Size Of Deoxyribonucleic Acid Research Articles

Mathematical model to reduce loop mediated isothermal amplification (LAMP) false-positive diagnosis.

Loop mediated isothermal amplification (LAMP) is a nucleic acid amplification technique performed under isothermal conditions. The output of this amplification technique includes multiple different sizes of deoxyribonucleic acid (DNA) structures which are identified by a banding pattern on gel electrophoresis plots. Although this is a specific amplification technique, the complexity of the primer design and amplification still lead to the issue of obtaining false‐positive results, especially when a positive reading is determined solely by whether there is any banding pattern in the gel electrophoresis plot. Here, we first performed extensive LAMP experiments and evaluated the DNA structures using microchip electrophoresis. We then developed a mathematical model derived from the various components that make up an entire LAMP structure to predict the full LAMP structure size in base pairs. This model can be implemented by users to make predictions for specific, DNA size dependent, banding patterns on their gel electrophoresis plots. Each prediction is specific to the target sequence and primers used and therefore reduces incorrect diagnosis errors through identifying true‐positive and false‐positive results. This model was accurately tested with multiple primer sets in house and was also translatable to different DNA and RNA types in previously published literature. The mathematical model can ultimately be used to reduce false‐positive LAMP diagnosis errors for applications ranging from tuberculosis diagnostics to E. coli to numerous other infectious diseases.

Probing the Mesh Formed by the Semirigid Polyelectrolytes

We correlate conformation and dynamics of the semirigid polyelectrolytes deoxyribonucleic acid (DNA) and hyaluronic acid (HA) in the semidilute regime, across a broad concentration range (10 −3 − 10 2 g/L). Salt-free polyelec- trolytes are distinct from uncharged polymers as they presumably form a rather rigid, isotropic mesh. The polyelectrolyte characteristic mesh size is known as de Gennes correlation length ξ. We directly probed the mesh formed by DNA and HA, by employing fluorescence correlation spectroscopy (FCS) to measure the diffusion coefficient of fluorescently labeled DNA fragments added in trace amounts. For the salt-free solutions we found that the DNA or HA mesh size has to be 2−3 times larger than the fragments for them to start to diffuse freely (as if in the dilute solution). For a tighter mesh (concentrations 0.1−1 g/L), the fragment diffusion coefficient is only half the free diffusion value. Conversely, fragments show the free diffusion coefficientas if there is no meshin DNA or HA in 10 mM buffer. This complies well with the fact that the ξ fades for polyelectrolytes with added salt. The diffusion coefficient falls off further when the mesh size gets smaller than the fragment size (above 1 g/L) and a similar value is reached in buffer as well as in pure water, respectively, at the highest measured concentrations (10 g/L). We also performed small-angle X-ray scattering (SAXS) on HA and DNA (range 3−130 g/L) to complement our previous dielectric spectroscopy (DS) studies (range 0.01−5 g/L). Combined, these methods provide reference values of the de Gennes length ξ across the range studied by FCS.

Characterization of effect of repeated freeze and thaw cycles on stability of genomic DNA using pulsed field gel electrophoresis.

In this study, we systematically investigated the effects of repeated cycles of freeze/thaw on stability of genomic Deoxyribonucleic acid (DNA) samples as evaluated by changes in DNA size, concentration, and freeze/thaw protocols. The DNA was isolated using standard extraction procedures including phenol/chloroform and commercially available Gentra Puregene and Qiagen QIAmp kits. Changes in DNA were monitored over 18 cycles of freezing and thawing utilizing several freeze/thaw protocols. DNA samples from multiple subjects prepared from whole blood samples were examined by pulsed field gel electrophoresis (PFGE), and shown to have different average molecular sizes and size distribution patterns depending on the extraction method. Results of freeze/thaw experiments, analyzed by PFGE, showed progressive DNA degradation of the samples, with DNA sizes larger than 100 kb most sensitive to freeze/thaw degradation. Increasing the DNA concentration of stored samples from 10 μg/mL to 100 μg/mL had a somewhat protective effect on DNA stability. Variations in freeze/thaw protocols did not have a significant impact on DNA stability during repeated freeze/thaw cycles. At freeze/thaw cycle 18, average molecular size and size distribution of all DNA samples tested approached 25 kb, regardless of their initial average size and size distributions. This study provides insight on DNA degradation during freeze/thaw cycles and offers guidance to storage and handling of DNA samples.

