified on Southern blots. When unlabeled DNA from strain ATCC 33152 was cleaved by endonuclease BamHI, the DNA fragments cross-hybridizing with the labeled DNA from all of the other species and genera tested (or with Escherichia coli 16 + 23 S RNA) had a size of 21.4 and 16.2 kilobase pairs (major bands) and 28.0, 12.8, and 10.1 kilobase pairs (minor bands). BamHI restriction fragments of L. pneumophila DNA deprived of the cross-hybridizing fragments were pooled and used as a probe for the detection of L. pneumophila. This probe proved to be specific for L. pneumophila in colony and dot hybridization. It can potentially be used for the detection of L. pneumophila in clinical and water samples. The procedure described can be readily applied to the preparation of probes specific for phylogenetically isolated bacterial species other than L. pneumophila. Images PMID:3980693

  15. Allostery through protein-induced DNA bubbles

    NASA Astrophysics Data System (ADS)

    Traverso, Joseph J.; Manoranjan, Valipuram S.; Bishop, A. R.; Rasmussen, Kim Ø.; Voulgarakis, Nikolaos K.

    2015-03-01

    Allostery through DNA is increasingly recognized as an important modulator of DNA functions. Here, we show that the coalescence of protein-induced DNA bubbles can mediate allosteric interactions that drive protein aggregation. We propose that such allostery may regulate DNA's flexibility and the assembly of the transcription machinery. Mitochondrial transcription factor A (TFAM), a dual-function protein involved in mitochondrial DNA (mtDNA) packaging and transcription initiation, is an ideal candidate to test such a hypothesis owing to its ability to locally unwind the double helix. Numerical simulations demonstrate that the coalescence of TFAM-induced bubbles can explain experimentally observed TFAM oligomerization. The resulting melted DNA segment, approximately 10 base pairs long, around the joints of the oligomers act as flexible hinges, which explains the efficiency of TFAM in compacting DNA. Since mitochondrial polymerase (mitoRNAP) is involved in melting the transcription bubble, TFAM may use the same allosteric interaction to both recruit mitoRNAP and initiate transcription.

  16. Leaf tissue sampling and DNA extraction protocols.

    PubMed

    Semagn, Kassa

    2014-01-01

    Taxonomists must be familiar with a number of issues in collecting and transporting samples using freezing methods (liquid nitrogen and dry ice), desiccants (silica gel and blotter paper), and preservatives (CTAB, ethanol, and isopropanol), with each method having its own merits and limitations. For most molecular studies, a reasonably good quality and quantity of DNA is required, which can only be obtained using standard DNA extraction protocols. There are many DNA extraction protocols that vary from simple and quick ones that yield low-quality DNA but good enough for routine analyses to the laborious and time-consuming standard methods that usually produce high quality and quantities of DNA. The protocol to be chosen will depend on the quality and quantity of DNA needed, the nature of samples, and the presence of natural substances that may interfere with the extraction and subsequent analysis. The protocol described in this chapter has been tested for extracting DNA from eight species and provided very good quality and quantity of DNA for different applications, including those genotyping methods that use restriction enzymes. PMID:24415469

  17. Allostery through protein-induced DNA bubbles

    DOE PAGESBeta

    Traverso, Joseph J.; Manoranjan, Valipuram S.; Bishop, A. R.; Rasmussen, Kim Ø.; Voulgarakis, Nikolaos K.

    2015-03-12

    Allostery through DNA is increasingly recognized as an important modulator of DNA functions. Here, we show that the coalescence of protein-induced DNA bubbles can mediate allosteric interactions that drive protein aggregation. We propose that such allostery may regulate DNA's flexibility and the assembly of the transcription machinery. Mitochondrial transcription factor A (TFAM), a dual-function protein involved in mitochondrial DNA (mtDNA) packaging and transcription initiation, is an ideal candidate to test such a hypothesis owing to its ability to locally unwind the double helix. Numerical simulations demonstrate that the coalescence of TFAM-induced bubbles can explain experimentally observed TFAM oligomerization. The resultingmore » melted DNA segment, approximately 10 base pairs long, around the joints of the oligomers act as flexible hinges, which explains the efficiency of TFAM in compacting DNA. Since mitochondrial polymerase (mitoRNAP) is involved in melting the transcription bubble, TFAM may use the same allosteric interaction to both recruit mitoRNAP and initiate transcription.« less

  18. Allostery through protein-induced DNA bubbles

    SciTech Connect

    Traverso, Joseph J.; Manoranjan, Valipuram S.; Bishop, A. R.; Rasmussen, Kim Ø.; Voulgarakis, Nikolaos K.

    2015-03-12

    Allostery through DNA is increasingly recognized as an important modulator of DNA functions. Here, we show that the coalescence of protein-induced DNA bubbles can mediate allosteric interactions that drive protein aggregation. We propose that such allostery may regulate DNA's flexibility and the assembly of the transcription machinery. Mitochondrial transcription factor A (TFAM), a dual-function protein involved in mitochondrial DNA (mtDNA) packaging and transcription initiation, is an ideal candidate to test such a hypothesis owing to its ability to locally unwind the double helix. Numerical simulations demonstrate that the coalescence of TFAM-induced bubbles can explain experimentally observed TFAM oligomerization. The resulting melted DNA segment, approximately 10 base pairs long, around the joints of the oligomers act as flexible hinges, which explains the efficiency of TFAM in compacting DNA. Since mitochondrial polymerase (mitoRNAP) is involved in melting the transcription bubble, TFAM may use the same allosteric interaction to both recruit mitoRNAP and initiate transcription.

  19. The DNA damage checkpoint allows recombination between divergent DNA sequences in budding yeast

    PubMed Central

    George, Carolyn M.; Lyndaker, Amy M.; Alani, Eric

    2011-01-01

    In the early steps of homologous recombination, single-stranded DNA (ssDNA) from a broken chromosome invades homologous sequence located in a sister or homolog donor. In genomes that contain numerous repetitive DNA elements or gene paralogs, recombination can potentially occur between non-allelic/divergent (homeologous) sequences that share sequence identity. Such recombination events can lead to lethal chromosomal deletions or rearrangements. However, homeologous recombination events can be suppressed through rejection mechanisms that involve recognition of DNA mismatches in heteroduplex DNA by mismatch repair factors, followed by active unwinding of the heteroduplex DNA by helicases. Because factors required for heteroduplex rejection are hypothesized to be targets and/or effectors of the DNA damage response (DDR), a cell cycle control mechanism that ensures timely and efficient repair, we tested whether the DDR, and more specifically, the RAD9 gene, had a role in regulating rejection. We performed these studies using a DNA repair assay that measures repair by single-strand annealing (SSA) of a double-strand break (DSB) using homeologous DNA templates. We found that repair of homeologous DNA sequences, but not identical sequences, induced a RAD9- dependent cell cycle delay in the G2 stage of the cell cycle. Repair through a divergent DNA template occurred more frequently in RAD9 compared to rad9Δ strains. However, repair in rad9Δ mutants could be restored to wild-type levels if a G2 delay was induced by nocodazole. These results suggest that cell cycle arrest induced by the Rad9-dependent DDR allows repair between divergent DNA sequences despite the potential for creating deleterious genome rearrangements, and illustrates the importance of additional cellular mechanisms that act to suppress recombination between divergent DNA sequences. PMID:21978436

  20. The effects of 4-MEI on cell proliferation, DNA breaking and DNA fragmentation.

    PubMed

    Tazehkand, M Norizadeh; Moridikia, A; Hajipour, O; Valipour, E; Timocin, T; Topaktas, M; Yilmaz, M B

    2016-01-01

    4-Methylimidazole (4-MEI) is a color widely found in cola drinks, roasted foods, grilled meats, coffee and other foods. This study was aimed to investigate the 4-MEI effects on the cell proliferation, purified circular DNA and DNA from cells of rats treated with the 4-MEI.In this study, mouse 3T3-L1 cell line was treated with 4-MEI at concentrations of 300, 450, 600 and 750 µg/mL for 24 hours and 48 hours periods, after that cytotoxic effect of the 4-MEI was studied by MTT test. Also, the effect of 4-MEI on purified circular DNA (pET22b) was investigated by treating of the DNA with 4-MEI concentrations of 300, 450, 600 and 750 µg/ml. DNA was extracted from liver cells of rats that have been treated with 4-MEI doses of 25 and 50 mg/kg for 10 week and it was subjected to agarose gel electrophoreses analyses.4-MEI significantly inhibited cell proliferation of 3T3-L1 cell line at highest concentration for 24 h and at all concentration for 48 h treatment time. DNA fragmentation assay showed that 4-MEI at 50 mg/kg concentration clearly produced characteristic DNA smear and no DNA laddering (200bp) was observed when mouse was exposed to 4-MEI. The results obtained from plasmid DNA damaging assay showed that 4-MEI has noeffect on the DNA, because the electrophoretic pattern of DNA treated with 4-MEI showed three bands on agarose gel electrophoresis as it was for untreated control. 4-MEI showed cytotoxic effect on 3T3-L1 cells but no effect on plasmid DNA breaking. According to DNA fragmentation assay 4-MEI has necrosis effects on mouse liver cells (Tab. 1, Fig. 4, Ref. 27). PMID:27546537

  1. DNA in Nanoscale Electronics

    NASA Astrophysics Data System (ADS)

    Slinker, Jason

    2012-10-01

    DNA, the quintessential molecule of life, possesses a number of attractive properties for use in nanoscale circuits. Charge transport (CT) through DNA itself is of both fundamental and practical interest. Fundamentally, DNA has a unique configuration of π-stacked bases in a well ordered, double helical structure. Given its unparalleled importance to life processes and its arrangement of conjugated subunits, DNA has been a compelling target of conductivity studies. In addition, further understanding of DNA CT will elucidate the biological implications of this process and advance its use in sensing technologies. We have investigated the fundamentals of DNA CT by measuring the electrochemistry of DNA monolayers under biologically-relevant conditions. We have uncovered both fundamental kinetic parameters to distinguish between competing models of operation as well as the practical implications of DNA CT for sensing. Furthermore, we are leveraging our studies of DNA conductivity for the manufacture of nanoscale circuits. We are investigating the electrical properties and self-assembly of DNA nanowires containing artificial base pair surrogates, which can be prepared through low cost and high throughput automated DNA synthesis. This unique and economically viable approach will establish a new paradigm for the scalable manufacture of nanoscale semiconductor devices.

  2. DNA structure and function.

    PubMed

    Travers, Andrew; Muskhelishvili, Georgi

    2015-06-01

    The proposal of a double-helical structure for DNA over 60 years ago provided an eminently satisfying explanation for the heritability of genetic information. But why is DNA, and not RNA, now the dominant biological information store? We argue that, in addition to its coding function, the ability of DNA, unlike RNA, to adopt a B-DNA structure confers advantages both for information accessibility and for packaging. The information encoded by DNA is both digital - the precise base specifying, for example, amino acid sequences - and analogue. The latter determines the sequence-dependent physicochemical properties of DNA, for example, its stiffness and susceptibility to strand separation. Most importantly, DNA chirality enables the formation of supercoiling under torsional stress. We review recent evidence suggesting that DNA supercoiling, particularly that generated by DNA translocases, is a major driver of gene regulation and patterns of chromosomal gene organization, and in its guise as a promoter of DNA packaging enables DNA to act as an energy store to facilitate the passage of translocating enzymes such as RNA polymerase. PMID:25903461

  3. DNA diagnosis of human genetic individuality.

    PubMed

    Pena, S D; Prado, V F; Epplen, J T

    1995-11-01

    DNA studies of the human genome have shown polymorphic variation at thousands of sites, defining an absolute genetic uniqueness for each individual. There are many circumstances in which it may be desirable to diagnose this molecular individuality, as for instance, in criminal investigations or paternity testing. Several techniques can be used for this DNA diagnosis and we can choose among them the one that best suits the specific problem at hand. In this review we describe the main methodologies in current use to investigate human DNA polymorphisms, discussing the best application of each option, as well as their advantages and disadvantages. PMID:8751139

  4. DNA-DNA hybridization assay for detection of Salmonella spp. in foods.

    PubMed Central

    Fitts, R; Diamond, M; Hamilton, C; Neri, M

    1983-01-01

    We have developed a DNA-DNA hybridization test for the presence of Salmonella spp. in foods. This test requires an initial pre-enrichment of food samples in nutrient broth but does not require selective enrichment. Samples of food cultures are collected on membrane filters and assayed by molecular hybridization to labeled probes. The probes consist of DNA sequences which are unique to the genus Salmonella and are widely distributed in the genus. A diverse panel of foods was assayed successfully by this methodology. Images PMID:6360046

  5. Human DNA repair genes.

    PubMed

    Wood, R D; Mitchell, M; Sgouros, J; Lindahl, T

    2001-02-16

    Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process. PMID:11181991

  6. Molecular cloning of Renibacterium salmoninarum DNA fragments.

    PubMed

    Etchegaray, J P; Martínez, M A; Krauskopf, M; León, G

    1991-03-15

    A Renibacterium salmoninarum enriched recombinant DNA library was constructed to isolate DNA fragments which could be used as probes to detect gene sequences specific for the causative agent of bacterial kidney disease in salmonid fish. One fragment of 149 base pairs was isolated and its specificity and sequence determined. This probe may prove useful in the design of diagnostic tests for the disease in asymptomatic fish and ova. PMID:2044941

  7. Formation of DNA triple helices inhibits DNA unwinding by the SV40 large T-antigen helicase.

    PubMed Central

    Peleg, M; Kopel, V; Borowiec, J A; Manor, H

    1995-01-01

    Previous studies have indicated that d(TC)n.d(GA)n microsatellites may serve as arrest signals for mammalian DNA replication through the ability of such sequences to form DNA triple helices and thereby inhibit replication enzymes. To further test this hypothesis, we examined the ability of d(TC)i.d(GA)i.d(TC)i triplexes to inhibit DNA unwinding in vitro by a model eukaryotic DNA helicase, the SV40 large T-antigen. DNA substrates that were able to form triplexes, and non-triplex-forming control substrates, were tested. We found that the presence of DNA triplexes, as assayed by endonuclease S1 and osmium tetroxide footprinting, significantly inhibited DNA unwinding by T-antigen. Strong inhibition was observed not only at acidic pH values, in which the triplexes were most stable, but also at physiological pH values in the range 6.9-7.2. Little or no inhibition was detected at pH 8.7. Based on these results, and on previous studies of DNA polymerases, we suggest that DNA triplexes may form in vivo and cause replication arrest through a dual inhibition of duplex unwinding by DNA helicases and of nascent strand synthesis by DNA polymerases. DNA triplexes also have the potential to inhibit recombination and repair processes in which helicases and polymerases are involved. Images PMID:7753619

  8. DNA/SNLA commonality program

    SciTech Connect

    Keller, D. V.; Watts, A. J.; Rice, D. A.; Powe, J.; Beezhold, W.

    1980-01-01

    The purpose of the Commonality program, initiated by DNA in 1978, was to evaluate e-beam material testing procedures and techniques by comparing material stress and spall data from various US and UK e-beam facilities and experimenters. As part of this joint DNA/SNL/UK Commonality effort, Sandia and Ktech used four different electron-beam machines to investigate various aspects of e-beam energy deposition in three materials. The deposition duration and the deposition profiles were varied, and the resulting stresses were measured. The materials studied were: (1) a low-Z material (A1), (2) a high-Z material (Ta), and (3) a typical porous material, a cermet. Aluminium and tantalum were irradiated using the DNA Blackjack 3 accelerator (60 ns pulse width), the DNA Blackjack 3' accelerator (30 ns pulse width), and the SNLA REHYD accelerator (100 ns pulse width). Propagating stresses were measured using x-cut quartz gauges, carbon gauges, and laser interferometry techniques. Data to determine the influence of deposition duration were obtained over a wide range of energy loadings. The cermet material was studied using the SNLA REHYD and HERMES II accelerators. The e-beam from REHYD generated propagating stresses which were monitored with quartz gauges as a function of sample thickness and energy loadings. The HERMES II accelerator was used to uniformly heat the cermet to determine the Grueneisen parameter and identify the incipient spall condition. Results of these experiments are presented.

  9. Integrity and Biological Activity of DNA after UV Exposure

    NASA Astrophysics Data System (ADS)

    Lyon, Delina Y.; Monier, Jean-Michel; Dupraz, Sébastien; Freissinet, Caroline; Simonet, Pascal; Vogel, Timothy M.

    2010-04-01

    The field of astrobiology lacks a universal marker with which to indicate the presence of life. This study supports the proposal to use nucleic acids, specifically DNA, as a signature of life (biosignature). In addition to its specificity to living organisms, DNA is a functional molecule that can confer new activities and characteristics to other organisms, following the molecular biology dogma, that is, DNA is transcribed to RNA, which is translated into proteins. Previous criticisms of the use of DNA as a biosignature have asserted that DNA molecules would be destroyed by UV radiation in space. To address this concern, DNA in plasmid form was deposited onto different surfaces and exposed to UVC radiation. The surviving DNA was quantified via the quantitative polymerase chain reaction (qPCR). Results demonstrate increased survivability of DNA attached to surfaces versus non-adsorbed DNA. The DNA was also tested for biological activity via transformation into the bacterium Acinetobacter sp. and assaying for antibiotic resistance conferred by genes encoded by the plasmid. The success of these methods to detect DNA and its gene products after UV exposure (254 nm, 3.5 J/m2s) not only supports the use of the DNA molecule as a biosignature on mineral surfaces but also demonstrates that the DNA retained biological activity.

