Single-stranded DNA binding protein facilitates specific enrichment of circular DNA molecules using rolling circle amplification

Many techniques in molecular biology require the use of pure nucleic acids in general and circular DNA (plasmid or mitochondrial) in particular. We have developed a method to separate these circular molecules from a mixture containing different species of nucleic acids using rolling circle amplification (RCA). RCA of plasmid or genomic DNA using random hexamers and bacteriophage Phi29 DNA polymerase has become increasingly popular for the amplification of template DNA in DNA sequencing protocols. Recently, we reported that the mutant single-stranded DNA binding protein (SSB) from Thermus thermophilus (TthSSB) HB8 eliminates nonspecific DNA products in RCA reactions. We developed this method for separating circular nucleic acids from a mixture having different species of nucleic acids. Use of the mutant TthSSB resulted in an enhancement of plasmid or mitochondrial DNA content in the amplified product by approximately 500×. The use of mutant TthSSB not only promoted the amplification of circular target DNA over the background but also could be used to enhance the amplification of circular targets over linear targets.     

Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification

Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 10⁶-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria.     

Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami

DNA origami requires long scaffold DNA to be aligned with the guidance of short staple DNA strands. Scaffold DNA is produced in Escherichia coli as a form of the M13 bacteriophage by rolling circle amplification (RCA). This study shows that RCA can be reconfigured by reducing phage protein V (pV) expression, improving the production throughput of scaffold DNA by at least 5.66-fold. The change in pV expression was executed by modifying the untranslated region sequence and monitored using a reporter green fluorescence protein fused to pV. In a separate experiment, pV expression was controlled by an inducer. In both experiments, reduced pV expression was correlated with improved M13 bacteriophage production. High-cell-density cultivation was attempted for mass scaffold DNA production, and the produced scaffold DNA was successfully folded into a barrel shape without compromising structural quality. This result suggested that scaffold DNA production throughput can be significantly improved by reprogramming the RCA in E. coli.     

滚环DNA扩增的原理、应用和展望

滚环DNA扩增 (rollingcircleDNAamplification ,RCA)是一种等温信号扩增方法 ,其线性扩增倍数为 1 0 ⁵,指数化扩增能力大于 10⁹,产生的扩增产物连接在固相支持物 (如玻片、微孔板等 )表面的DNA引物或抗体上。RCA是一种适合在芯片上 (on chip)进行信号扩增的新技术 ,它既能提供研究分析的敏感性和特异性 ,又能保持立体分析的多元性。RCA亦是一种痕量的分子检测方法 ,可用于极其微量的生物大分子和生物标志的检测与研究     

Genetic Control of Immune Responses to HIV-1 env DNA Vaccine

We investigated the genetic control of immunoglobulin production and the delayed-type hypersensitivity (DTH) response produced by an HIV-specific DNA vaccine using several strains of mice. Murine antigen-specific immunoglobulin production was determined by ELISA. The DTH response was assessed in terms of the footpad swelling reaction. All strains of mice, except for B10.RIII and B10.T(6R), exhibited strong immunoglobulin production and footpad swelling in response to the DNA vaccine. In vitro treatment of lymphoid cells with monoclonal antibodies showed that the footpad swelling response was mediated by CD4⁺8^? and Ia— T cells. However, CD8⁺ T cells did not suppress footpad swelling. There was no difference in the induction of HIV-specific immunoglobulin production or DTH response induced by the DNA vaccine among the strains, suggesting that HIV-specific DNA vaccine is useful for immunizing various populations against HIV-1.     