Advanced Vectors for Gene Delivery

Gene therapy is an important tool in the treatment of many diseases involving malfunctioning of deoxyribo nucleic acid (DNA). Development of various techniques for efficiently transferring and expression of recombinant DNA or ribonucleic acid (RNA) at target site is a significant challenge. Viral vectors which have been used classically have a number of restrictions due to their induced toxicity and immunogenicity, the limited size of DNA that can be carried, the lack of specificity and the high cost of production. To overcome these problems nonviral vectors like nanoparticles, dendrimers, molecular conjugates, liposomes, microbubbles etc. are being explored. This review encompasses the details of these nonviral vectors. Keywords: Efficiency, gene delivery, nonviral, transfection, vector, viral, Nanoparticles, Dendriplexes, Molecular Conjugates, Microbubbles

Electrochemical behavior of neomycin at DNA-modified gold electrodes

Deoxyribonucleic acid (DNA) modified gold electrodes are prepared by the dry adsorptive method and the electrochemical behavior of neomycin and the influence of Pb(II) are studied by cyclic voltammetry, chronocoulometry, differential pulse voltammetry. It is found that in 0.01 M phosphate-buffered saline (PBS) buffer solutions (pH 7.3) at DNA/Au electrode neomycin exhibits an irreversible cathodic peak ( E p = 0.489 V), which is more positive and less sensitive compared with that at bare gold electrodes ( E p = 0.423 V). In the presence of Pb(II) the peak shifts toward positive with its height increasing. Moreover, the peak height is linear to neomycin concentration over the range of 0.15–57 μM. The interaction of Pb(II)–neomycin complex with calf thymus DNA is also studied by calculating the binding constants ( K) of the Pb(II)–neomycin complex to DNA and binding site size ( s) from voltammetric data (1.0 × 10 7 M −1 and 4 bp, respectively).

Classification of the Legionnaires' disease bacterium: An interim report

Deoxyribonucleic acid (DNA) from strains of the Legionnaires' disease bacterium (LDB) was characterized in order to aid in the proper classification of this organism. The genome size of LDB DNA was estimated at 2.5×109 daltons by reassociation kinetics; a guanine-plus-cytosine content of LDB of 39% was established by optical thermal denaturation and buoyant density ultracentrifugation measurements. DNA relatedness studies on 12 strains of the LDB indicated that they were all members of the same species. DNA relatedness studies have thus far failed to show that the LDB is significantly related to any other organism, including all members of Enterobacteriaceae,Pasteurella multocida, Francisella tularensis, Rochalimaea quintana, Vibrio species,Staphylococcus epidermidis, andFlavobacterium meningosepticum.

Differential Association of F′ Plasmid and R Plasmid Deoxyribonucleic Acid with a Rapidly Sedimenting Fraction of a Proteus mirabilis Lysate