  10. Integrity and biological activity of DNA after UV exposure.

    PubMed

    Lyon, Delina Y; Monier, Jean-Michel; Dupraz, Sébastien; Freissinet, Caroline; Simonet, Pascal; Vogel, Timothy M

    2010-04-01

    The field of astrobiology lacks a universal marker with which to indicate the presence of life. This study supports the proposal to use nucleic acids, specifically DNA, as a signature of life (biosignature). In addition to its specificity to living organisms, DNA is a functional molecule that can confer new activities and characteristics to other organisms, following the molecular biology dogma, that is, DNA is transcribed to RNA, which is translated into proteins. Previous criticisms of the use of DNA as a biosignature have asserted that DNA molecules would be destroyed by UV radiation in space. To address this concern, DNA in plasmid form was deposited onto different surfaces and exposed to UVC radiation. The surviving DNA was quantified via the quantitative polymerase chain reaction (qPCR). Results demonstrate increased survivability of DNA attached to surfaces versus non-adsorbed DNA. The DNA was also tested for biological activity via transformation into the bacterium Acinetobacter sp. and assaying for antibiotic resistance conferred by genes encoded by the plasmid. The success of these methods to detect DNA and its gene products after UV exposure (254 nm, 3.5 J/m(2)s) not only supports the use of the DNA molecule as a biosignature on mineral surfaces but also demonstrates that the DNA retained biological activity. PMID:20446869

  11. Studying DNA Looping by Single-Molecule FRET

    PubMed Central

    Le, Tung T.; Kim, Harold D.

    2014-01-01

    Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA. PMID:24998459

  12. Researchers Get Closer to Test Predicting Colon Cancer's Return

    MedlinePlus

    ... Get Closer to Test Predicting Colon Cancer's Return DNA-based screen would aid treatment decisions for people ... News) -- A blood test that detects bits of DNA shed from colon cancers may someday help doctors ...

  13. Validation of the FAM19A4/mir124-2 DNA methylation test for both lavage- and brush-based self-samples to detect cervical (pre)cancer in HPV-positive women

    PubMed Central

    De Strooper, Lise M.A.; Verhoef, Viola M.J.; Berkhof, Johannes; Hesselink, Albertus T.; de Bruin, Helena M.E.; van Kemenade, Folkert J.; Bosgraaf, Remko P.; Bekkers, Ruud L.M.; Massuger, Leon F.A.G.; Melchers, Willem J.G.; Steenbergen, Renske D.M.; Snijders, Peter J.F.; Meijer, Chris J.L.M.; Heideman, Daniëlle A.M.

    2016-01-01

    Objectives DNA methylation analysis of cancer-related genes is a promising tool for HPV-positive women to identify those with cervical (pre)cancer (CIN3+) in need of treatment. However, clinical performance of methylation markers can be influenced by the sample type utilized. We describe a multiplex quantitative methylation-specific PCR that targets FAM19A4 and mir124-2 loci, to detect CIN3+ using both HPV-positive lavage- and brush self-samples. Methods We determined methylation thresholds for clinical classification using HPV-positive training sets comprising lavage self-samples of 182 women (including 40 with CIN3+) and brush self-samples of 224 women (including 61 with CIN3+). Subsequently, independent HPV-positive validation sets of 389 lavage self-samples (including 78 with CIN3+), and 254 brush self-samples (including 72 with CIN3+) were tested using the preset thresholds. Furthermore, the clinical performance of combined methylation analysis and HPV16/18 genotyping was determined. Results Training set analysis revealed similar FAM19A4 and mir124-2 thresholds for both self-sample types to yield highest CIN3+ sensitivity at 70% specificity. Validation set analysis resulted in a CIN3+ sensitivity of 70.5% (95%CI: 60.4–80.6) at a specificity of 67.8% (95%CI: 62.7–73.0) for lavage self-samples, and a CIN3+ sensitivity of 69.4% (95%CI: 58.8–80.1) at a 76.4% (95%CI: 70.2–82.6) specificity for brush self-samples. In combination with HPV16/18 genotyping, CIN3+ sensitivity and specificity were 88.5% (95%CI: 81.4–95.6) and 46.0% (95%CI: 40.4–51.5) for lavage self-samples, and 84.7% (95%CI: 76.4–93.0) and 54.9% (95%CI: 47.7–62.2) for brush self-samples. Conclusions FAM19A4/mir124-2 methylation analysis performs equally well in HPV-positive lavage- and brush self-samples to identify women with CIN3+. In combination with HPV16/18 genotyping, significantly higher CIN3+ sensitivities are obtained, at decreased specificity. PMID:26921784

  14. DNA methylation in plants.

    PubMed

    Vanyushin, B F

    2006-01-01

    DNA in plants is highly methylated, containing 5-methylcytosine (m5C) and N6-methyladenine (m6A); m5C is located mainly in symmetrical CG and CNG sequences but it may occur also in other non-symmetrical contexts. m6A but not m5C was found in plant mitochondrial DNA. DNA methylation in plants is species-, tissue-, organelle- and age-specific. It is controlled by phytohormones and changes on seed germination, flowering and under the influence of various pathogens (viral, bacterial, fungal). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication. DNA replication is accompanied by the appearance of under-methylated, newly formed DNA strands including Okazaki fragments; asymmetry of strand DNA methylation disappears until the end of the cell cycle. A model for regulation of DNA replication by methylation is suggested. Cytosine DNA methylation in plants is more rich and diverse compared with animals. It is carried out by the families of specific enzymes that belong to at least three classes of DNA methyltransferases. Open reading frames (ORF) for adenine DNA methyltransferases are found in plant and animal genomes, and a first eukaryotic (plant) adenine DNA methyltransferase (wadmtase) is described; the enzyme seems to be involved in regulation of the mitochondria replication. Like in animals, DNA methylation in plants is closely associated with histone modifications and it affects binding of specific proteins to DNA and formation of respective transcription complexes in chromatin. The same gene (DRM2) in Arabidopsis thaliana is methylated both at cytosine and adenine residues; thus, at least two different, and probably interdependent, systems of DNA modification are present in plants. Plants seem to have a restriction-modification (R-M) system. RNA-directed DNA methylation has been observed in plants; it involves de novo methylation of almost all cytosine residues in a region of siRNA-DNA

  15. 49 CFR 40.13 - How do DOT drug and alcohol tests relate to non-DOT tests?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... drugs, and a laboratory is prohibited from making a DOT urine specimen available for a DNA test or other... a blood or urine specimen collected by the employee's physician or a DNA test result purporting...

  16. 49 CFR 40.13 - How do DOT drug and alcohol tests relate to non-DOT tests?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... drugs, and a laboratory is prohibited from making a DOT urine specimen available for a DNA test or other... a blood or urine specimen collected by the employee's physician or a DNA test result purporting...

  17. 49 CFR 40.13 - How do DOT drug and alcohol tests relate to non-DOT tests?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... drugs, and a laboratory is prohibited from making a DOT urine specimen available for a DNA test or other... a blood or urine specimen collected by the employee's physician or a DNA test result purporting...

  18. 49 CFR 40.13 - How do DOT drug and alcohol tests relate to non-DOT tests?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... drugs, and a laboratory is prohibited from making a DOT urine specimen available for a DNA test or other... a blood or urine specimen collected by the employee's physician or a DNA test result purporting...

  19. 49 CFR 40.13 - How do DOT drug and alcohol tests relate to non-DOT tests?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... drugs, and a laboratory is prohibited from making a DOT urine specimen available for a DNA test or other... a blood or urine specimen collected by the employee's physician or a DNA test result purporting...

  20. Development of an Automated DNA Detection System Using an Electrochemical DNA Chip Technology

    NASA Astrophysics Data System (ADS)

    Hongo, Sadato; Okada, Jun; Hashimoto, Koji; Tsuji, Koichi; Nikaido, Masaru; Gemma, Nobuhiro

    A new compact automated DNA detection system Genelyzer™ has been developed. After injecting a sample solution into a cassette with a built-in electrochemical DNA chip, processes from hybridization reaction to detection and analysis are all operated fully automatically. In order to detect a sample DNA, electrical currents from electrodes due to an oxidization reaction of electrochemically active intercalator molecules bound to hybridized DNAs are detected. The intercalator is supplied as a reagent solution by a fluid supply unit of the system. The feasibility test proved that the simultaneous typing of six single nucleotide polymorphisms (SNPs) associated with a rheumatoid arthritis (RA) was carried out within two hours and that all the results were consistent with those by conventional typing methods. It is expected that this system opens a new way to a DNA testing such as a test for infectious diseases, a personalized medicine, a food inspection, a forensic application and any other applications.

  1. Low-Dose Formaldehyde Delays DNA Damage Recognition and DNA Excision Repair in Human Cells

    PubMed Central

    Luch, Andreas; Frey, Flurina C. Clement; Meier, Regula; Fei, Jia; Naegeli, Hanspeter

    2014-01-01

    Objective Formaldehyde is still widely employed as a universal crosslinking agent, preservative and disinfectant, despite its proven carcinogenicity in occupationally exposed workers. Therefore, it is of paramount importance to understand the possible impact of low-dose formaldehyde exposures in the general population. Due to the concomitant occurrence of multiple indoor and outdoor toxicants, we tested how formaldehyde, at micromolar concentrations, interferes with general DNA damage recognition and excision processes that remove some of the most frequently inflicted DNA lesions. Methodology/Principal Findings The overall mobility of the DNA damage sensors UV-DDB (ultraviolet-damaged DNA-binding) and XPC (xeroderma pigmentosum group C) was analyzed by assessing real-time protein dynamics in the nucleus of cultured human cells exposed to non-cytotoxic (<100 μM) formaldehyde concentrations. The DNA lesion-specific recruitment of these damage sensors was tested by monitoring their accumulation at local irradiation spots. DNA repair activity was determined in host-cell reactivation assays and, more directly, by measuring the excision of DNA lesions from chromosomes. Taken together, these assays demonstrated that formaldehyde obstructs the rapid nuclear trafficking of DNA damage sensors and, consequently, slows down their relocation to DNA damage sites thus delaying the excision repair of target lesions. A concentration-dependent effect relationship established a threshold concentration of as low as 25 micromolar for the inhibition of DNA excision repair. Conclusions/Significance A main implication of the retarded repair activity is that low-dose formaldehyde may exert an adjuvant role in carcinogenesis by impeding the excision of multiple mutagenic base lesions. In view of this generally disruptive effect on DNA repair, we propose that formaldehyde exposures in the general population should be further decreased to help reducing cancer risks. PMID:24722772

  2. Development and Validation of Environmental DNA (eDNA) Markers for Detection of Freshwater Turtles.

    PubMed

    Davy, Christina M; Kidd, Anne G; Wilson, Chris C

    2015-01-01

    Environmental DNA (eDNA) is a potentially powerful tool for detection and monitoring of rare species, including threatened native species and recently arrived invasive species. Here, we develop DNA primers for a suite of nine sympatric freshwater turtles, and use it to test whether turtle eDNA can be successfully detected in samples from aquaria and an outdoor pond. We also conduct a cost comparison between eDNA detection and detection through traditional survey methods, using data from field surveys at two sites in our target area. We find that eDNA from turtles can be detected using both conventional polymerase chain reaction (PCR) and quantitative PCR (qPCR), and that the cost of detection through traditional survey methods is 2-10X higher than eDNA detection for the species in our study range. We summarize necessary future steps for application of eDNA surveys to turtle monitoring and conservation and propose specific cases in which the application of eDNA could further the conservation of threatened turtle species. PMID:26200348

  3. Partial uracil-DNA-glycosylase treatment for screening of ancient DNA.

    PubMed

    Rohland, Nadin; Harney, Eadaoin; Mallick, Swapan; Nordenfelt, Susanne; Reich, David

    2015-01-19

    The challenge of sequencing ancient DNA has led to the development of specialized laboratory protocols that have focused on reducing contamination and maximizing the number of molecules that are extracted from ancient remains. Despite the fact that success in ancient DNA studies is typically obtained by screening many samples to identify a promising subset, ancient DNA protocols have not, in general, focused on reducing the time required to screen samples. We present an adaptation of a popular ancient library preparation method that makes screening more efficient. First, the DNA extract is treated using a protocol that causes characteristic ancient DNA damage to be restricted to the terminal nucleotides, while nearly eliminating it in the interior of the DNA molecules, allowing a single library to be used both to test for ancient DNA authenticity and to carry out population genetic analysis. Second, the DNA molecules are ligated to a unique pair of barcodes, which eliminates undetected cross-contamination from this step onwards. Third, the barcoded library molecules include incomplete adapters of short length that can increase the specificity of hybridization-based genomic target enrichment. The adapters are completed just before sequencing, so the same DNA library can be used in multiple experiments, and the sequences distinguished. We demonstrate this protocol on 60 ancient human samples. PMID:25487342

  4. Development and Validation of Environmental DNA (eDNA) Markers for Detection of Freshwater Turtles

    PubMed Central

    Davy, Christina M.; Kidd, Anne G.; Wilson, Chris C.

    2015-01-01

    Environmental DNA (eDNA) is a potentially powerful tool for detection and monitoring of rare species, including threatened native species and recently arrived invasive species. Here, we develop DNA primers for a suite of nine sympatric freshwater turtles, and use it to test whether turtle eDNA can be successfully detected in samples from aquaria and an outdoor pond. We also conduct a cost comparison between eDNA detection and detection through traditional survey methods, using data from field surveys at two sites in our target area. We find that eDNA from turtles can be detected using both conventional polymerase chain reaction (PCR) and quantitative PCR (qPCR), and that the cost of detection through traditional survey methods is 2–10X higher than eDNA detection for the species in our study range. We summarize necessary future steps for application of eDNA surveys to turtle monitoring and conservation and propose specific cases in which the application of eDNA could further the conservation of threatened turtle species. PMID:26200348

  5. Mutagenic DNA repair in enterobacteria

    SciTech Connect

    Sedgwick, S.G. ); Chao Ho; Woodgate, R. )

    1991-09-01

    Sixteen species of enterobacteria have been screened for mutagenic DNA repair activity. In Escherichia coli, mutagenic DNA repair is encoded by the umuDC operon. Synthesis of UmuD and UmuC proteins is induced as part of the SOS response to DNA damage, and after induction, the UmuD protein undergoes an autocatalytic cleavage to produce the carboxy-terminal UmuD{prime} fragment needed for induced mutagenesis. The presence of a similar system in other species was examined by using a combined approach of inducible-mutagenesis assays, cross-reactivity to E. coli UmuD and UmuD{prime} antibodies to test for induction and cleavage of UmuD-like proteins, and hybridization with E. coli and Salmonella typhimurium u mu DNA probes to map umu-like genes. The results indicate a more widespread distribution of mutagenic DNA repair in other species than was previously thought. They also show that umu loci can be more complex in other species than in E. coli. Differences in UV-induced mutability of more than 200-fold were seen between different species of enteric bacteria and even between multiple natural isolates of E. coli, and yet some of the species which display a poorly mutable phenotype still have umu-like genes and proteins. It is suggested that umuDC genes can be curtailed in their mutagenic activities but that they may still participate in some other, unknown process which provides the continued stimulus for their retention.