Molecules involved in cell–cell adhesion during development

A peculiarity of nitrosamines is the high degree of cell and organ specificity in inducing tumors. There is substantial evidence that the initiation of the carcinogenesis process by carcinogens of this group is linked to the metabolic competence of the target tissue or cell to convert these carcinogens into mutagenic metabolites and to the binding of those metabolites to cellular DNA. Alkylation occurs in the DNA at the N-1, N-3, and N-7 positions of adenine; the N-3, N-7, and O⁶ of guanine; the N-3, and O² of cytosine; and the N-3, O⁴, and O² of thymine; and the phosphate groups. The initial proportion of each DNA adduct depends upon the alkylating agent used. The various DNA adducts are lost to a variable extent from DNA in vivo by spontaneous release of bases and Or by specific DNA repair processes. Studies conducted in vitro and in vivo indicate that alkylation at the oxygen atoms of DNA bases is more critical than alkylation at other positions in the mutagenesis and carcinogenesis induced by N-nitroso compounds. In particular, tissues in which tumors occur more frequently after a pulse dose of nitrosamine are those in which O⁶-alkylguanine persists longest in DNA, presumably resulting in an increased probability that a miscoding event (mutation) will take place during DNA synthesis. The more rapid removal of O⁶-methylguanine from the DNA of liver (as compared with cxtrahepatic tissues) of rats has been associated with the absence of tumor production in this organ by a single dose of dimethylnitrosamine; however, a significant incidence of liver tumors is observed if the same dose is given 24 hr after partial hepatectomy, and tumors arc induced by such a dose of dimethyl-nitrosamine in the liver of hamsters, which has a low capacity to remove O⁶-methylguanine from its DNA. These data also indicate that the rate of disappearance of 7-methylguanine from the liver or cxtrahepatic tissues is independent of the dose of dimethylnitrosamine; whereas O⁶-methylguanine is lost from DNA more rapidly after a low dose of this nitrosamine. It has been shown that in liver the removal of O⁶-methylguanine, but not of other DNA adducts, from DNA can be affected by pretreating the animals with N-nitroso compounds. The modulation of DNA repair processes observed after a single dose and after chronic treatment with nitrosamines is discussed in relation to the tissue-specific carcinogenic effect of this group of carcinogens.     

Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification

Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R² = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays.     

Isothermal strand-displacement amplification applications for high-throughput genomics

Amplification of source DNA is a nearly universal requirement for molecular biology applications. The primary methods currently available to researchers are limited to in vivo amplification in Escherichia coli hosts and the polymerase chain reaction. Rolling-circle DNA replication is a well-known method for synthesis of phage genomes and recently has been applied as rolling circle amplification (RCA) of specific target sequences as well as circular vectors used in cloning. Here, we demonstrate that RCA using random hexamer primers with 29 DNA polymerase can be used for strand-displacement amplification of different vector constructs containing a variety of insert sizes to produce consistently uniform template for end-sequencing reactions. We show this procedure to be especially effective in a high-throughput plasmid production sequencing process. In addition, we demonstrate that whole bacterial genomes can be effectively amplified from cells or small amounts of purified genomic DNA without apparent bias for use in downstream applications, including whole genome shotgun sequencing.     

Separation of recombinant antibodies from DNA using divalent cations

New downstream methods for the purification of antibodies are required to meet the demands of current and future antibody applications, e.g. for mass production as cancer therapeutic. The standard chromatographic methods suffer from high material costs and mass transfer limitations. In this study, we established and characterized a method for DNA precipitation for antibody purification using divalent cations, particularly CaCl₂, using four different antibodies. By implementing high‐throughput screening using a factorial design plan, we determined that CaCl₂ concentration and PO₄^3? concentration were significant factors, while temperature and pH were not significant. We detected DNA precipitation as well as host‐cell protein (HCP) reduction. Two‐dimensional difference gel electrophoresis (2D‐DIGE) revealed that improved HCP removal does not occur via an unspecific random mechanism such as the enclosure of proteins in the precipitate. CaCl₂ precipitation of DNA and HCP can be combined with nonchromatographic methods such as precipitation and protein A affinity chromatography. This additional purification method not only enhances DNA removal, but also the removal of HCP and antibody multimers, which will reduce immunogenicity and increase homogeneity of the resulting drug.     

Effect of temperature and water activity on the production of fumonisins by Aspergillus niger and different Fusariumspecies

Background

Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing.

Results

The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate.

Conclusion

The sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.     

Dna Damage by Chemically Generated Singlet Oxygen

A naphthalenic endoperoxide was used as a non-photochemical source of singlet oxygen (¹O₂) to examine some interactions between this reactive oxygen species and DNA. High molecular weight DNA (ca. 10⁸ daltons) was exposed to 120 mol m^?31O₂ (cumulative concentration) and analyzed for interstrand crosslinkage by hydroxyl apatite chromatography following formamide denaturation. No evidence for ¹O₂-induced interstrand crosslinking was obtained. The capacity of ¹O₂ to generate strand breaks in single-stranded (ss) and double-stranded (ds) DNA was investigated by sucrose gradient centrifugation analysis of bacteriophage øX174 DNA. No direct strand breaks could be detected at neutral pH, whereas extensive strand breakage was observed after treatment with alkali. Possible biological consequences of ¹O₂ -exposure were assessed by examining the plaque-forming capacity of ss and ds øX 174 DNA molecules using wildtype Escherichia coli spheroplasts as recipients. Without any further treatment with heat or alkali, exposure to the endoperoxide resulted in a time- and dose-dependent inactivation, ss DNA being considerably more sensitive than ds DNA. From the present results and those reported earlier (Nieuwint et al.,²⁰) we infer that ¹O₂-induced inactivation of øX174 DNA is not due to DNA backbone breakage nor to interstrand crosslinking, but rather to some form of damage to the base or sugar moiety of the DNA, the exact nature of which remains to be elucidated.     