We have examined the association of an F' plasmid and an R plasmid in Proteus mirabilis with a rapidly sedimenting material that is generated by sodium dodecyl sulfate lysis and low speed centrifugation. Virtually all of the chromosomal deoxyribonucleic acid (DNA) and the F' plasmid DNA are associated with the rapidly sedimenting material after gentle lysis and centrifugation. A portion of R plasmid NR1 DNA (usually 5 to 25%) is not bound to the rapidly sedimenting material and is recovered in the supernatant fraction. This difference in binding is not related to the size of the plasmid DNA, since F' plasmids and R plasmids of different molecular weights showed the same behavior. R plasmid DNA labeled by a brief pulse of [(3)H]thymine is recovered in the supernatant fraction to a lower extent than the total R plasmid DNA. It would appear that R plasmid replication takes place in association with the rapidly sedimenting material. With prolongation of the [(3)H]thymine pulse, the [(3)H]thymine-labeled R plasmid DNA is recovered in the supernatant fraction with the same probability as the total R plasmid DNA. This finding indicates that a change in R plasmid attachment to the rapidly sedimenting material occurs some time after its replication. The differences observed in the replication of F' plasmids and R plasmids in P. mirabilis may be related to their different modes of association with the rapidly sedimenting material.

Association of a Penicillin Resistance Gene with a Tetracycline Resistance Plasmid (P TP–2 ) in Staphylococcus aureus

On transduction with a lysogenic strain of Straphylococcus aureus isolated from a clinical specimen and having tetracycline (TC)-penicillin (PC)-chloramphenicol (CP)-resistant plasmids, the three-drug-resistant strain was frequently obtained. By repeatedly transducing from this strain, a strain (TP-2) having stable resistance to TC and PC could be obtained. In transformation with the deoxyribonucleic acid (DNA) of TP-2 as donor, all of the transformants obtained by selecting with either TC or PC were both TC and PC resistant. According to electron microscopy study of the covalently closed circular DNA of TP-2, the plasmid DNA size was 1.37 +/- 0.03 mum (2.84 x 10(6) daltons). The plasmid (P(TP-2)) is presumed to be a new plasmid in which the PC resistance gene was integrated into the TC-resistant plasmid.

Mechanism of gap-filling during postreplication repair of ultraviolet damage in Haemophilus influenzae.

Deoxyribonucleic acid (DNA), pulse labeled after ultraviolet irradiation of excision-defective mutants of Haemophilus influenzae, is of lower single strand molecular weight than that of unirradiated cells but approaches the size of DNA from unirradiated cells upon further incubation in growth medium. This gap-filling process is controlled by the rec-1 gene. Gap-filling occurs normally in a temperature-sensitive DNA synthesis mutant at the restrictive temperature showing that normal semiconservative DNA synthesis is not necessary for gap-filling. To test for recombinational events after irradiation, the DNA synthesized after irradiation was radioactively labeled for a short time in medium containing 5-bromodeoxyuridine followed by incubation for various times in non-radioactive, 5-bromodeoxyuridine-containing medium. The DNA was denatured and analyzed isopycnically. The labeled DNA was initially "heavy," but later shifted toward lighter densities. This shift occurred in the temperature-sensitive DNA synthesis mutant at the restrictive temperature and in the recombination-defective mutant rec-2, but was not seen in the rec-1 mutant. The density shift can be interpreted as evidence that rather extensive exchanges occurred between parental DNA and the DNA made after irradiation. These results suggest that such exchanges are important for gap-filling in H. influenzae.

R-factor mutant capable of specifying hypersynthesis of penicillinase.

The physical characteristics of a mutant, R(M201-2), capable of conferring high and stable ampicillion resistance was analyzed. The R(M201-2) and its parent R-factor deoxyribonucleic acid (DNA) could be isolated as an extrachromosomal and covalently closed circular form. Their buoyant densities were both 1.712 g/cm(3), and their molecular weights were about 82 x 10(6) and 64 x 10(6), respectively, when measured by CsCl and sucrose density gradient analyses. The contour lengths by electron microscopy were 35.9 +/- 0.6 and 31.0 +/- 0.6 mum, respectively. By using the extracted R-factor DNA, the mutant and parent characters were transformable to another Escherichia coli strain. The mutant R factor showed an increased amount of DNA even after conjugal transfer to Proteus. An increase in the size of R-factor DNA was thus considered to be the cause of the high level of ampicillin resistance.