  6. Mitochondrial DNA variants in obesity.

    PubMed

    Knoll, Nadja; Jarick, Ivonne; Volckmar, Anna-Lena; Klingenspor, Martin; Illig, Thomas; Grallert, Harald; Gieger, Christian; Wichmann, Heinz-Erich; Peters, Annette; Wiegand, Susanna; Biebermann, Heike; Fischer-Posovszky, Pamela; Wabitsch, Martin; Völzke, Henry; Nauck, Matthias; Teumer, Alexander; Rosskopf, Dieter; Rimmbach, Christian; Schreiber, Stefan; Jacobs, Gunnar; Lieb, Wolfgang; Franke, Andre; Hebebrand, Johannes; Hinney, Anke

    2014-01-01

    Heritability estimates for body mass index (BMI) variation are high. For mothers and their offspring higher BMI correlations have been described than for fathers. Variation(s) in the exclusively maternally inherited mitochondrial DNA (mtDNA) might contribute to this parental effect. Thirty-two to 40 mtDNA single nucleotide polymorphisms (SNPs) were available from genome-wide association study SNP arrays (Affymetrix 6.0). For discovery, we analyzed association in a case-control (CC) sample of 1,158 extremely obese children and adolescents and 435 lean adult controls. For independent confirmation, 7,014 population-based adults were analyzed as CC sample of n = 1,697 obese cases (BMI ≥ 30 kg/m2) and n = 2,373 normal weight and lean controls (BMI<25 kg/m2). SNPs were analyzed as single SNPs and haplogroups determined by HaploGrep. Fisher's two-sided exact test was used for association testing. Moreover, the D-loop was re-sequenced (Sanger) in 192 extremely obese children and adolescents and 192 lean adult controls. Association testing of detected variants was performed using Fisher's two-sided exact test. For discovery, nominal association with obesity was found for the frequent allele G of m.8994G/A (rs28358887, p = 0.002) located in ATP6. Haplogroup W was nominally overrepresented in the controls (p = 0.039). These findings could not be confirmed independently. For two of the 252 identified D-loop variants nominal association was detected (m.16292C/T, p = 0.007, m.16189T/C, p = 0.048). Only eight controls carried the m.16292T allele, five of whom belonged to haplogroup W that was initially enriched among these controls. m.16189T/C might create an uninterrupted poly-C tract located near a regulatory element involved in replication of mtDNA. Though follow-up of some D-loop variants still is conceivable, our hypothesis of a contribution of variation in the exclusively maternally inherited mtDNA to the observed larger correlations for BMI between mothers and their

  7. Mitochondrial DNA Variants in Obesity

    PubMed Central

    Knoll, Nadja; Jarick, Ivonne; Volckmar, Anna-Lena; Klingenspor, Martin; Illig, Thomas; Grallert, Harald; Gieger, Christian; Wichmann, Heinz-Erich; Peters, Annette; Wiegand, Susanna; Biebermann, Heike; Fischer-Posovszky, Pamela; Wabitsch, Martin; Völzke, Henry; Nauck, Matthias; Teumer, Alexander; Rosskopf, Dieter; Rimmbach, Christian; Schreiber, Stefan; Jacobs, Gunnar; Lieb, Wolfgang; Franke, Andre; Hebebrand, Johannes; Hinney, Anke

    2014-01-01

    Heritability estimates for body mass index (BMI) variation are high. For mothers and their offspring higher BMI correlations have been described than for fathers. Variation(s) in the exclusively maternally inherited mitochondrial DNA (mtDNA) might contribute to this parental effect. Thirty-two to 40 mtDNA single nucleotide polymorphisms (SNPs) were available from genome-wide association study SNP arrays (Affymetrix 6.0). For discovery, we analyzed association in a case-control (CC) sample of 1,158 extremely obese children and adolescents and 435 lean adult controls. For independent confirmation, 7,014 population-based adults were analyzed as CC sample of n = 1,697 obese cases (BMI≥30 kg/m2) and n = 2,373 normal weight and lean controls (BMI<25 kg/m2). SNPs were analyzed as single SNPs and haplogroups determined by HaploGrep. Fisher's two-sided exact test was used for association testing. Moreover, the D-loop was re-sequenced (Sanger) in 192 extremely obese children and adolescents and 192 lean adult controls. Association testing of detected variants was performed using Fisher's two-sided exact test. For discovery, nominal association with obesity was found for the frequent allele G of m.8994G/A (rs28358887, p = 0.002) located in ATP6. Haplogroup W was nominally overrepresented in the controls (p = 0.039). These findings could not be confirmed independently. For two of the 252 identified D-loop variants nominal association was detected (m.16292C/T, p = 0.007, m.16189T/C, p = 0.048). Only eight controls carried the m.16292T allele, five of whom belonged to haplogroup W that was initially enriched among these controls. m.16189T/C might create an uninterrupted poly-C tract located near a regulatory element involved in replication of mtDNA. Though follow-up of some D-loop variants still is conceivable, our hypothesis of a contribution of variation in the exclusively maternally inherited mtDNA to the observed larger correlations for BMI between

  8. iDNA-Prot: identification of DNA binding proteins using random forest with grey model.

    PubMed

    Lin, Wei-Zhong; Fang, Jian-An; Xiao, Xuan; Chou, Kuo-Chen

    2011-01-01

    DNA-binding proteins play crucial roles in various cellular processes. Developing high throughput tools for rapidly and effectively identifying DNA-binding proteins is one of the major challenges in the field of genome annotation. Although many efforts have been made in this regard, further effort is needed to enhance the prediction power. By incorporating the features into the general form of pseudo amino acid composition that were extracted from protein sequences via the "grey model" and by adopting the random forest operation engine, we proposed a new predictor, called iDNA-Prot, for identifying uncharacterized proteins as DNA-binding proteins or non-DNA binding proteins based on their amino acid sequences information alone. The overall success rate by iDNA-Prot was 83.96% that was obtained via jackknife tests on a newly constructed stringent benchmark dataset in which none of the proteins included has ≥25% pairwise sequence identity to any other in a same subset. In addition to achieving high success rate, the computational time for iDNA-Prot is remarkably shorter in comparison with the relevant existing predictors. Hence it is anticipated that iDNA-Prot may become a useful high throughput tool for large-scale analysis of DNA-binding proteins. As a user-friendly web-server, iDNA-Prot is freely accessible to the public at the web-site on http://icpr.jci.edu.cn/bioinfo/iDNA-Prot or http://www.jci-bioinfo.cn/iDNA-Prot. Moreover, for the convenience of the vast majority of experimental scientists, a step-by-step guide is provided on how to use the web-server to get the desired results. PMID:21935457

  9. Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

    DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

  10. DNA profiles from fingermarks.

    PubMed

    Templeton, Jennifer E L; Linacre, Adrian

    2014-11-01

    Criminal investigations would be considerably improved if DNA profiles could be routinely generated from single fingermarks. Here we report a direct DNA profiling method that was able to generate interpretable profiles from 71% of 170 fingermarks. The data are based on fingermarks from all 5 digits of 34 individuals. DNA was obtained from the fingermarks using a swab moistened with Triton-X, and the fibers were added directly to one of two commercial DNA profiling kits. All profiles were obtained without increasing the number of amplification cycles; therefore, our method is ideally suited for adoption by the forensic science community. We indicate the use of the technique in a criminal case in which a DNA profile was generated from a fingermark on tape that was wrapped around a drug seizure. Our direct DNA profiling approach is rapid and able to generate profiles from touched items when current forensic practices have little chance of success. PMID:25391915

  11. Electrocatalysis in DNA Sensors.

    PubMed

    Furst, Ariel; Hill, Michael G; Barton, Jacqueline K

    2014-12-14

    Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology. PMID:25435647

  12. DNA Media Storage

    PubMed Central

    Bogard, Christy M.; Rouchka, Eric C.

    2010-01-01

    In 1994, University of Southern California computer scientist Dr. Leonard Adelman solved the Hamiltonian Path Problem using DNA as a computational mechanism. He proved the principle that DNA computing could be used to solve computationally complex problems. Because of the limitations in discovery time, resource requirements, and sequence mismatches, DNA computing has not yet become a commonly accepted practice. However, advancements are continually being discovered that are evolving the field of DNA Computing. Practical applications of DNA are not restricted to computation alone. This research presents a novel approach in which DNA could be used as a means of storing files. Through the use of Multiple Sequence Alignment combined with intelligent heuristics, the most probabilistic file contents can be determined with minimal errors. PMID:20622994

  13. DNA Media Storage.

    PubMed

    Bogard, Christy M; Rouchka, Eric C

    2007-09-01

    In 1994, University of Southern California computer scientist Dr. Leonard Adelman solved the Hamiltonian Path Problem using DNA as a computational mechanism. He proved the principle that DNA computing could be used to solve computationally complex problems. Because of the limitations in discovery time, resource requirements, and sequence mismatches, DNA computing has not yet become a commonly accepted practice. However, advancements are continually being discovered that are evolving the field of DNA Computing. Practical applications of DNA are not restricted to computation alone. This research presents a novel approach in which DNA could be used as a means of storing files. Through the use of Multiple Sequence Alignment combined with intelligent heuristics, the most probabilistic file contents can be determined with minimal errors. PMID:20622994

  14. Ribonucleotides in Bacterial DNA

    PubMed Central

    Schroeder, Jeremy W.; Randall, Justin R.; Matthews, Lindsay A.; Simmons, Lyle A.

    2014-01-01

    In all living cells, DNA is the storage medium for genetic information. Being quite stable, DNA is well-suited for its role in storage and propagation of information, but RNA is also covalently included in DNA through various mechanisms. Recent studies also demonstrate useful aspects of including ribonucleotides in the genome during repair. Therefore, our understanding of the consequences of RNA inclusion into bacterial genomic DNA is just beginning, but with its high frequency of occurrence the consequences and potential benefits are likely to be numerous and diverse. In this review, we discuss the processes that cause ribonucleotide inclusion in genomic DNA, the pathways important for ribonucleotide removal and the consequences that arise should ribonucleotides remain nested in genomic DNA. PMID:25387798

  15. Electrocatalysis in DNA Sensors

    PubMed Central

    Furst, Ariel; Hill, Michael G.; Barton, Jacqueline K.

    2014-01-01

    Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology. PMID:25435647

  16. Cell-free DNA screening and sex chromosome aneuploidies.

    PubMed

    Mennuti, Michael T; Chandrasekaran, Suchitra; Khalek, Nahla; Dugoff, Lorraine

    2015-10-01

    Cell-free DNA (cfDNA) testing is increasingly being used to screen pregnant women for fetal aneuploidies. This technology may also identify fetal sex and can be used to screen for sex chromosome aneuploidies (SCAs). Physicians offering this screening will need to be prepared to offer comprehensive prenatal counseling about these disorders to an increasing number of patients. The purpose of this article is to consider the source of information to use for counseling, factors in parental decision-making, and the performance characteristics of cfDNA testing in screening for SCAs. Discordance between ultrasound examination and cfDNA results regarding fetal sex is also discussed. PMID:26088741

  17. Replication of nanoscale DNA patterns

    NASA Astrophysics Data System (ADS)

    Maass, Corinna; Wang, Tong; Sha, Ruojie; Leunissen, Mirjam; Dreyfus, Remi; Seeman, Nadrian; Chaikin, Paul

    2011-03-01

    We present an artificial supramolecular system mimicking self- replication and information transmission strategies in nature, but without the aid of enzymes or equivalent biological mechanisms. Using DNA nanotechnology techniques, we can make DNA tiles with selective interactions based on complementary single-strand connections. A linear tile pattern distinguished by their connector sequences is transmitted to a subsequent generation of copies by connector hybridisation. Longitudinal pattern formation and transverse copy attachment are well separated by different melting temperatures. We have achieved a faithful transmission of the pattern information to the second replication generation. We use AFM imaging to test for pattern fidelity and gel electrophoresis for quantitative yield analysis. supported by a DAAD postdoc grant.

  18. DNA-based machines.

    PubMed

    Wang, Fuan; Willner, Bilha; Willner, Itamar

    2014-01-01

    The base sequence in nucleic acids encodes substantial structural and functional information into the biopolymer. This encoded information provides the basis for the tailoring and assembly of DNA machines. A DNA machine is defined as a molecular device that exhibits the following fundamental features. (1) It performs a fuel-driven mechanical process that mimics macroscopic machines. (2) The mechanical process requires an energy input, "fuel." (3) The mechanical operation is accompanied by an energy consumption process that leads to "waste products." (4) The cyclic operation of the DNA devices, involves the use of "fuel" and "anti-fuel" ingredients. A variety of DNA-based machines are described, including the construction of "tweezers," "walkers," "robots," "cranes," "transporters," "springs," "gears," and interlocked cyclic DNA structures acting as reconfigurable catenanes, rotaxanes, and rotors. Different "fuels", such as nucleic acid strands, pH (H⁺/OH⁻), metal ions, and light, are used to trigger the mechanical functions of the DNA devices. The operation of the devices in solution and on surfaces is described, and a variety of optical, electrical, and photoelectrochemical methods to follow the operations of the DNA machines are presented. We further address the possible applications of DNA machines and the future perspectives of molecular DNA devices. These include the application of DNA machines as functional structures for the construction of logic gates and computing, for the programmed organization of metallic nanoparticle structures and the control of plasmonic properties, and for controlling chemical transformations by DNA machines. We further discuss the future applications of DNA machines for intracellular sensing, controlling intracellular metabolic pathways, and the use of the functional nanostructures for drug delivery and medical applications. PMID:24647836

  19. DNA ELECTROPHORESIS AT SURFACES

    SciTech Connect

    RAFAILOVICH, MIRIAM; SOKOLOV, JONATHAN; GERSAPPE, DILIP

    2003-09-01

    During this year we performed two major projects: I. We developed a detailed theoretical model which complements our experiments on surface DNA electrophoresis. We found that it was possible to enhance the separation of DNA chains by imposing a chemical nanoscale pattern on the surface. This approach utilized the surface interaction effect of the DNA chains with the substrate and is a refinement to our previous method in which DNA chains were separated on homogeneous flat surfaces. By introducing the nano-patterns on the surface, the conformational changes of DNA chains of different lengths can be amplified, which results in the different friction strengths with the substrate surface. Our results also show that, when compared to the DNA electrophoresis performed on homogeneous flat surfaces, nanopatterned surfaces offer a larger window in choosing different surface interactions to achieve separation. II. In collaboration with a large international manufacturer of skin care products we also embarked on a project involving photo toxicity of titanium dioxide nanoparticles, which are a key ingredient in sunscreen and cosmetic lotions. The results clearly implicated the nanoparticles in catalyzing damage to chromosomal DNA. We then used this knowledge to develop a polymer/anti-oxidant coating which prevented the photocatalytic reaction on DNA while still retaining the UV absorptive properties of the nanoparticles. The standard gel electrophoresis was not sufficient in determining the extent of the DNA damage. The conclusions of this study were based predominantly on analysis obtained with the surface electrophoresis method.

  20. Tiny telomere DNA

    PubMed Central

    Ren, Jinsong; Qu, Xiaogang; Trent, John O.; Chaires, Jonathan B.

    2002-01-01

    We describe the design, synthesis and biophysical characterization of a novel DNA construct in which a folded quadruplex structure is joined to a standard double helix. Circular dichroism, gel electrophoresis, three-dimensional UV melting and differential scanning calorimetry were all used to characterize the structure. Rigorous molecular dynamics simulations were used to build a plausible atomic-level structural model of the DNA construct. This novel DNA construct provides a model for the duplex–quadruplex junction region at the end of chromosomal DNA and offers a system for the study of structure-selective ligand binding. PMID:12034817

  1. DNA Damage Response

    PubMed Central

    Giglia-Mari, Giuseppina; Zotter, Angelika; Vermeulen, Wim

    2011-01-01

    Structural changes to DNA severely affect its functions, such as replication and transcription, and play a major role in age-related diseases and cancer. A complicated and entangled network of DNA damage response (DDR) mechanisms, including multiple DNA repair pathways, damage tolerance processes, and cell-cycle checkpoints safeguard genomic integrity. Like transcription and replication, DDR is a chromatin-associated process that is generally tightly controlled in time and space. As DNA damage can occur at any time on any genomic location, a specialized spatio-temporal orchestration of this defense apparatus is required. PMID:20980439

  2. A comparison of DNA extraction methods using Petunia hybrida tissues.

    PubMed

    Tamari, Farshad; Hinkley, Craig S; Ramprashad, Naderia

    2013-09-01

    Extraction of DNA from plant tissue is often problematic, as many plants contain high levels of secondary metabolites that can interfere with downstream applications, such as the PCR. Removal of these secondary metabolites usually requires further purification of the DNA using organic solvents or other toxic substances. In this study, we have compared two methods of DNA purification: the cetyltrimethylammonium bromide (CTAB) method that uses the ionic detergent hexadecyltrimethylammonium bromide and chloroform-isoamyl alcohol and the Edwards method that uses the anionic detergent SDS and isopropyl alcohol. Our results show that the Edwards method works better than the CTAB method for extracting DNA from tissues of Petunia hybrida. For six of the eight tissues, the Edwards method yielded more DNA than the CTAB method. In four of the tissues, this difference was statistically significant, and the Edwards method yielded 27-80% more DNA than the CTAB method. Among the different tissues tested, we found that buds, 4 days before anthesis, had the highest DNA concentrations and that buds and reproductive tissue, in general, yielded higher DNA concentrations than other tissues. In addition, DNA extracted using the Edwards method was more consistently PCR-amplified than that of CTAB-extracted DNA. Based on these results, we recommend using the Edwards method to extract DNA from plant tissues and to use buds and reproductive structures for highest DNA yields. PMID:23997658

  3. Implementing Prenatal Diagnosis Based on Cell-Free Fetal DNA: Accurate Identification of Factors Affecting Fetal DNA Yield

    PubMed Central

    Barrett, Angela N.; Zimmermann, Bernhard G.; Wang, Darrell; Holloway, Andrew; Chitty, Lyn S.