A high amount of satellite DNA in the genome of Lupinus angustifolius L.

Embryo DNA, isolated from ungerminated seeds of Lupinus angustifolius L., contains an exceptionally high amount of guanine-cytosine-rich satellite DNA. The thermal denaturation curve of total embryo DNA is biphasic with an inflexion point at 62% denaturation, indicating the presence of satellite DNA. The satellite fraction could be separated from the mainband DNA by three successive preparative CsCl-gradient centrifugations. The densities of the DNA fractions are 1.7045 g cm^-3 and 1.6925 g cm^-3, respectively. The percentages of guanine-cytosine calculated from these densities are comparable to the percentages of GC calculated from the melting temperatures. Finally, ressociation studies prove that foldback DNA and highly repeated sequences are much more frequent in the satellite DNA fraction than in the mainband DNA.Abbreviation C _o t the product of the DNA concentration (mol nucleotides l^-1) and the time (s) of incubation in a DNA reassociation reaction - GC guanine-cytosine - np nucleotide parirs - T temperature interval between 16 and 84% denaturation     

Magnesium reduces nickel inhibition of DNA polymerization

The activities of DNA polymerization and DNA ligation in extract of Chinese hamster ovary cells were both stimulated by MgCl₂. DNA polymerization was stimulated by MgCl₂ above 0.25 mM, whereas, MgCl₂ above 2 mM was required to stimulate DNA ligation. The activity of DNA polymerization maintained a plateau at MgCl₂ 1–12 mM, whereas DNA ligation reached a maximal activity at MgCl₂ 6 mM and decreased thereafter. NiCl₂ 0.1-0.2 mM also had a stimulatory effect on DNA polymerization, but was much less potent than MgCl₂. However, nickel ion (Ni²⁺) had no detectable stimulating effect on the activity of DNA ligation. In the presence of MgCl₂, the activities of DNA polymerization and DNA ligation decreased with increasing concentration of NiCl₂. Ni₂₊ inhibition of DNA polymerization was reduced by increasing the concentration of MgCl2, but increasing the concentration of MgCl₂ did not reduce Ni²⁺ inhibition of DNA ligation. Preincubating cell extract with MgCl₂ decreased the Ni²⁺ inhibition of DNA polymerization but not DNA ligation. These results suggest that Ni²⁺ may compete with magnesium ion (Mg²⁺) to reduce DNA polymerization, but this mechanism seems not applicable to Ni²⁺ inhibition of DNA ligation.     

DNA plasmid production in different host strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

We compared plasmid DNA production in 13 strains of Escherichia coli in shake flasks using media containing glucose or glycerol. DNA yield from either carbon source showed small correlation with maximum growth rate. Three strains, SCS1-L, BL21 and MC4100, were selected for a controlled exponential fed-batch process at a growth rate of 0.14 h⁻¹ to an optical density of about 70, followed by a four-hour heat treatment. Prior to heat treatment, SCS1-L generated 15.4 mg DNA/g, BL21 generated 11.0 mg DNA/g and MC4100 generated 7.9 mg DNA/g, while after heat treatment the strains attained DNA yields, respectively, of 18.0, 15.0 and 6.8 mg/g. The strains also varied in their percentage of supercoiled DNA after heat treatment, with SCS1-L averaging 66% supercoiled, BL21 17% and MC4100 40%. We further investigated the two strains that yielded the highest percentage of supercoiled DNA (SCS1-L and MC4100) at a higher growth rate of 0.28 h⁻¹. At this condition, a slightly lower DNA yield was generated faster, and the percentage of supercoiled DNA increased. Heat treatment improved DNA yield, and surprisingly did so to a greater extent at the higher growth rate. As a consequence of these factors, higher growth rates might be advantageous for DNA production.     