Conjugal deoxyribonucleic acid replication by Escherichia coli K-12: effect of nalidixic acid.

During the conjugal transfer of the R64-11 plasmid at 42 C from donor cells thermosensitive for vegetative deoxyribonucleic acid (DNA) synthesis to recipient minicells, the plasmids are conjugally replicated in the donor cells. This conjugal replication is inhibited by nalidixic acid, and the degree of inhibition is comparable to the reduction in the amount of plasmid DNA transferred to the recipient minicells in the presence of the drug. In addition, the size of DNA transferred to the minicells and the fraction of conjugally replicated DNA in the donor cells that can be isolated as closed-circular plasmid DNA under alkaline conditions are both reduced by nalidixic acid. When the drug is added to a mating that is underway, the rate of conjugal replication is immediately reduced. This change is accompanied by a reduction in the amount of conjugally replicated DNA in the donor cells that can be isolated as closed-circular plasmid DNA. Furthermore, conjugally replicated plasmid DNA that is not associated with the donor cell membrane becomes membrane bound after the addition of nalidixic acid.

Transformation and deoxyribonucleic acid size: extent of degradation on entry varies with size of donor.

The fate of label introduced as donor deoxyribonucleic acid (DNA) into competent cells of Diplococcus pneumoniae was determined immediately after entry at 25 C, as a function of the size of the donor DNA. Part of the label is found to be acid soluble, part has been incorporated into chromosomal DNA, apparently through reincorporation of degraded donor DNA, and part is found in single strands of length smaller than that of the input donor DNA strands. The last fraction apparently constitutes the precursor for integration of intact donor genetic markers and is referred to as the intact fraction. For large donor DNA the intact fraction contains over 80% of the total intracellular label, but the median strand length has been reduced to 2.2 x 10(6) daltons. For small donor molecules (1 x 10(5) to 6 x 10(5) daltons per strand) the fraction intact increases with donor size from 10 to 50% of the total intracellular label, and the median strand length of this fraction is half that of the donor strands. By combining these results with earlier data on the size dependence of the yield of transformants per unit of total intracellular donor label, we have calculated the probability that a marker in the intact fraction will be integrated, as a function of the length of the donor strand after entry. This probability has a linear dependence on strand length for activities below 40% of maximum, and extrapolates to zero activity at 77,000 daltons per strand.

Competence Mutant of Haemophilus influenzae with Abnormal Ratios of Marker Efficiencies in Transformation

In studies of competence-deficient mutants of Haemophilus influenzae which absorb deoxyribonucleic acid (DNA) but fail to produce transformants, it was observed that in some mutants the residual transforming activity for different markers varied widely, i.e., produced a ratio effect. One of these mutants, com(-56), was studied intensively to determine the cause of the residual efficiency of transformation and the reason for the ratio effect. The residual frequency of transformation was higher for markers considered single-site mutations (like naladixic acid resistance), whereas the least efficient markers tested were those conferring resistance to high levels of streptomycin or novobiocin which are more complex than single-site mutations. Measurement of frequencies of cotransformation indicated that overall genetic linkage was reduced. Transfection was fairly efficient with phage S2 DNA, but not prophage DNA. Donor marker activity could be detected in transformed cell lysates, but not linked to recipient markers in recombinant molecules. Sucrose gradient analysis of such lysates revealed that donor material was associated with recipient DNA in at least normal quantities, but lacked detectable genetic activity. Material from donor DNA labeled with heavy isotopes was incorporated into recipient chromosomal fragments having a density indistinguishable from normal density, unlike the hybrid density recombinant material found in normal cells. No excessive solubilization or nicking of unincorporated donor was detected. It is postulated that this strain contains a hyperactive nuclease, which reduces the effective size of the input DNA during the integration process.

Effect of ultra-violet light on the shape and size of deoxyribonucleic acid.