    2011-01-01

    Objective Cell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing cell-stabilizing agents, storage temperature, interval to sample processing and DNA extraction method used. Methods Microfluidic digital PCR was performed to precisely quantify male (fetal) DNA, total DNA and long DNA fragments (indicative of maternal cellular DNA). Real-time qPCR was used to assay for the presence of male SRY signal in samples. Results Total cell-free DNA quantity increased significantly with time in samples stored in K3EDTA tubes, but only minimally in cell stabilizing tubes. This increase was solely due to the presence of additional long fragment DNA, with no change in quantity of fetal or short DNA, resulting in a significant decrease in proportion of cell-free fetal DNA over time. Storage at 4°C did not prevent these changes. Conclusion When samples can be processed within eight hours of blood draw, K3EDTA tubes can be used. Prolonged transfer times in K3EDTA tubes should be avoided as the proportion of fetal DNA present decreases significantly; in these situations the use of cell stabilising tubes is preferable. The DNA extraction kit used may influence success rate of diagnostic tests. PMID:21998643

  4. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    SciTech Connect

    SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

    2004-03-24

    Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNA populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.

  5. Cytomegalovirus in urine: detection of viral DNA by sandwich hybridization.

    PubMed

    Virtanen, M; Syvänen, A C; Oram, J; Söderlund, H; Ranki, M

    1984-12-01

    A cytomegalovirus (CMV)-specific sandwich hybridization test was constructed by using two adjacent BamHI DNA fragments of CMV DNA as reagents. The fragments were cloned into two different vectors. One of the recombinants was attached to the filter, and the other was the labeled probe. When present in the sample, CMV DNA mediated labeling of the filter by hybridizing to both the filter-bound DNA and the probe. The sandwich hybridization test was applied for the detection of CMV DNA from urine. DNA was released from virus by 2% Sarkosyl, concentrated by 2-butanol extraction and isopropanol precipitation, denatured, and finally subjected to the sandwich hybridization test. As a result, 70 to 90% of the original viral DNA could be recovered and demonstrated by the quantitative hybridization reaction. Urine could be stored at room temperature in Sarkosyl for at least 2 days without affecting the detectability of CMV. The clinical applicability of the test was evaluated by studying urine samples from four infants excreting CMV. Sandwich hybridization demonstrated the presence of CMV DNA in all of the specimens. These contained originally 10(5) to 10(8) CMV DNA molecules per ml. PMID:6097598

  6. Comparison of four commercial DNA extraction kits for the recovery of Bacillus spp. spore DNA from spiked powder samples.

    PubMed

    Mölsä, Markos; Kalin-Mänttäri, Laura; Tonteri, Elina; Hemmilä, Heidi; Nikkari, Simo

    2016-09-01

    Bacillus spp. include human pathogens such as Bacillus anthracis, the causative agent of anthrax and a biothreat agent. Bacillus spp. form spores that are physically highly resistant and may remain active over sample handling. We tested four commercial DNA extraction kits (QIAamp DNA Mini Kit, RTP Pathogen Kit, ZR Fungal/Bacterial DNA MiniPrep, and genesig Easy DNA/RNA Extraction kit) for sample inactivation and DNA recovery from two powders (icing sugar and potato flour) spiked with Bacillus thuringiensis spores. The DNA was analysed using a B. thuringiensis-specific real-time PCR assay. The detection limit was 3×10(1)CFU of spiked B. thuringiensis spores with the QIAamp DNA Mini, RTP Pathogen, and genesig Easy DNA/RNA Extraction kits, and 3×10(3)CFU with the ZR Fungal/Bacterial DNA MiniPrep kit. The results showed that manual extraction kits are effective and safe for fast and easy DNA extraction from powder samples even in field conditions. Adding a DNA filtration step to the extraction protocol ensures the removal of Bacillus spp. spores from DNA samples without affecting sensitivity. PMID:27435532

  7. How to limit false positives in environmental DNA and metabarcoding?

    PubMed

    Ficetola, Gentile Francesco; Taberlet, Pierre; Coissac, Eric

    2016-05-01

    Environmental DNA (eDNA) and metabarcoding are boosting our ability to acquire data on species distribution in a variety of ecosystems. Nevertheless, as most of sampling approaches, eDNA is not perfect. It can fail to detect species that are actually present, and even false positives are possible: a species may be apparently detected in areas where it is actually absent. Controlling false positives remains a main challenge for eDNA analyses: in this issue of Molecular Ecology Resources, Lahoz-Monfort et al. () test the performance of multiple statistical modelling approaches to estimate the rate of detection and false positives from eDNA data. Here, we discuss the importance of controlling for false detection from early steps of eDNA analyses (laboratory, bioinformatics), to improve the quality of results and allow an efficient use of the site occupancy-detection modelling (SODM) framework for limiting false presences in eDNA analysis. PMID:27062589

  8. Simplified field preservation of tissues for subsequent DNA analyses.

    PubMed

    Michaud, Corinne L; Foran, David R

    2011-07-01

    Successful DNA-based identification of mass disaster victims depends on acquiring tissues that are not highly degraded. In this study, multiple protocols for field preservation of tissues for later DNA analysis were tested. Skin and muscle samples were collected from decaying pig carcasses. Tissues were preserved using cold storage, desiccation, or room temperature storage in preservative solutions for up to 6 months. DNA quality was assessed through amplification of successively larger segments of nuclear DNA. Solution-based storage, including a DMSO/NaCl/EDTA mixture, alcohols, and RNAlater preserved DNA of the highest quality, refrigeration was intermediate, and desiccation was least effective. Tissue type and extent of decomposition significantly affected stored DNA quality. Overall, the results indicate that any tissue preservation attempt is far superior to delaying or forgoing preservation efforts, and that simple, inexpensive methods can be highly effective in preserving DNA, thus should be initiated as quickly as possible. PMID:21480896

  9. A method to study in vivo stability of DNA nanostructures☆

    PubMed Central

    Surana, Sunaina; Bhatia, Dhiraj; Krishnan, Yamuna

    2013-01-01

    DNA nanostructures are rationally designed, synthetic, nanoscale assemblies obtained from one or more DNA sequences by their self-assembly. Due to the molecularly programmable as well as modular nature of DNA, such designer DNA architectures have great potential for in cellulo and in vivo applications. However, demonstrations of functionality in living systems necessitates a method to assess the in vivo stability of the relevant nanostructures. Here, we outline a method to quantitatively assay the stability and lifetime of various DNA nanostructures in vivo. This exploits the property of intact DNA nanostructures being uptaken by the coelomocytes of the multicellular model organism Caenorhabditis elegans. These studies reveal that the present fluorescence based assay in coelomocytes of C. elegans is an useful in vivo test bed for measuring DNA nanostructure stability. PMID:23623822

  10. Replicative DNA polymerases.

    PubMed

    Johansson, Erik; Dixon, Nicholas

    2013-06-01

    In 1959, Arthur Kornberg was awarded the Nobel Prize for his work on the principles by which DNA is duplicated by DNA polymerases. Since then, it has been confirmed in all branches of life that replicative DNA polymerases require a single-stranded template to build a complementary strand, but they cannot start a new DNA strand de novo. Thus, they also depend on a primase, which generally assembles a short RNA primer to provide a 3'-OH that can be extended by the replicative DNA polymerase. The general principles that (1) a helicase unwinds the double-stranded DNA, (2) single-stranded DNA-binding proteins stabilize the single-stranded DNA, (3) a primase builds a short RNA primer, and (4) a clamp loader loads a clamp to (5) facilitate the loading and processivity of the replicative polymerase, are well conserved among all species. Replication of the genome is remarkably robust and is performed with high fidelity even in extreme environments. Work over the last decade or so has confirmed (6) that a common two-metal ion-promoted mechanism exists for the nucleotidyltransferase reaction that builds DNA strands, and (7) that the replicative DNA polymerases always act as a key component of larger multiprotein assemblies, termed replisomes. Furthermore (8), the integrity of replisomes is maintained by multiple protein-protein and protein-DNA interactions, many of which are inherently weak. This enables large conformational changes to occur without dissociation of replisome components, and also means that in general replisomes cannot be isolated intact. PMID:23732474

  11. Bac clones generated from sheared dna

    SciTech Connect

    Osoegawa, Kazutoyo; Vessere, Gery M.; Shu, Chung Li; Hoskins,Roger A.; Abad, Jose P.; de Pablos, Beatriz; Villasante, Alfredo; deJong, Pieter J.

    2006-08-09

    BAC libraries generated from restriction-digested genomic DNA display representational bias and lack some sequences. To facilitate completion of genome projects, procedures have been developed to create BACs from DNA physically sheared to create fragments extending up to 200kb. The DNA fragments were repaired to create blunt ends and ligated to a new BAC vector. This approach has been tested by generating BAC libraries from Drosophila DNA, with insert lengths of 50 kb to 150 kb. The libraries lack chimeric clone problems as determined by mapping paired BAC-end sequences of one library to the D. melanogaster genome sequence. The utility of ''sheared'' libraries was demonstrated by closure of a previous clone gap and by isolation of clones from telomeric regions, which were notably absent from previous Drosophila BAC libraries.

  12. DMEAS: DNA methylation entropy analysis software

    PubMed Central

    He, Jianlin; Sun, Xinxi; Shao, Xiaojian; Liang, Liji; Xie, Hehuang

    2013-01-01

    Summary: DMEAS is the first user-friendly tool dedicated to analyze the distribution of DNA methylation patterns for the quantification of epigenetic heterogeneity. It supports the analysis of both locus-specific and genome-wide bisulfite sequencing data. DMEAS progressively scans the mapping results of bisulfite sequencing reads to extract DNA methylation patterns for contiguous CpG dinucleotides. It determines the DNA methylation level and calculates methylation entropy for genomic segments to enable the quantitative assessment of DNA methylation variations observed in cell populations. Availability and implementation: DMEAS program, user guide and all the testing data are freely available from http://sourceforge.net/projects/dmeas/files/ Contact: davidxie@vt.edu Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:23749987

  13. Many Ways to Loop DNA

    PubMed Central

    Griffith, Jack D.

    2013-01-01

    In the 1960s, I developed methods for directly visualizing DNA and DNA-protein complexes using an electron microscope. This made it possible to examine the shape of DNA and to visualize proteins as they fold and loop DNA. Early applications included the first visualization of true nucleosomes and linkers and the demonstration that repeating tracts of adenines can cause a curvature in DNA. The binding of DNA repair proteins, including p53 and BRCA2, has been visualized at three- and four-way junctions in DNA. The trombone model of DNA replication was directly verified, and the looping of DNA at telomeres was discovered. PMID:24005675

  14. The use of Hemastix and the subsequent lack of DNA recovery using the Promega DNA IQ system.

    PubMed

    Poon, Hiron; Elliott, Jim; Modler, Jeff; Frégeau, Chantal

    2009-11-01

    Following implementation of our automated process incorporating the Promega DNA IQ system as a DNA extraction method, a large number of blood-containing exhibits failed to produce DNA. These exhibits had been tested with the Hemastix reagent strip, commonly used by police investigators and forensic laboratories as a screening test for blood. Some exhibits were even tainted green following transfer of the presumptive test reagents onto the samples. A series of experiments were carried out to examine the effect of the Hemastix chemistries on the DNA IQ system. Our results indicate that one or more chemicals imbedded in the Hemastix reagent strip severely reduce the ability to recover DNA from any suspected stain using the DNA IQ magnetic bead technology. The 3,3',5,5'-tetramethylbenzidine (TMB) used as the reporting dye appears to interact with the magnetic beads to prevent DNA recovery. Hydrogen peroxide does not seem to be involved. The Hemastix chemistries do not interfere in any way with DNA extraction performed using phenol-chloroform. The incompatibility of the Hemastix chemistries on the DNA IQ system forced us to adopt an indirect approach using filter paper to carry out the presumptive test. PMID:19804523

  15. Nanoparticle bridge DNA biosensor

    NASA Astrophysics Data System (ADS)

    Huang, Hong-Wen

    A new DNA sensing method is demonstrated in which DNA hybridization events lead to the formation of nanoparticle satellites that bridge two electrodes and are detected electrically. The hybridization events are exclusively carried out only on specific locations, the surfaces of C-ssDNA modified 50 nm GNPs. The uniqueness of this work is that only a small number of T-ccDNA molecules (<10) is required to form the nanoparticle satellites, allowing ultra-sensitive DNA sensing. The principle of this new DNA sensing technique has been demonstrated using target DNA and three-base-pair-mismatched DNA in 20nM concentrations. Three single-stranded DNA (ssDNA) system is used in our experiment which includes Capture-ssDNA (C-ssDNA), Target-ssDNA (T-ssDNA) and Probe-ssDNA (P-ssDNA). Both C-ssDNA and P-ssDNA are modified by a thiol group and can hybridize with different portions of T-ssDNA. T-ssDNA requires no modification in three ssDNA system, which is beneficial in many applications. C-ssDNA modified 50nm gold nanoparticle (C-50au) and P-ssDNA modified 30nm gold nanoparticle (P-30au) are prepared through the reaction of thiol-gold chemical bonding between thiolated ssDNA and gold nanoparticle (GNP) (C-ssDNA with 50nm GNP, P-ssDNA with 30nm GNP). We controllably place the C-50au only on the SiO2 band surface (˜ 90nm width) between two gold electrodes (source and drain electrodes) by forming positively- and negatively-charged self-assembled monolayers (SAMs) on SiO2 and gold surface, respectively. DNA modified GNP is negatively charged due to ionization of phosphate group on DNA back bone. C-50au therefore is negatively charged and can only be attracted toward SiO2 area (repelled by negatively charged gold electrode surface). The amine group of positively-charged SAMs on SiO2 surface is then passivated by converting to non-polar methyl functional group after C-50au placement. P-30au is first hybridized with T-ssDNA in the solution phase (T-P- 30au formed) and is introduced

  16. Simulated radioactive decontamination of biological samples using a portable DNA extraction instrument for rapid DNA profiling.

    PubMed

    Frégeau, Chantal J; Dalpé, Claude

    2016-02-01

    A portable DNA extraction instrument was evaluated for its ability to decontaminate blood and saliva samples deposited on different surfaces (metal, plastic and glass) contaminated with stable isotopes of cobalt (Co), cesium (Cs), and strontium (Sr) as equivalents to their radiogenic (60)Co, (137)Cs, and (90)Sr isotopes, respectively, that could be released during a nuclear weapon accident or a radiological dispersal device (RDD) detonation. Despite the very high contamination levels tested in this study, successful removal of greater than 99.996% of the Co, Cs, Sr contaminants was achieved based on inductively coupled plasma-mass spectrometry (ICP-MS) and neutron activation analyses carried out on all liquids (including DNA eluates) and solid waste produced during automated DNA extraction. The remaining amounts of Co, Cs and Sr in the DNA eluates, when converted to dose rates (corresponding to (60)Co, (137)Cs and (90)Sr), were determined to be below the recommended dose limits for the general public in most of the scenarios tested. The presence of Co, Cs and Sr contaminants in the cell lysates had no adverse impact on the binding of DNA onto the magnetic DNA IQ™ beads. DNA yields were similar to uncontaminated controls. The remaining Co, Cs and Sr in the DNA eluates did not interfere with real-time PCR DNA quantification. In addition, the quality of the AmpFlSTR(®) Identifiler(®) profiles derived in 26min using an accelerated protocol was very good and comparable to controls. This study emphasizes the use of an accelerated process involving a portable DNA extraction instrument to significantly reduce radioactive dose rates to allow contaminated samples to be processed safely in a forensic mobile laboratory to expedite the identification of individuals potentially involved in the dispersal of nuclear or other radioactive materials. PMID:26773226

  17. Non-equilibrium Dynamics of DNA Nanotubes

    NASA Astrophysics Data System (ADS)

    Hariadi, Rizal Fajar

    Can the fundamental processes that underlie molecular biology be understood and simulated by DNA nanotechnology? The early development of DNA nanotechnology by Ned Seeman was driven by the desire to find a solution to the protein crystallization problem. Much of the later development of the field was also driven by envisioned applications in computing and nanofabrication. While the DNA nanotechnology community has assembled a versatile tool kit with which DNA nanostructures of considerable complexity can be assembled, the application of this tool kit to other areas of science and technology is still in its infancy. This dissertation reports on the construction of non-equilibrium DNA nanotube dynamic to probe molecular processes in the areas of hydrodynamics and cytoskeletal behavior. As the first example, we used DNA nanotubes as a molecular probe for elongational flow measurement in different micro-scale flow settings. The hydrodynamic flow in the vicinity of simple geometrical objects, such as a rigid DNA nanotube, is amenable to rigorous theoretical investigation. We measured the distribution of elongational flows produced in progressively more complex settings, ranging from the vicinity of an orifice in a microfluidic chamber to within a bursting bubble of Pacific ocean water. This information can be used to constrain theories on the origin of life in which replication involves a hydrodynamically driven fission process, such as the coacervate fission proposed by Oparin. A second theme of this dissertation is the bottom-up construction of a de novo artificial cytoskeleton with DNA nanotubes. The work reported here encompasses structural, locomotion, and control aspects of non-equilibrium cytoskeletal behavior. We first measured the kinetic parameters of DNA nanotube assembly and tested the accuracy of the existing polymerization models in the literature. Toward recapitulation of non-equilibrium cytoskeletal dynamics, we coupled the polymerization of DNA

  18. Curating DNA specimens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA data are used in a variety of ethnobiological disciplines including archaeology, conservation, ecology, medicinal plants and natural products research, taxonomy and systematics, crop evolution and domestication, and genetic diversity. It frequently is convenient to store and share DNA among coop...

  19. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  20. MICROWAVE RESONANCES IN DNA

    EPA Science Inventory

    This report describes spectroscopic studies of DNA which were undertaken to better understand a physical basis for microwave absorption by this molecule. hree types of studies are described. ) The low frequency scattered light spectrum of DNA was studied by two methods. irst, Ram...

  1. Replicating repetitive DNA.

    PubMed

    Tognetti, Silvia; Speck, Christian

    2016-05-27

    The function and regulation of repetitive DNA, the 'dark matter' of the genome, is still only rudimentarily understood. Now a study investigating DNA replication of repetitive centromeric chromosome segments has started to expose a fascinating replication program that involves suppression of ATR signalling, in particular during replication stress. PMID:27230530

  2. Hydrogels: DNA bulks up

    NASA Astrophysics Data System (ADS)

    Labean, Thom

    2006-10-01

    Since the 1940s DNA has been known as the genetic material connected to heredity, and from the early 1980s it has also been considered as a potential structural material for nanoscale construction. Now, a hydrogel made entirely of DNA brings this molecule into the realm of bulk materials.

  3. Translesion DNA synthesis

    PubMed Central

    Vaisman, Alexandra; McDonald, John P.; Woodgate, Roger

    2014-01-01

    All living organisms are continually exposed to agents that damage their DNA, which threatens the integrity of their genome. As a consequence, cells are equipped with a plethora of DNA repair enzymes to remove the damaged DNA. Unfortunately, situations nevertheless arise where lesions persist, and these lesions block the progression of the cell’s replicase. Under these situations, cells are forced to choose between recombination-mediated “damage avoidance” pathways, or use a specialized DNA polymerase (pol) to traverse the blocking lesion. The latter process is referred to as Translesion DNA Synthesis (TLS). As inferred by its name, TLS not only results in bases being (mis)incorporated opposite DNA lesions, but also downstream of the replicase-blocking lesion, so as to ensure continued genome duplication and cell survival. Escherichia coli and Salmonella typhimurium possess five DNA polymerases, and while all have been shown to facilitate TLS under certain experimental conditions, it is clear that the LexA-regulated and damage-inducible pols II, IV and V perform the vast majority of TLS under physiological conditions. Pol V can traverse a wide range of DNA lesions and performs the bulk of mutagenic TLS, whereas pol II and pol IV appear to be more specialized TLS polymerases. PMID:26442823

  4. Characterization of muntjac DNA

    SciTech Connect

    Davis, R.C.

    1981-05-27

    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange.

  5. DNA as information.

    PubMed

    Wills, Peter R

    2016-03-13

    This article reviews contributions to this theme issue covering the topic 'DNA as information' in relation to the structure of DNA, the measure of its information content, the role and meaning of information in biology and the origin of genetic coding as a transition from uninformed to meaningful computational processes in physical systems. PMID:26857666

  6. DNA-cell conjugates

    DOEpatents

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  7. Assessing the Utility of Soil DNA Extraction Kits for Increasing DNA Yields and Eliminating PCR Inhibitors from Buried Skeletal Remains.

    PubMed

    Hebda, Lisa M; Foran, David R

    2015-09-01

    DNA identification of human remains is often necessary when decedents are skeletonized; however, poor DNA recovery and polymerase chain reaction (PCR) inhibition are frequently encountered, a situation exacerbated by burial. In this research, the utility of integrating soil DNA isolation kits into buried skeletal DNA analysis was evaluated and compared to a standard human DNA extraction kit and organic extraction. The soil kits successfully extracted skeletal DNA at quantities similar to standard methods, although the two kits tested, which differ mechanistically, were not equivalent. Further, the PCR inhibitors calcium and humic acid were effectively removed using the soil kits, whereas collagen was less so. Finally, concordant control region sequences were obtained from human skeletal remains using all four methods. Based on these comparisons, soil DNA isolation kits, which quickened the extraction process, proved to be a viable extraction technique for skeletal remains that resulted in positive identification of a decedent. PMID:26258388

  8. Environmental DNA (eDNA) sampling improves occurrence and detection estimates of invasive burmese pythons.

    PubMed

    Hunter, Margaret E; Oyler-McCance, Sara J; Dorazio, Robert M; Fike, Jennifer A; Smith, Brian J; Hunter, Charles T; Reed, Robert N; Hart, Kristen M

    2015-01-01

    Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors

  9. Environmental DNA (eDNA) Sampling Improves Occurrence and Detection Estimates of Invasive Burmese Pythons

    PubMed Central

    Hunter, Margaret E.; Oyler-McCance, Sara J.; Dorazio, Robert M.; Fike, Jennifer A.; Smith, Brian J.; Hunter, Charles T.; Reed, Robert N.; Hart, Kristen M.

    2015-01-01

    Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors

  10. Environmental DNA (eDNA) sampling improves occurrence and detection estimates of invasive Burmese pythons

    USGS Publications Warehouse

    Hunter, Margaret E.; Oyler-McCance, Sara J.; Dorazio, Robert M.; Fike, Jennifer A.; Smith, Brian J.; Hunter, Charles T.; Reed, Robert N.; Hart, Kristen M.

    2015-01-01

    Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors

  11. Premeltons in DNA.

    PubMed

    Sobell, Henry M

    2016-03-01

    Premeltons are examples of emergent-structures (i.e., structural-solitons) that arise spontaneously in DNA due to the presence of nonlinear-excitations in its structure. They are of two kinds: B-B (or A-A) premeltons form at specific DNA-regions to nucleate site-specific DNA melting. These are stationary and, being globally-nontopological, undergo breather-motions that allow drugs and dyes to intercalate into DNA. B-A (or A-B) premeltons, on the other hand, are mobile, and being globally-topological, act as phase-boundaries transforming B- into A-DNA during the structural phase-transition. They are not expected to undergo breather motions. A key feature of both types of premeltons is the presence of an intermediate structural-form in their central regions (proposed as being a transition-state intermediate in DNA-melting and in the B- to A-transition), which differs from either A- or B-DNA. Called beta-DNA, this is both metastable and hyperflexible--and contains an alternating sugar-puckering pattern along the polymer backbone combined with the partial unstacking (in its lower energy-forms) of every-other base-pair. Beta-DNA is connected to either B- or to A-DNA on either side by boundaries possessing a gradation of nonlinear structural-change, these being called the kink and the antikink regions. The presence of premeltons in DNA leads to a unifying theory to understand much of DNA physical chemistry and molecular biology. In particular, premeltons are predicted to define the 5' and 3' ends of genes in naked-DNA and DNA in active-chromatin, this having important implications for understanding physical aspects of the initiation, elongation and termination of RNA-synthesis during transcription. For these and other reasons, the model will be of broader interest to the general-audience working in these areas. The model explains a wide variety of data, and carries with it a number of experimental predictions--all readily testable--as will be described in this review

  12. Problem-Solving Test: Pyrosequencing

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2013-01-01

    Terms to be familiar with before you start to solve the test: Maxam-Gilbert sequencing, Sanger sequencing, gel electrophoresis, DNA synthesis reaction, polymerase chain reaction, template, primer, DNA polymerase, deoxyribonucleoside triphosphates, orthophosphate, pyrophosphate, nucleoside monophosphates, luminescence, acid anhydride bond,…

  13. Problem-Solving Test: Expression Cloning of the Erythropoietin Receptor

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2008-01-01

    Terms to be familiar with before you start to solve the test: cytokines, cytokine receptors, cDNA library, cDNA synthesis, poly(A)[superscript +] RNA, primer, template, reverse transcriptase, restriction endonucleases, cohesive ends, expression vector, promoter, Shine-Dalgarno sequence, poly(A) signal, DNA helicase, DNA ligase, topoisomerases,…

  14. Advances in DNA photonics

    NASA Astrophysics Data System (ADS)

    Heckman, Emily M.; Aga, Roberto S.; Fehrman Cory, Emily M.; Ouchen, Fahima; Lesko, Alyssa; Telek, Brian; Lombardi, Jack; Bartsch, Carrie M.; Grote, James G.

    2012-10-01

    In this paper we present our current research in exploring a DNA biopolymer for photonics applications. A new processing technique has been adopted that employs a modified soxhlet-dialysis (SD) rinsing technique to completely remove excess ionic contaminants from the DNA biopolymer, resulting in a material with greater mechanical stability and enhanced performance reproducibility. This newly processed material has been shown to be an excellent material for cladding layers in poled polymer electro-optic (EO) waveguide modulator applications. Thin film poling results are reported for materials using the DNA biopolymer as a cladding layer, as are results for beam steering devices also using the DNA biopolymer. Finally, progress on fabrication of a Mach Zehnder EO modulator with DNA biopolymer claddings using nanoimprint lithography techniques is reported.

  15. Archaeal DNA replication.

    PubMed

    Kelman, Lori M; Kelman, Zvi

    2014-01-01

    DNA replication is essential for all life forms. Although the process is fundamentally conserved in the three domains of life, bioinformatic, biochemical, structural, and genetic studies have demonstrated that the process and the proteins involved in archaeal DNA replication are more similar to those in eukaryal DNA replication than in bacterial DNA replication, but have some archaeal-specific features. The archaeal replication system, however, is not monolithic, and there are some differences in the replication process between different species. In this review, the current knowledge of the mechanisms governing DNA replication in Archaea is summarized. The general features of the replication process as well as some of the differences are discussed. PMID:25421597

  16. Detection of Neospora caninum DNA by the polymerase chain reaction.

    PubMed

    Payne, S; Ellis, J

    1996-04-01

    Neospora caninum is a cyst-forming coccidian parasite which is now recognised as a major cause of abortion and neonatal mortality in cattle and other livestock. This study describes the primary DNA structure of the transcribed spacer region of the rDNA of N. caninum. Of importance is that the sequence data generated have been used to develop a species-specific PCR test for N. caninum DNA, which will prove valuable in epidemiology studies on neosporosis. PMID:8773521

  17. DNA Align Editor: DNA Alignment Editor Tool

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The SNPAlignEditor is a DNA sequence alignment editor that runs on Windows platforms. The purpose of the program is to provide an intuitive, user-friendly tool for manual editing of multiple sequence alignments by providing functions for input, editing, and output of nucleotide sequence alignments....

  18. Repair of gaps in retroviral DNA integration intermediates.

    PubMed

    Yoder, K E; Bushman, F D

    2000-12-01

    Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3' DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5' two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex. PMID:11070016

  19. DNA from soil mirrors plant taxonomic and growth form diversity.

    PubMed

    Yoccoz, N G; Bråthen, K A; Gielly, L; Haile, J; Edwards, M E; Goslar, T; Von Stedingk, H; Brysting, A K; Coissac, E; Pompanon, F; Sønstebø, J H; Miquel, C; Valentini, A; De Bello, F; Chave, J; Thuiller, W; Wincker, P; Cruaud, C; Gavory, F; Rasmussen, M; Gilbert, M T P; Orlando, L; Brochmann, C; Willerslev, E; Taberlet, P

    2012-08-01

    Ecosystems across the globe are threatened by climate change and human activities. New rapid survey approaches for monitoring biodiversity would greatly advance assessment and understanding of these threats. Taking advantage of next-generation DNA sequencing, we tested an approach we call metabarcoding: high-throughput and simultaneous taxa identification based on a very short (usually <100 base pairs) but informative DNA fragment. Short DNA fragments allow the use of degraded DNA from environmental samples. All analyses included amplification using plant-specific versatile primers, sequencing and estimation of taxonomic diversity. We tested in three steps whether degraded DNA from dead material in soil has the potential of efficiently assessing biodiversity in different biomes. First, soil DNA from eight boreal plant communities located in two different vegetation types (meadow and heath) was amplified. Plant diversity detected from boreal soil was highly consistent with plant taxonomic and growth form diversity estimated from conventional above-ground surveys. Second, we assessed DNA persistence using samples from formerly cultivated soils in temperate environments. We found that the number of crop DNA sequences retrieved strongly varied with years since last cultivation, and crop sequences were absent from nearby, uncultivated plots. Third, we assessed the universal applicability of DNA metabarcoding using soil samples from tropical environments: a large proportion of species and families from the study site were efficiently recovered. The results open unprecedented opportunities for large-scale DNA-based biodiversity studies across a range of taxonomic groups using standardized metabarcoding approaches. PMID:22507540

  20. Engineered DNA sequence syntax inspector.

    PubMed

    Hsiau, Timothy Hwei-Chung; Anderson, J Christopher

    2014-02-21

    DNAs encoding polypeptides often contain design errors that cause experiments to prematurely fail. One class of design errors is incorrect or missing elements in the DNA, here termed syntax errors. We have identified three major causes of syntax errors: point mutations from sequencing or manual data entry, gene structure misannotation, and unintended open reading frames (ORFs). The Engineered DNA Sequence Syntax Inspector (EDSSI) is an online bioinformatics pipeline that checks for syntax errors through three steps. First, ORF prediction in input DNA sequences is done by GeneMark; next, homologous sequences are retrieved by BLAST, and finally, syntax errors in the protein sequence are predicted by using the SIFT algorithm. We show that the EDSSI is able to identify previously published examples of syntactical errors and also show that our indel addition to the SIFT program is 97% accurate on a test set of Escherichia coli proteins. The EDSSI is available at http://andersonlab.qb3.berkeley.edu/Software/EDSSI/ . PMID:24364864

  1. Papillomavirus DNA Complementation in Vivo

    PubMed Central

    Hu, Jiafen; Cladel, Nancy M.; Budgeon, Lynn; Balogh, Karla K.; Christensen, Neil D.

    2009-01-01

    Recent phylogenic studies indicate that DNA recombination could have occurred in ancient papillomaviruses types. However, no experimental data is available to demonstrate this event because of the lack of human papillomavirus infection models. We have used the cottontail rabbit papillomavirus (CRPV)/rabbit model to study pathogenesis and immunogenicity of different mutant genomes in vivo. Although the domestic rabbit is not a natural host for CRPV infection, it is possible to initiate infection with naked CRPV DNA cloned into a plasmid and monitor papilloma outgrowth on these animals. Taking advantage of a large panel of mutants based on a CRPV strain (Hershey CRPV), we tested the hypothesis that two non-viable mutant genomes could induce papillomas by either recombination or complementation. We found that co-infection with a dysfunctional mutant with an E2 transactivation domain mutation and another mutant with an E7 ATG knock out generated papillomas in rabbits. DNA extracted from these papillomas contained genotypes from both parental genomes. Three additional pairs of dysfunctional mutants also showed similar results. Individual wild type genes were also shown to rescue the function of corresponding dysfunctional mutants. Therefore, we suggest that complementation occurred between these two non-viable mutant PV genomes in vivo. PMID:19379784

  2. DNA Microarray-Based Diagnostics.

    PubMed

    Marzancola, Mahsa Gharibi; Sedighi, Abootaleb; Li, Paul C H

    2016-01-01

    The DNA microarray technology is currently a useful biomedical tool which has been developed for a variety of diagnostic applications. However, the development pathway has not been smooth and the technology has faced some challenges. The reliability of the microarray data and also the clinical utility of the results in the early days were criticized. These criticisms added to the severe competition from other techniques, such as next-generation sequencing (NGS), impacting the growth of microarray-based tests in the molecular diagnostic market.Thanks to the advances in the underlying technologies as well as the tremendous effort offered by the research community and commercial vendors, these challenges have mostly been addressed. Nowadays, the microarray platform has achieved sufficient standardization and method validation as well as efficient probe printing, liquid handling and signal visualization. Integration of various steps of the microarray assay into a harmonized and miniaturized handheld lab-on-a-chip (LOC) device has been a goal for the microarray community. In this respect, notable progress has been achieved in coupling the DNA microarray with the liquid manipulation microsystem as well as the supporting subsystem that will generate the stand-alone LOC device.In this chapter, we discuss the major challenges that microarray technology has faced in its almost two decades of development and also describe the solutions to overcome the challenges. In addition, we review the advancements of the technology, especially the progress toward developing the LOC devices for DNA diagnostic applications. PMID:26614075

  3. Transport Distance of Invertebrate Environmental DNA in a Natural River

    PubMed Central

    Deiner, Kristy; Altermatt, Florian

    2014-01-01

    Environmental DNA (eDNA) monitoring is a novel molecular technique to detect species in natural habitats. Many eDNA studies in aquatic systems have focused on lake or ponds, and/or on large vertebrate species, but applications to invertebrates in river systems are emerging. A challenge in applying eDNA monitoring in flowing waters is that a species' DNA can be transported downstream. Whether and how far eDNA can be detected due to downstream transport remains largely unknown. In this study we tested for downstream detection of eDNA for two invertebrate species, Daphnia longispina and Unio tumidus, which are lake dwelling species in our study area. The goal was to determine how far away from the source population in a lake their eDNA could be detected in an outflowing river. We sampled water from eleven river sites in regular intervals up to 12.3 km downstream of the lake, developed new eDNA probes for both species, and used a standard PCR and Sanger sequencing detection method to confirm presence of each species' eDNA in the river. We detected D. longispina at all locations and across two time points (July and October); whereas with U. tumidus, we observed a decreased detection rate and did not detect its eDNA after 9.1 km. We also observed a difference in detection for this species at different times of year. The observed movement of eDNA from the source amounting to nearly 10 km for these species indicates that the resolution of an eDNA sample can be large in river systems. Our results indicate that there may be species' specific transport distances for eDNA and demonstrate for the first time that invertebrate eDNA can persist over relatively large distances in a natural river system. PMID:24523940

  4. Studying DNA in the Classroom.

    ERIC Educational Resources Information Center

    Zarins, Silja

    1993-01-01

    Outlines a workshop for teachers that illustrates a method of extracting DNA and provides instructions on how to do some simple work with DNA without sophisticated and expensive equipment. Provides details on viscosity studies and breaking DNA molecules. (DDR)

  5. Simple & Safe Genomic DNA Isolation.

    ERIC Educational Resources Information Center

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A procedure for purifying DNA using either bacteria or rat liver is presented. Directions for doing a qualitative DNA assay using diphenylamine and a quantitative DNA assay using spectroscopy are included. (KR)

  6. The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing clamp loaders to the appropriate polarity of DNA

    PubMed Central

    Hayner, Jaclyn N.; Douma, Lauren G.; Bloom, Linda B.