SEVERAL investigators1,2 have found that the viscosity of solutions of deoxyribonucleic acid is reduced by irradiation with ultra-violet light of 254 mµ. This change could be the result either of main-chain scission which reduces the length of the molecules or of an increase in flexibility which allows the molecule to take up a more coiled configuration and thereby reduces the volume occupied in solution, as measured by the radius of gyration.

Anion exchange chromatography and molecular size of deoxyribonucleic acid.

IN conjunction with investigations on deoxyribonucleic acid isolated from white blood cells of patients with leukaemia (Polli, E. E., Rosoff, M., di Mayorca, G., and Cavalieri, L. F. (unpublished) and ref. 1), an interesting correlation was found between the molecular parameters of the acid and their chromatographic profiles obtained by fractionation on columns of ‘Ecteola’2.

Evidence from light-scattering studies for a dimeric structure for deoxyribonucleic acid in solution.

The investigation of the size and shape of the sodium salt of deoxyribonucleic acid (DNA) in solution has attracted much attention both because of its inherent interest and its great biological importance. But the complications of its polyelectrolyte character and its very great size, both of which contribute to the marked intermolecular interactions, make the interpretation of the experimental results difficult. The least ambiguous method, light scattering, indicates that the nucleic acid derived from calf thymus is best regarded as a stiffened random coil with a molecular weight of about 6 x 106, and using the formulae appropriate to coiling polymer chains, Doty, Reichmann, Rice & Thomas (1954) have shown that the results from measurements of viscosity and sedimentation are consistent with this value. Crick & Watson (1953, 1954) have proposed recently that the analytical data for DNA and the X-ray diffraction diagrams by Franklin & Gosling (1953) obtained forDNA fibres can best be explained by a model in which two strands of nucleic acid intertwine to form a double helix held together by hydrogen bonds between the amino and hydroxyl groups of the purines and pyrimidines. X-ray diagrams indicating the presence of such structures have also been obtained from DNA in native materials (Wilkins, Stokes & Wilson, 1953). Since it is unlikely that such a specific structure can be formed in the process of producing the fibres, it follows on this hypothesis that DNA in solution must already be associated as a double molecule. To test this hypothesis we examined by light scattering the size and shape of DNA in concentrations of urea which we had previously shown to dissociate hydrogen-bonded aggregates of dyes (Alexander & Stacey, 1952) and ofpolyvinyl alcohol (Stacey & Alexander, 1954a). The dissociation by 4M urea of the haemoglobin molecule into two halves by the breaking of hydrogen bonds was clearly demonstrated in the ultracentrifuge by Steinhardt (1938). The results ofpreliminary experiments (Stacey & Alexander, 1954b) indicated that there was a dissociation of the DNA molecule, although it appeared to retain its linear dimensions. Greenstein & Jenrette (1941) had observed that urea decreases the viscosity ofsolution ofDNA, and this observation was confirmed by Conway & Butler (1952), who interpreted this change as a loss of the rigidity of the DNA molecule and also found indications for a halving of the molecular weight. The ionization of the amino groups of cytosine and adenine which are involved in the hydrogen bonds should also produce a dissociation of the twin spiral structure, and for this reason the effect of adding acid on the size and shape of DNA has also been examined. Cecil & Ogston (1948) found in the ultracentrifuge that in solutions of nucleic acid acidified to pH 3*5 a second heterogeneous component appeared with a lower sedimentation constant and increased rate ofboundary spreading, but Doty, Bunce & Reichmann (1953) have shown that it is possible to titrate DNA to pH 2-6 and back to pH 5-8 without degradation. From the change in the angular distribution of the light scattering they concluded that the well-known decrease in viscosity on acidification was due to the contraction of the coil to a much more compact structure. Pouyet & Sadron (1954) have challenged this interpretation because they find no change in the light scattering on adding acid to pH 3-8 and suggest DNA is rather more rigid. We have studied the light scattering of nucleic acid from herring sperm, which appears to have the same properties as the more extensively investigated DNA from calf thymus.