    2014-01-01

    Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3′OH (3′DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3′DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5′phosphate (5′P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5′DNA). The 5′P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3′DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5′DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3′DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role. PMID:25159615

  7. New incompatibilities uncovered using the Promega DNA IQ™ chemistry.

    PubMed

    Laurin, Nancy; Célestin, Florence; Clark, Meagan; Wilkinson, Della; Yamashita, Brian; Frégeau, Chantal

    2015-12-01

    Over the years, the Promega DNA IQ™ System was proven an effective technology for the production of clean DNA from a wide variety of casework specimens. The capture of DNA using the DNA IQ™ paramagnetic beads, however, was shown to be affected by a few specific chemicals that could be present on exhibits submitted to the laboratory. In this study, various blood and latent fingerprint enhancement reagents/methods, marker pens and adhesive tapes, applied at the crime scene or in the forensic laboratory on casework exhibits or used to collect biological material, were tested for their compatibility with the DNA IQ™ technology. Although no impact on DNA recovery was observed for most reagents, the MAGNA™ Jet Black fingerprint powder and three 3M Scotch(®) adhesive tapes were shown to severely or completely inhibit DNA binding onto the DNA IQ™ beads. The effect of MAGNA™ Jet Black on DNA recovery could be counteracted by separating the magnetic powder from the lysates by centrifugation or filtration, prior to DNA extraction. High quality STR profiles were obtained from samples subjected to MAGNA™ Jet Black suggesting it does not impact DNA integrity. PMID:26295931

  8. Incorporation of reporter-labeled nucleotides by DNA polymerases.

    PubMed

    Anderson, Jon P; Angerer, Bernhard; Loeb, Lawrence A

    2005-02-01

    The incorporation of fluorescently labeled nucleotides into DNA by DNA polymerases has been used extensively for tagging genes and for labeling DNA. However, we lack studies comparing polymerase efficiencies for incorporating different fluorescently labeled nucleotides. We analyzed the incorporation of fluorescent deoxynucleoside triphosphates by 10 different DNA polymerases, representing a cross-section of DNA polymerases from families A, B, and reverse transcriptase. The substitution of one or more different reporter-labeled nucleotides for the cognate nucleotides was initially investigated by using an in vitro polymerase extension filter-binding assay with natural DNA as a template. Further analysis on longer DNA fragments containing one or more nucleotide analogs was performed using a newly developed extension cut assay. The results indicate that incorporation of fluorescent nucleotides is dependent on the DNA polymerase, fluorophore, linker between the nucleotide and the fluorophore, and position for attachment of the linker and the cognate nucleotide. Of the polymerases tested, Taq and Vent exo DNA polymerases were most efficient at incorporating a variety of fluorescently labeled nucleotides. This study suggests that it should be feasible to copy DNA with reactions mixtures that contain all four fluorescently labeled nucleotides allowing for high-density labeling of DNA. PMID:15727132

  9. Myeloperoxidase-induced genomic DNA-centered radicals.

    PubMed

    Gomez-Mejiba, Sandra E; Zhai, Zili; Gimenez, Maria S; Ashby, Michael T; Chilakapati, Jaya; Kitchin, Kirk; Mason, Ronald P; Ramirez, Dario C

    2010-06-25

    Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis. PMID:20406811

  10. Myeloperoxidase-induced Genomic DNA-centered Radicals*

    PubMed Central

    Gomez-Mejiba, Sandra E.; Zhai, Zili; Gimenez, Maria S.; Ashby, Michael T.; Chilakapati, Jaya; Kitchin, Kirk; Mason, Ronald P.; Ramirez, Dario C.

    2010-01-01

    Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis. PMID:20406811

  11. Evaluation of a Modified DNA Extraction Method for Isolation of Cell-Free Fetal DNA from Maternal Serum

    PubMed Central

    Keshavarz, Zeinab; Moezzi, Leili; Ranjbaran, Reza; Aboualizadeh, Farzaneh; Behzad-Behbahani, Abbas; Abdullahi, Masooma; Sharifzadeh, Sedigheh

    2015-01-01

    Background: Discovery of short cell free fetal DNA (cffDNA) fragments in maternal plasma has created major changes in the field of prenatal diagnosis. The use of cffDNA to set up noninvasive prenatal test is limited due to the low concentration of fetal DNA in maternal plasma therefore, employing a high efficiency extraction method leads to more accurate results. The aim of this study was to evaluate the efficiency of Triton/Heat/Phenol (THP) protocol in comparison with the QIAamp DNA Blood mini Kit for cffDNA purification. Methods: In order to evaluate the efficiency of THP protocol, DNA of Rhesus D (RhD) negative pregnant women's plasma was collected, then real-time PCR for RHD exon 7 was performed. The Ct value data of real time PCR obtained by two different methods were compared and after delivery serology test on cord blood was done to validate the real time PCR results. Results: The results indicated significant differences between two extraction methods (p=0.001). The mean±SD of Ct-value using THP protocol was 33.8±1.6 and 36.1±2.47 using QIAamp DNA Blood mini Kit. Conclusion: Our finding demonstrated that THP protocol was more effective than the QIAamp DNA Blood mini Kits for cffDNA extraction and lead to decrease the false negative results. PMID:26140187

  12. Quantitive DNA Fiber Mapping

    SciTech Connect

    Lu, Chun-Mei; Wang, Mei; Greulich-Bode, Karin M.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-01-28

    Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.

  13. Reversible DNA compaction.

    PubMed

    González-Pérez, Alfredo

    2014-01-01

    In this review we summarize and discuss the different methods we can use to achieve reversible DNA compaction in vitro. Reversible DNA compaction is a natural process that occurs in living cells and viruses. As a result these process long sequences of DNA can be concentrated in a small volume (compacted) to be decompacted only when the information carried by the DNA is needed. In the current work we review the main artificial compacting agents looking at their suitability for decompaction. The different approaches used for decompaction are strongly influenced by the nature of the compacting agent that determines the mechanism of compaction. We focus our discussion on two main artificial compacting agents: multivalent cations and cationic surfactants that are the best known compacting agents. The reversibility of the process can be achieved by adding chemicals like divalent cations, alcohols, anionic surfactants, cyclodextrins or by changing the chemical nature of the compacting agents via pH modifications, light induced conformation changes or by redox-reactions. We stress the relevance of electrostatic interactions and self-assembly as a main approach in order to tune up the DNA conformation in order to create an on-off switch allowing a transition between coil and compact states. The recent advances to control DNA conformation in vitro, by means of molecular self-assembly, result in a better understanding of the fundamental aspects involved in the DNA behavior in vivo and serve of invaluable inspiration for the development of potential biomedical applications. PMID:24444152

  14. Tracking Mitochondrial DNA In Situ.

    PubMed

    Ligasová, Anna; Koberna, Karel

    2016-01-01

    The methods of the detection of (1) non-labeled and (2) BrdU-labeled mitochondrial DNA (mtDNA) are described. They are based on the production of singlet oxygen by monovalent copper ions and the subsequent induction of DNA gaps. The ends of interrupted DNA serve as origins for the labeling of mtDNA by DNA polymerase I or they are utilized by exonuclease that degrades DNA strands, unmasking BrdU in BrdU-labeled DNA. Both methods are sensitive approaches without the need of additional enhancement of the signal or the use of highly sensitive optical systems. PMID:26530676

  15. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  16. DNA-PK assay

    DOEpatents

    Anderson, Carl W.; Connelly, Margery A.

    2004-10-12

    The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

  17. DNA Mismatch Repair

    PubMed Central

    MARINUS, M. G.

    2014-01-01

    DNA mismatch repair functions to correct replication errors in newly synthesized DNA and to prevent recombination between related, but not identical (homeologous), DNA sequences. The mechanism of mismatch repair is best understood in Escherichia coli and is the main focus of this review. The early genetic studies of mismatch repair are described as a basis for the subsequent biochemical characterization of the system. The effects of mismatch repair on homologous and homeologous recombination are described. The relationship of mismatch repair to cell toxicity induced by various drugs is included. The VSP (Very Short Patch) repair system is described in detail. PMID:26442827

  18. Focus: DNA probes

    SciTech Connect

    Not Available

    1986-11-01

    Progress in the development of DNA probes for the identification and quantitation of specific genetic sequences in biological samples is reviewed. Current research efforts in the development of DNA probes for the diagnosis of a wide variety of bacterial, viral, and other infectious diseases, such as herpes simplex and cytomegalovirus, and inherited genetic diseases such as cystic fibrosis and sickle cell anemia are discussed. Progress in development of DNA probe assays for cancer diagnosis, detection of Salmonella food poisoning, tissue typing (detection of histocompatibility antigens), mutagen screening, and animal diseases, among other applications is included.

  19. Usefulness of microchip electrophoresis for the analysis of mitochondrial DNA in forensic and ancient DNA studies.

    PubMed

    Alonso, Antonio; Albarran, Cristina; Martín, Pablo; García, Pilar; Capilla, Javier; García, Oscar; de la Rua, Concepción; Izaguirre, Neskuts; Pereira, Filipe; Pereira, Luisa; Amorim, António; Sancho, Manuel

    2006-12-01

    We evaluate the usefulness of a commercially available microchip CE (MCE) device in different genetic identification studies performed with mitochondrial DNA (mtDNA) targets, including the haplotype analysis of HVR1 and HVR2 and the study of interspecies diversity of cytochrome b (Cyt b) and 16S ribosomal RNA (16S rRNA) mitochondrial genes in forensic and ancient DNA samples. The MCE commercial system tested in this study proved to be a fast and sensitive detection method of length heteroplasmy in cytosine stretches produced by 16 189T>C transitions in HVR1 and by 309.1 and 309.2 C-insertions in HVR2. Moreover, the quantitative analysis of PCR amplicons performed by LIF allowed normalizing the amplicon input in the sequencing reactions, improving the overall quality of sequence data. These quantitative data in combination with the quantification of genomic mtDNA by real-time PCR has been successfully used to evaluate the PCR efficiency and detection limit of full sequencing methods of different mtDNA targets. The quantification of amplicons also provided a method for the rapid evaluation of PCR efficiency of multiplex-PCR versus singleplex-PCR to amplify short HV1 amplicons (around 100 bp) from severely degraded ancient DNA samples. The combination of human-specific (Cyt b) and universal (16S rRNA) mtDNA primer sets in a single PCR reaction followed by MCE detection offers a very rapid and simple screening test to differentiate between human and nonhuman hair forensic samples. This method was also very efficient with degraded DNA templates from forensic hair and bone samples, because of its applicability to detect small amplicon sizes. Future possibilities of MCE in forensic DNA typing, including nuclear STRs and SNP profiling are suggested. PMID:17120261

  20. Methylation of cell-free circulating DNA in the diagnosis of cancer

    PubMed Central

    Warton, Kristina; Samimi, Goli

    2015-01-01

    A range of molecular alterations found in tumor cells, such as DNA mutations and DNA methylation, is reflected in cell-free circulating DNA (circDNA) released from the tumor into the blood, thereby making circDNA an ideal candidate for the basis of a blood-based cancer diagnosis test. In many cancer types, mutations driving tumor development and progression are present in a wide range of oncogenes and tumor suppressor genes. However, even when a gene is consistently mutated in a particular cancer, the mutations can be spread over very large regions of its sequence, making evaluation difficult. This diversity of sequence changes in tumor DNA presents a challenge for the development of blood tests based on DNA mutations for cancer diagnosis. Unlike mutations, DNA methylation that can be consistently measured, as it tends to occur in specific regions of the DNA called CpG islands. Since DNA methylation is reflected within circDNA, detection of tumor-specific DNA methylation in patient plasma is a feasible approach for the development of a blood-based test. Aberrant circDNA methylation has been described in most cancer types and is actively being investigated for clinical applications. A commercial blood test for colorectal cancer based on the methylation of the SEPT9 promoter region in circDNA is under review for approval by the Federal Drug Administration (FDA) for clinical use. In this paper, we review the state of research in circDNA methylation as an application for blood-based diagnostic tests in colorectal, breast, lung, pancreatic and ovarian cancers, and we consider some of the future directions and challenges in this field. There are a number of potential circDNA biomarkers currently under investigation, and experience with SEPT9 shows that the time to clinical translation can be relatively rapid, supporting the promise of circDNA as a biomarker. PMID:25988180

  1. Interaction of DNA and DNA-anti-DNA complexes to fibronectin

    SciTech Connect

    Gupta, R.C.; Simpson, W.A.; Raghow, R.; Hasty, K.

    1986-03-01

    Fibronectin (Fn) is a large multidomain glycoprotein found in the basement membrane, on cell surface and in plasma. The interactions of Fn with DNA may be significant in glomerular deposition of DNA-anti-DNA complexes in patients with systemic lupus erythematosus (SLE). The authors examined the binding of DNA and DNA-anti-DNA complexes to Fn by a solid phase assay in which Fn was coated to microtiter plates and reacted with (/sup 3/H)DNA or DNA complexes with a monoclonal anti-DNA antibody. The optimal interaction of DNA with Fn occurs at <0.1M NaCl suggesting that the binding is charge dependent; the specificity of this binding was shown by competitive inhibition and locking experiments using anti-Fn. The binding was maximum at pH 6.5 and in the absence of Ca/sup 2 +/. The addition of Clq enhanced the binding of DNA and DNA-anti-DNA complexes to Fn, whereas heparan sulfate inhibited such binding. The monomeric or aggregated IgC did not bind Fn but aggregated IgG bound to Fn in the presence of Clq. Furthermore, DNA-anti-DNA complexes in sera from active SLE patients bound Fn which was enhanced in the presence of Clq; DNase abolished this binding indicating that the interaction of these complexes was mediated by DNA. These observations may partially explain the molecular mechanism(s) of the deposition of DNA-anti-DNA complexes in basement membrane.

  2. Sequence-specific DNA nicking endonucleases.

    PubMed

    Xu, Shuang-yong

    2015-08-01

    A group of small HNH nicking endonucleases (NEases) was discovered recently from phage or prophage genomes that nick double-stranded DNA sites ranging from 3 to 5 bp in the presence of Mg2+ or Mn2+. The cosN site of phage HK97 contains a gp74 nicking site AC↑CGC, which is similar to AC↑CGR (R=A/G) of N.ϕGamma encoded by Bacillus phage Gamma. A minimal nicking domain of 76 amino acid residues from N.ϕGamma could be fused to other DNA binding partners to generate chimeric NEases with new specificities. The biological roles of a few small HNH endonucleases (HNHE, gp74 of HK97, gp37 of ϕSLT, ϕ12 HNHE) have been demonstrated in phage and pathogenicity island DNA packaging. Another group of NEases with 3- to 7-bp specificities are either natural components of restriction systems or engineered from type IIS restriction endonucleases. A phage group I intron-encoded HNH homing endonucleases, I-PfoP3I was found to nick DNA sites of 14-16 bp. I-TslI encoded by T7-like ΦI appeared to nick DNA sites with a 9-bp core sequence. DNA nicking and labeling have been applied to optical mapping to aid genome sequence assembly and detection of large insertion/deletion mutations in genomic DNA of cancer cells. Nicking enzyme-mediated amplification reaction has been applied to rapid diagnostic testing of influenza A and B in clinical setting and for construction of DNA-based Boolean logic gates. The clustered regularly interspaced short palindromic repeats-ribonucleoprotein complex consisting of engineered Cas9 nickases in conjunction with tracerRNA:crRNA or a single-guide RNA have been successfully used in genome modifications. PMID:26352356

  3. Expansion of the DNA Alphabet beyond Natural DNA Recognition.

    PubMed

    Tateishi-Karimata, Hisae; Sugimoto, Naoki

    2016-07-15

    Simple and inexpensive DNA fibres: New, stable DNA structures are created by the binding of a small molecule to poly(A). Because these DNA fibres are formed from inexpensive materials by using very simple methods, DNA materials suitable for practical use such as information storage should be possible in the near future. PMID:27061868

  4. Plasma Cell-Free DNA in Paediatric Lymphomas

    PubMed Central

    Mussolin, Lara; Burnelli, Roberta; Pillon, Marta; Carraro, Elisa; Farruggia, Piero; Todesco, Alessandra; Mascarin, Maurizio; Rosolen, Angelo

    2013-01-01

    Background: Extracellular circulating DNA (cfDNA) can be found in small amounts in plasma of healthy individuals. Increased levels of cfDNA have been reported in patients with cancer of breast, cervix, colon, liver and it was shown that cfDNA can originate from both tumour and non-tumour cells. Objectives: Levels of cfDNA of a large series of children with lymphoma were evaluated and analyzed in relation with clinical characteristics. Methods: plasma cfDNA levels obtained at diagnosis in 201 paediatric lymphoma patients [43 Hodgkin lymphomas (HL), 45 anaplastic large cell lymphomas (ALCL), 88 Burkitt lymphomas (BL), 17 lymphoblastic (LBL), 8 diffuse large B cell lymphoma (DLBCL)] and 15 healthy individuals were determined using a quantitative PCR assay for POLR2 gene and, in addition, for NPM-ALK fusion gene in ALCL patients. Wilcoxon rank sum test was used to compare plasma levels among different patient subgroups and controls and to analyze relationship between levels of cfDNA and clinical characteristics. Results: Levels of cfDNA in lymphoma patients were significantly higher compared with controls (p<0.0001). CfDNA was associated with median age (p=0.01) in HL, and with stage in ALCL (p=0.01). In HL patients high cfDNA levels were correlated with poor prognosis (p=0.03). In ALCL we found that most of the cfDNA (77%) was non-tumor DNA. Conclusion: level of plasma cfDNA might constitute an important non-invasive tool at diagnosis in lymphoma patients' management; in particular in patients with HL, cfDNA seems to be a promising prognostic biomarker. PMID:23678368

  5. Free DNA in serum and plasma from normal adults.

    PubMed Central

    Steinman, C R

    1975-01-01

    Circulating DNA has been associated with several human disorders, including the nephritis of systemic lupus erythematosus (SLE), in which it is thought to play an etiological role. However, it remains unclear whether its appearance in the circulation is truly pathological. Several reports, each generally based on a single assay method, have disagreed as to whether DNA may circulate in normals. Some, but not all, of this disagreement may be explained by the recently described appearance of DNA in serum, but not plasma, apparently as the result of release from leukocytes in vitro. In the present report an attempt is made to clarify this problem. Normal plasma and serum samples were examined by four assays for DNA that were newly modified to enhance their specificity and/or sensitivity. Plasma DNA was undetectable by all four methods, the most sensitive of which could detect 0.05 mug/ml of native DNA (nDNA) or 0.1 mug/ml of single-stranded DNA (ssDNA). Serum DNA was present in 14 of 16 samples tested in variable concentrations with an estimated mean of 1.9 mug/ml. It is concluded that the appearance of DNA in adult human plasma is a pathological event. Presumably, previous reports describing detection of DNA in normal plasma were based on the measurement of non-DNAase-sensitive interfering substance. Furthermore, it is emphasized that the use of serum in studies dependent on sensitive assays for DNA (or anti-DNA antibody) introduces an ambiguity that may be avoided by substitution of carefully collected plasma for serum. PMID:1150882

  6. DNA computing in microreactors

    NASA Astrophysics Data System (ADS)

    Wagler, Patrick; van Noort, Danny; McCaskill, John S.

    2001-11-01

    The goal of this research is to improve the modular stability and programmability of DNA-based computers and in a second step towards optical programmable DNA computing. The main focus here is on hydrodynamic stability. Clockable microreactors can be connected in various ways to solve combinatorial optimisation problems, such as Maximum Clique or 3-SAT. This work demonstrates by construction how one micro-reactor design can be programmed to solve any instance of Maximum Clique up to its given maximum size (N). It reports on an implementation of the architecture proposed previously. This contrasts with conventional DNA computing where the individual sequence of biochemical operations depends on the specific problem. In this pilot study we are tackling a graph for the Maximum Clique problem with NDNA solution space will be presented, which is symbolized by a set of bit-strings (words).

  7. DNA sequencing conference, 2

    SciTech Connect

    Cook-Deegan, R.M.; Venter, J.C.; Gilbert, W.; Mulligan, J.; Mansfield, B.K.

    1991-06-19

    This conference focused on DNA sequencing, genetic linkage mapping, physical mapping, informatics and bioethics. Several were used to study this sequencing and mapping. This article also discusses computer hardware and software aiding in the mapping of genes.

  8. Close encounters with DNA

    PubMed Central

    Maffeo, C.; Yoo, J.; Comer, J.; Wells, D. B.; Luan, B.; Aksimentiev, A.

    2014-01-01

    Over the past ten years, the all-atom molecular dynamics method has grown in the scale of both systems and processes amenable to it and in its ability to make quantitative predictions about the behavior of experimental systems. The field of computational DNA research is no exception, witnessing a dramatic increase in the size of systems simulated with atomic resolution, the duration of individual simulations and the realism of the simulation outcomes. In this topical review, we describe the hallmark physical properties of DNA from the perspective of all-atom simulations. We demonstrate the amazing ability of such simulations to reveal the microscopic physical origins of experimentally observed phenomena and we review the frustrating limitations associated with imperfections of present atomic force fields and inadequate sampling. The review is focused on the following four physical properties of DNA: effective electric charge, response to an external mechanical force, interaction with other DNA molecules and behavior in an external electric field. PMID:25238560

  9. Making DNA Fingerprints.

    ERIC Educational Resources Information Center

    Nunley, Kathie F.

    1996-01-01

    Presents an activity to simulate electrophoresis using everyday items. Uses adding machine paper to construct a set of DNA fingerprints that can be used to solve crime cases designed by students in any biology class. (JRH)

  10. Harnessing DNA intercalation.

    PubMed

    Persil, Ozgül; Hud, Nicholas V

    2007-10-01

    Numerous small molecules are known to bind to DNA through base pair intercalation. Fluorescent dyes commonly used for nucleic acid staining, such as ethidium, are familiar examples. Biological and physical studies of DNA intercalation have historically been motivated by mutation and drug discovery research. However, this same mode of binding is now being harnessed for the creation of novel molecular assemblies. Recent studies have used DNA scaffolds and intercalators to construct supramolecular assemblies that function as fluorescent 'nanotags' for cell labeling. Other studies have demonstrated how intercalators can be used to promote the formation of otherwise unstable nucleic acid assemblies. These applications illustrate how intercalators can be used to facilitate and expand DNA-based nanotechnology. PMID:17825446

  11. DNA damage tolerance.

    PubMed

    Branzei, Dana; Psakhye, Ivan

    2016-06-01

    Accurate chromosomal DNA replication is fundamental for optimal cellular function and genome integrity. Replication perturbations activate DNA damage tolerance pathways, which are crucial to complete genome duplication as well as to prevent formation of deleterious double strand breaks. Cells use two general strategies to tolerate lesions: recombination to a homologous template, and trans-lesion synthesis with specialized polymerases. While key players of these processes have been outlined, much less is known on their choreography and regulation. Recent advances have uncovered principles by which DNA damage tolerance is regulated locally and temporally - in relation to replication timing and cell cycle stage -, and are beginning to elucidate the DNA dynamics that mediate lesion tolerance and influence chromosome structure during replication. PMID:27060551

  12. Multiplex analysis of DNA

    DOEpatents

    Church, George M.; Kieffer-Higgins, Stephen

    1992-01-01

    This invention features vectors and a method for sequencing DNA. The method includes the steps of: a) ligating the DNA into a vector comprising a tag sequence, the tag sequence includes at least 15 bases, wherein the tag sequence will not hybridize to the DNA under stringent hybridization conditions and is unique in the vector, to form a hybrid vector, b) treating the hybrid vector in a plurality of vessels to produce fragments comprising the tag sequence, wherein the fragments differ in length and terminate at a fixed known base or bases, wherein the fixed known base or bases differs in each vessel, c) separating the fragments from each vessel according to their size, d) hybridizing the fragments with an oligonucleotide able to hybridize specifically with the tag sequence, and e) detecting the pattern of hybridization of the tag sequence, wherein the pattern reflects the nucleotide sequence of the DNA.

  13. Patterning nanocrystals using DNA

    SciTech Connect

    Williams, Shara Carol

    2003-09-01

    One of the goals of nanotechnology is to enable programmed self-assembly of patterns made of various materials with nanometer-sized control. This dissertation describes the results of experiments templating arrangements of gold and semiconductor nanocrystals using 2'-deoxyribonucleic acid (DNA). Previously, simple DNA-templated linear arrangements of two and three nanocrystals structures have been made.[1] Here, we have sought to assemble larger and more complex nanostructures. Gold-DNA conjugates with 50 to 100 bases self-assembled into planned arrangements using strands of DNA containing complementary base sequences. We used two methods to increase the complexity of the arrangements: using branched synthetic doublers within the DNA covalent backbone to create discrete nanocrystal groupings, and incorporating the nanocrystals into a previously developed DNA lattice structure [2][3] that self-assembles from tiles made of DNA double-crossover molecules to create ordered nanoparticle arrays. In the first project, the introduction of a covalently-branched synthetic doubler reagent into the backbone of DNA strands created a branched DNA ''trimer.'' This DNA trimer templated various structures that contained groupings of three and four gold nanoparticles, giving promising, but inconclusive transmission electron microscopy (TEM) results. Due to the presence of a variety of possible structures in the reaction mixtures, and due to the difficulty of isolating the desired structures, the TEM and gel electrophoresis results for larger structures having four particles, and for structures containing both 5 and 10 nm gold nanoparticles were inconclusive. Better results may come from using optical detection methods, or from improved sample preparation. In the second project, we worked toward making two-dimensional ordered arrays of nanocrystals. We replicated and improved upon previous results for making DNA lattices, increasing the size of the lattices to a length greater than

  14. Das DNA-Puzzle

    NASA Astrophysics Data System (ADS)

    Kirchner, Stefan

    Im Jahre 1953 wurde von James Watson und Francis Crick erstmalig der strukturelle Aufbau der sogenannten DNA (Desoxyribonukleinsäure) beschrieben, welche das Erbgut jedes Lebewesens enthält. Der wesentliche Teil des Erbguts wird dabei durch eine sehr lange Folge der vier Basen Adenin (A), Cytosin (C), Guanin (G) und Thymin (T) codiert. Seit einigen Jahren ist es möglich, die Folge der vier Basen zu einer gegebenen DNA zu bestimmen. Biologen bezeichnen diesen Vorgang als Sequenzierung.

  15. DNA-Nanoparticle Tinkertoys.

    PubMed

    Chandrasekaran, Arun Richard

    2016-06-16

    Nanoparticle superlattices can be self-assembled by using DNA linkers, which gives control over their size, shape, and composition. Recently, such programmable atom equivalents have been tailored to respond to chemical stimuli and result in specific crystalline lattices. Moreover, the molecular recognition properties and the robustness of designed DNA nanostructures have been used in combination with metallic nanoparticles for the production of the elusive diamond superlattice. PMID:27080095

  16. Ribonucleotide triggered DNA damage and RNA-DNA damage responses

    PubMed Central

    Wallace, Bret D; Williams, R Scott

    2014-01-01

    Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage. PMID:25692233

  17. Probing Protein-DNA Interactions by Unzipping DNA

    NASA Astrophysics Data System (ADS)

    Wang, Michelle

    2003-03-01

    Protein-DNA interactions are essential to cellular processes. In replication, transcription, recombination, DNA repair, and DNA packaging, proteins bind to DNA as activators or repressors, to recruit other proteins, or to carry out various catalytic activities. I will present Unzipping Force Analysis of Protein Association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule, we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding "event" revealed information about the spatial location and dynamic nature of the protein-DNA complex.

  18. DNA biosensors that reason.

    PubMed

    Sainz de Murieta, Iñaki; Rodríguez-Patón, Alfonso

    2012-08-01

    Despite the many designs of devices operating with the DNA strand displacement, surprisingly none is explicitly devoted to the implementation of logical deductions. The present article introduces a new model of biosensor device that uses nucleic acid strands to encode simple rules such as "IF DNA_strand(1) is present THEN disease(A)" or "IF DNA_strand(1) AND DNA_strand(2) are present THEN disease(B)". Taking advantage of the strand displacement operation, our model makes these simple rules interact with input signals (either DNA or any type of RNA) to generate an output signal (in the form of nucleotide strands). This output signal represents a diagnosis, which either can be measured using FRET techniques, cascaded as the input of another logical deduction with different rules, or even be a drug that is administered in response to a set of symptoms. The encoding introduces an implicit error cancellation mechanism, which increases the system scalability enabling longer inference cascades with a bounded and controllable signal-noise relation. It also allows the same rule to be used in forward inference or backward inference, providing the option of validly outputting negated propositions (e.g. "diagnosis A excluded"). The models presented in this paper can be used to implement smart logical DNA devices that perform genetic diagnosis in vitro. PMID:22406690

  19. Ancient dirt DNA

    NASA Astrophysics Data System (ADS)

    Willerslev, E.

    2007-12-01

    In the past two decades, ancient DNA research has progressed from the retrieval of small fragments of mitochondrial DNA from a few late Holocene specimens, to large-scale studies of ancient populations, phenotypically important nuclear loci, and even whole genomic studies of extinct species. However, the field is still regularly marred by erroneous reports, which underestimate the extent of contamination within laboratories and samples themselves. An improved understanding of these processes and the effects of damage on ancient DNA templates has started to provide a more robust basis for research. Recent methodological advances have included the discoveries of DNA preserved in ancient sediments, coprolites, and fossil ice (Ancient Dirt DNA). These findings promise to make possible the reconstructions of entire ecosystems through time and allow for studies of past population genetics in cases where fossils are rare. The advantages and pitfalls connected to the Ancient Dirt DNA approach will be discussed as will recently obtained data relating to Greenland environmental history, long-term bacterial survival and the first human migration into the Americas.

  20. Variations in brain DNA

    PubMed Central

    Avila, Jesús; Gómez-Ramos, Alberto; Soriano, Eduardo

    2014-01-01

    It is assumed that DNA sequences are conserved in the diverse cell types present in a multicellular organism like the human being. Thus, in order to compare the sequences in the genome of DNA from different individuals, nucleic acid is commonly isolated from a single tissue. In this regard, blood cells are widely used for this purpose because of their availability. Thus blood DNA has been used to study genetic familiar diseases that affect other tissues and organs, such as the liver, heart, and brain. While this approach is valid for the identification of familial diseases in which mutations are present in parental germinal cells and, therefore, in all the cells of a given organism, it is not suitable to identify sporadic diseases in which mutations might occur in specific somatic cells. This review addresses somatic DNA variations in different tissues or cells (mainly in the brain) of single individuals and discusses whether the dogma of DNA invariance between cell types is indeed correct. We will also discuss how single nucleotide somatic variations arise, focusing on the presence of specific DNA mutations in the brain. PMID:25505410

  1. Vibrational structure of DNA

    NASA Astrophysics Data System (ADS)

    Gómez C, S.; Rey-González, R. R.

    2003-10-01

    DNA has been object of more extensive research in last years. Human genome may be the main work. On the other hand, DNA has been used as physical system in opposite to its biological character. Examples of this are electronic, thermally and Ramman spectroscopy studies, in others. However, some DNA physical features are unclear, they deserve more work in a effort to understand them. In this work we are interesting on the vibrational properties of DNA. We model it as a lineal chain constituted by three different mass. We use two different constant forces into the Dynamical Matrix formalism. Two masses represent the real mass of DNA bases plus the glucose mass and the third represents the phosphate mass. In this model, DNA unit cell is composed by four masses The dispersion relation shows one acoustical and three optical branches. Also, there is a wide gap between the first and second optical branches. These features are confirmed by the density of states. Also we consider disorder effects in the proposal to do a more realistic model. In this case our results suggest a behavior as diatomic chain where the central and wide gap is preserved.

  2. Detection of Streptococcus mutans Genomic DNA in Human DNA Samples Extracted from Saliva and Blood

    PubMed Central

    Vieira, Alexandre R.; Deeley, Kathleen B.; Callahan, Nicholas F.; Noel, Jacqueline B.; Anjomshoaa, Ida; Carricato, Wendy M.; Schulhof, Louise P.; DeSensi, Rebecca S.; Gandhi, Pooja; Resick, Judith M.; Brandon, Carla A.; Rozhon, Christopher; Patir, Asli; Yildirim, Mine; Poletta, Fernando A.; Mereb, Juan C.; Letra, Ariadne; Menezes, Renato; Wendell, Steven; Lopez-Camelo, Jorge S.; Castilla, Eduardo E.; Orioli, Iêda M.; Seymen, Figen; Weyant, Robert J.; Crout, Richard; McNeil, Daniel W.; Modesto, Adriana; Marazita, Mary L.

    2011-01-01

    Caries is a multifactorial disease, and studies aiming to unravel the factors modulating its etiology must consider all known predisposing factors. One major factor is bacterial colonization, and Streptococcus mutans is the main microorganism associated with the initiation of the disease. In our studies, we have access to DNA samples extracted from human saliva and blood. In this report, we tested a real-time PCR assay developed to detect copies of genomic DNA from Streptococcus mutans in 1,424 DNA samples from humans. Our results suggest that we can determine the presence of genomic DNA copies of Streptococcus mutans in both DNA samples from caries-free and caries-affected individuals. However, we were not able to detect the presence of genomic DNA copies of Streptococcus mutans in any DNA samples extracted from peripheral blood, which suggests the assay may not be sensitive enough for this goal. Values of the threshold cycle of the real-time PCR reaction correlate with higher levels of caries experience in children, but this correlation could not be detected for adults. PMID:21731912

  3. 76 FR 72950 - Draft Guidance for Industry: Use of Nucleic Acid Tests on Pooled and Individual Samples From...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-28

    ... (HBV) deoxyribonucleic acid (DNA) and recommendations for product testing and disposition, donor...- licensed NAT to screen blood donors for HBV DNA. FDA is also providing these blood establishments...

  4. Reversible Condensation of DNA using a Redox-Active Surfactant

    PubMed Central

    Hays, Melissa E.; Jewell, Christopher M.; Lynn, David M.; Abbott, Nicholas L.

    2008-01-01

    We report characterization of aqueous solutions of dilute Lambda phage DNA containing the redox-active surfactant (11-ferrocenylundecyl)trimethylammonium bromide (FTMA) as a function of the oxidation state of the FTMA. FTMA undergoes a reversible one-electron oxidation from a reduced state that forms micelles in aqueous solution to an oxidized state (containing the ferrocenium cation) that does not selfassociate in solution. This investigation sought to test the hypothesis that FTMA can be used to achieve reversible control over the conformation of DNA-surfactant complexes in solution. Whereas DNA adopts extended coil conformations in aqueous solutions, our measurements revealed that addition of reduced FTMA (2–5μM) to aqueous solutions of DNA (5 μM in nucleotide units) resulted in coexistence of extended coils and compact globules in solution. At higher concentrations of reduced FTMA (up to 30μM), the DNA was present as compact globules only. In contrast, oxidized FTMA had no measurable effect on the conformation of DNA, allowing DNA to maintain an extended coil state up to a concentration of 75μM oxidized FTMA. We further demonstrate that it is possible to chemically or electrochemically transform the oxidation state of FTMA in preformed complexes of FTMA and DNA, thus achieving in situ control over the conformations of the DNA in solution. These results provide guidance for the design of surfactant systems that permit active control of DNA-surfactant interactions. PMID:17428073

  5. Polymorphism of the SOD1-DNA aggregation species can be modulated by DNA.

    PubMed

    Jiang, Wei; Zhang, Bo; Yin, Jun; Liu, Liang; Wang, Li; Liu, Changlin

    2008-12-01

    Proteinaceous aggregates rich in copper, zinc superoxide dismutase (SOD1) have been found in both in vivo and in vitro models. We have shown that double-stranded DNA that acts as a template accelerates the in vitro formation of wild-type SOD1 aggregates. Here, we examined the polymorphism of templated-SOD1 aggregates generated in vitro upon association with DNA under different conditions. Electron microscopy imaging indicates that this polymorphism is capable of being manipulated by the shapes, structures, and doses of the DNAs tested. The nanometer- and micrometer-scale aggregates formed under acidic conditions and under neutral conditions containing ascorbate fall into three classes: aggregate monomers, oligomeric aggregates, and macroaggregates. The aggregate monomers observed at given DNA doses exhibit a polymorphism that is markedly corresponded to the coiled shapes of linear DNA and structures of plasmid DNA. On the other hand, the regularly branched structures observed under both atomic force microscopy and optical microscope indicate that the DNAs tested are simultaneously condensed into a nanoparticle with a specific morphology during SOD1 aggregation, revealing that SOD1 aggregation and DNA condensation are two concurrent phenomena. The results might provide the basis of therapeutic approaches to suppress the formation of toxic protein oligomers or aggregates by screening the toxicity of the protein aggregates with various sizes and morphologies. PMID:18690666

  6. Supramolecular Complexes of DNA

    NASA Astrophysics Data System (ADS)

    Zuber, G.; Scherman, D.

    Deoxyribose nucleic acid or DNA is a linear polymer in the form of a double strand, synthesised by sequential polymerisation of a large number of units chosen from among the nucleic bases called purines (adenosine A and guanosine G) and pyrimidines (cytosine C and thymidine T). DNA contains all the genetic information required for life. It exists in the form of a limited number (a few dozen) of very big molecules, called chromosomes. This genetic information is first of all transcribed. In this process, a restricted fragment of the DNA called a gene is copied in the form of ribonucleic acid, or RNA. This RNA is itself a polymer, but with a single strand in which the sequence of nucleic acids is schematically analogous to the sequence on one of the two strands of the transcribed DNA. Finally, this RNA is translated into a protein, yet another linear polymer. The proteins make up the main part of the active constituents ensuring the survival of the cell. Any loss of information, either by mutation or by deletion of the DNA, will cause an imbalance in the cell's metabolism that may in turn lead to incurable pathologies. Several strategies have been developed to reduce the consequences of such genetic deficiencies or, more generally, to act, by amplifying or suppressing them, on the mechanisms leading from the reading of the genetic information to the production of proteins: Strategies aiming to introduce synthetic DNA or RNA, which selectively block the expression of certain genes, are now being studied by an increasing number of research scientists and pharmacologists. They use antisense oligodeoxyribonucleotides or interfering oligoribonucleotides and they already have clinical applications. This kind of therapy is often called gene pharmacology. Other, more ambitious strategies aim to repair in situ mutated or incomplete DNA within the chromosomes themselves, by introducing short sequences of DNA or RNA which recognise and take the place of mutations. This is the

  7. Characterization of the Host Factors Required for Hepadnavirus Covalently Closed Circular (ccc) DNA Formation

    PubMed Central

    Zhou, Tianlun; Block, Timothy M.; Guo, Ju-Tao

    2012-01-01

    Synthesis of the covalently closed circular (ccc) DNA is a critical, but not well-understood step in the life cycle of hepadnaviruses. Our previous studies favor a model that removal of genome-linked viral DNA polymerase occurs in the cytoplasm and the resulting deproteinized relaxed circular DNA (DP-rcDNA) is subsequently transported into the nucleus and converted into cccDNA. In support of this model, our current study showed that deproteinization of viral double-stranded linear (dsl) DNA also took place in the cytoplasm. Furthermore, we demonstrated that Ku80, a component of non-homologous end joining DNA repair pathway, was essential for synthesis of cccDNA from dslDNA, but not rcDNA. In an attempt to identify additional host factors regulating cccDNA biosynthesis, we found that the DP-rcDNA was produced in all tested cell lines that supported DHBV DNA replication, but cccDNA was only synthesized in the cell lines that accumulated high levels of DP-rcDNA, except for NCI-H322M and MDBK cells, which failed to synthesize cccDNA despite of the existence of nuclear DP-rcDNA. The results thus imply that while removal of the genome-linked viral DNA polymerase is most likely catalyzed by viral or ubiquitous host function(s), nuclear factors required for the conversion of DP-rcDNA into cccDNA and/or its maintenance are deficient in the above two cell lines, which could be useful tools for identification of the elusive host factors essential for cccDNA biosynthesis or maintenance. PMID:22912842

  8. USE OF DNA TECHNOLOGY IN FORENSIC DENTISTRY

    PubMed Central

    da Silva, Ricardo Henrique Alves; Sales-Peres, Arsenio; de Oliveira, Rogério Nogueira; de Oliveira, Fernando Toledo; Sales-Peres, Sílvia Helena de Carvalho

    2007-01-01

    The established importance of Forensic Dentistry for human identification, mainly when there is little remaining material to perform such identification (e.g., in fires, explosions, decomposing bodies or skeletonized bodies), has led dentists working with forensic investigation to become more familiar with the new molecular biology techniques. The currently available DNA tests have high reliability and are accepted as legal proofs in courts. This article presents a literature review referring to the main studies on Forensic Dentistry that involve the use of DNA for human identification, and makes an overview of the evolution of this technology in the last years, highlighting the importance of molecular biology in forensic sciences. PMID:19089123

  9. Phylogenetic reconstruction in the Order Nymphaeales: ITS2 secondary structure analysis and in silico testing of maturase k (matK) as a potential marker for DNA bar coding

    PubMed Central

    2012-01-01

    Background The Nymphaeales (waterlilly and relatives) lineage has diverged as the second branch of basal angiosperms and comprises of two families: Cabombaceae and Nymphaceae. The classification of Nymphaeales and phylogeny within the flowering plants are quite intriguing as several systems (Thorne system, Dahlgren system, Cronquist system, Takhtajan system and APG III system (Angiosperm Phylogeny Group III system) have attempted to redefine the Nymphaeales taxonomy. There have been also fossil records consisting especially of seeds, pollen, stems, leaves and flowers as early as the lower Cretaceous. Here we present an in silico study of the order Nymphaeales taking maturaseK (matK) and internal transcribed spacer (ITS2) as biomarkers for phylogeny reconstruction (using character-based methods and Bayesian approach) and identification of motifs for DNA barcoding. Results The Maximum Likelihood (ML) and Bayesian approach yielded congruent fully resolved and well-supported trees using a concatenated (ITS2+ matK) supermatrix aligned dataset. The taxon sampling corroborates the monophyly of Cabombaceae. Nuphar emerges as a monophyletic clade in the family Nymphaeaceae while there are slight discrepancies in the monophyletic nature of the genera Nymphaea owing to Victoria-Euryale and Ondinea grouping in the same node of Nymphaeaceae. ITS2 secondary structures alignment corroborate the primary sequence analysis. Hydatellaceae emerged as a sister clade to Nymphaeaceae and had a basal lineage amongst the water lilly clades. Species from Cycas and Ginkgo were taken as outgroups and were rooted in the overall tree topology from various methods. Conclusions MatK genes are fast evolving highly variant regions of plant chloroplast DNA that can serve as potential biomarkers for DNA barcoding and also in generating primers for angiosperms with identification of unique motif regions. We have reported unique genus specific motif regions in the Order Nymphaeles from matK dataset

  10. DNA Knots: Theory and Experiments

    NASA Astrophysics Data System (ADS)

    Sumners, D. W.

    Cellular DNA is a long, thread-like molecule with remarkably complex topology. Enzymes that manipulate the geometry and topology of cellular DNA perform many vital cellular processes (including segregation of daughter chromosomes, gene regulation, DNA repair, and generation of antibody diversity). Some enzymes pass DNA through itself via enzyme-bridged transient breaks in the DNA; other enzymes break the DNA apart and reconnect it to different ends. In the topological approach to enzymology, circular DNA is incubated with an enzyme, producing an enzyme signature in the form of DNA knots and links. By observing the changes in DNA geometry (supercoiling) and topology (knotting and linking) due to enzyme action, the enzyme binding and mechanism can often be characterized. This paper will discuss some personal research history, and the tangle model for the analysis of site-specific recombination experiments on circular DNA.

  11. Forensic DNA-typing of dog hair: DNA-extraction and PCR amplification.

    PubMed

    Pfeiffer, I; Völkel, I; Täubert, H; Brenig, B

    2004-05-10

    The forensic application of DNA-typing for the identification of dog hair provides objective evidence in the characterisation of traces found at crime scenes. During the past few years forensic dog identity testing has been improved considerably using multiplex PCR systems. However, DNA-typing from samples of one up to 10 dog hairs is often problematic in forensic science. A single dog hair contains very small quantities of DNA or the hair sample consists of hairs with roots of bad quality or even of broken hairshafts without roots. Here we describe an experimental study about dog hairs by means of a Ca(2+) improved DNA-extraction method, quantification and amplification. PMID:15062955

  12. DNA integrity of onion root cells under catechol influence.

    PubMed

    Petriccione, Milena; Forte, Valentina; Valente, Diego; Ciniglia, Claudia

    2013-07-01

    Catechol is a highly toxic organic pollutant, usually abundant in the waste effluents of industrial processes and agricultural activities. The environmental sources of catechol include pesticides, wood preservatives, tanning lotion, cosmetic creams, dyes, and synthetic intermediates. Genotoxicity of catechol at a concentration range 5 × 10(-1)-5 mM was evaluated by applying random amplified polymorphic DNA (RAPD) and time-lapse DNA laddering tests using onion (Allium cepa) root cells as the assay system. RAPD analysis revealed polymorphisms in the nucleotidic sequence of DNA that reflected the genotoxic potential of catechol to provoke point mutations, or deletions, or chromosomal rearrangements. Time-lapse DNA laddering test provided evidence that catechol provoked DNA necrosis and apoptosis. Acridine orange/ethidium bromide staining could distinguish apoptotic from necrotic cells in root cells of A. cepa. PMID:23307075

  13. Elongation method for electronic structure calculations of random DNA sequences.

    PubMed

    Orimoto, Yuuichi; Liu, Kai; Aoki, Yuriko

    2015-10-30

    We applied ab initio order-N elongation (ELG) method to calculate electronic structures of various deoxyribonucleic acid (DNA) models. We aim to test potential application of the method for building a database of DNA electronic structures. The ELG method mimics polymerization reactions on a computer and meets the requirements for linear scaling computational efficiency and high accuracy, even for huge systems. As a benchmark test, we applied the method for calculations of various types of random sequenced A- and B-type DNA models with and without counterions. In each case, the ELG method maintained high accuracy with small errors in energy on the order of 10(-8) hartree/atom compared with conventional calculations. We demonstrate that the ELG method can provide valuable information such as stabilization energies and local densities of states for each DNA sequence. In addition, we discuss the "restarting" feature of the ELG method for constructing a database that exhaustively covers DNA species. PMID:26337429

  14. Geometry of a complex formed by double strand break repair proteins at a single DNA end: recruitment of DNA-PKcs induces inward translocation of Ku protein.

    PubMed

    Yoo, S; Dynan, W S

    1999-12-15

    Ku protein and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are essential components of the double-strand break repair machinery in higher eukaryotic cells. Ku protein binds to broken DNA ends and recruits DNA-PKcs to form an enzymatically active complex. To characterize the arrangement of proteins in this complex, we developed a set of photocross-linking probes, each with a single free end. We have previously used this approach to characterize the contacts in an initial Ku-DNA complex, and we have now applied the same technology to define the events that occur when Ku recruits DNA-PKcs. The new probes allow the binding of one molecule of Ku protein and one molecule of DNA-PKcs in a defined position and orientation. Photocross-linking reveals that DNA-PKcs makes direct contact with the DNA termini, occupying an approximately 10 bp region proximal to the free end. Characterization of the Ku protein cross-linking pattern in the presence and absence of DNA-PKcs suggests that Ku binds to form an initial complex at the DNA ends, and that recruitment of DNA-PKcs induces an inward translocation of this Ku molecule by about one helical turn. The presence of ATP had no effect on protein-DNA contacts, suggesting that neither DNA-PK-mediated phosphorylation nor a putative Ku helicase activity plays a role in modulating protein conformation under the conditions tested. PMID:10572166

  15. Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC)

    PubMed Central

    Sherwood, James L.; Corcoran, Claire; Brown, Helen; Sharpe, Alan D.; Musilova, Milena; Kohlmann, Alexander

    2016-01-01

    Introduction Non-invasive mutation testing using circulating tumour DNA (ctDNA) is an attractive premise. This could enable patients without available tumour sample to access more treatment options. Materials & Methods Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits. Results 2 hr incubation time and double plasma centrifugation (2000 x g) reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA). Reduced “contamination” and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT) (Streck), after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield. Conclusion This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous. PMID:26918901

  16. Comparison of different protocols for the extraction of microbial DNA from reef corals

    PubMed Central

    Santos, H.F.; Carmo, F.L.; Leite, D.C.A.; Jesus, H.E.; Maalouf, P. De Carvalho; Almeida, C.; Soriano, A.U.; Altomari, D.; Suhett, L.; Vólaro, V.; Valoni, E.; Francisco, M.; Vieira, J.; Rocha, R.; Sardinha, B.L.; Mendes, L.B.; João, R.R.; Lacava, B.; Jesus, R.F.; Sebastian, G.V.; Pessoa, A.; van Elsas, J.D.; Rezende, R.P.; Pires, D.O.; Duarte, G.; Castro, C.B.; Rosado, A.S.; Peixoto, R.S.

    2012-01-01

    This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals. PMID:24031859