Rapid generation of long tandem DNA repeat arrays by homologous recombination in yeast to study their function in mammalian genomes

We describe here a method to rapidly convert any desirable DNA fragment, as small as 100 bp, into long tandem DNA arrays up to 140 kb in size that are inserted into a microbe vector. This method includes rolling-circle phi29 amplification (RCA) of the sequence in vitro and assembly of the RCA products in vivo by homologous recombination in the yeast Saccharomyces cerevisiae. The method was successfully used for a functional analysis of centromeric and pericentromeric repeats and construction of new vehicles for gene delivery to mammalian cells. The method may have general application in elucidating the role of tandem repeats in chromosome organization and dynamics. Each cycle of the protocol takes ~ two weeks to complete.     

Nuclear DNA Ligase and Its Action on Chromatin DNA in Neuronal, Glial and Liver Nuclei Isolated from Adult Guinea Pigs

Abstract: DNA ligase activities were measured in neuron-rich and glial nuclear preparations and liver nuclei isolated from adult guinea pigs. The enzymatic properties of cerebral and liver nuclear DNA ligases were studied with isolated nuclei and nuclear extracts. ATP (K_m= 46–48 μM) and bivalent cation (Mg²⁺ or Mn²⁺) were required for the maximal activities in cerebral and liver nuclei. β-Mercaptoethanol did not affect the activities, but N-ethylmaleimide and p-chloromercuribenzoate completely inhibited the activities. Deoxyadenosine-5′-triphosphate partially inhibited the activities in both cerebral and liver nuclei. An interdependent effect of Na⁺ and Mg²⁺ on the enzyme activities was observed. A high concentration (200 mM) of Na⁺ activated both enzymes and shifted to the acid side the optimal pH for both enzymes. DNA ligase was more easily extracted with lower concentrations of NaCl from liver nuclei than from cerebral nuclei, but the extraction curves from both nuclear species reached a plateau level (92% of total activities of nuclear enzymes) at 200 mM-NaCl. Apparent K_m for the substrate [³²P]phosphoryl DNA was determined according to a modification of the Michaelis-Menten equation, which was applied for the case where an unknown amount of substrate nicks in chromatin DNA coexisted with the nicks in exogenous substrate DNA. Neuronal and glial nuclear enzymes had similar K_m values (about 20 μg of [³²P]phosphoryl DNA/ml), but the liver nuclear enzyme had a higher K_m value (54 μg of [³²P]phosphoryl DNA/ml). The modified Michaelis-Menten equation provided the amounts of nicks available as substrate in chromatin DNA of isolated nuclei. Neuronal and glial nuclei contained 1.5 and 0.29 pmol of nicks/μg of nuclear DNA, respectively, in contrast to an intermediate amount of nicks in liver nuclei (0.63 pmol/μg of nuclear DNA). DNA ligase activity in neuronal nuclei [312 units (fmol of 5′-phosphomonoester converted into a phosphatase-resistant form per min at 37°C) per μg of nuclear DNA] was 11-fold higher than that in glial nuclei [28.7 units/μg of nuclear DNA]. Liver nuclei contained an intermediate activity [54.7 units/μg of nuclear DNA].     

Mineralization of pentachlorophenol in a contaminated soil by Pseudomonas sp UG30 cells encapsulated in κ-carrageenan

A fermentation process in Escherichia coli for production of supercoiled plasmid DNA for use as a DNA vaccine was developed using an automated feed-back control nutrient feeding strategy based on dissolved oxygen (DO) and pH. The process was further automated through a computer-aided data processing system to regulate the cell growth rate by controlling interactively both the nutrient feed rate and agitation speed based on DO. The process increased the total yield of the plasmid DNA by approximately 10-fold as compared to a manual fed-batch culture. The final cell yield from the automated process reached 60 g L⁻¹ of dry cell weight (OD₆₀₀ = 120) within 24 h. A plasmid DNA yield of 100 mg L⁻¹ (1.7 mg g⁻¹ cell weight) was achieved by using an alkaline cell lysis method. Plasmid yield was confirmed using High Performance Liquid Chromatography (HPLC) analysis. Because cells had been grown under carbon-limiting conditions in the automated process, acetic acid production was minimal (below 0.01 g L⁻¹) throughout the fed-batch stage. In contrast, in the manual process, an acid accumulation rate as high as 0.36 g L⁻¹ was observed, presumably due to the high nutrient feed rates used to maintain a maximum growth rate. The manual fed-batch process produced a low cell density averaging 10–12 g L⁻¹ (OD₆₀₀ = 25–30) and plasmid yields of 5–8 mg L⁻¹ (approximately 0.7 mg g⁻¹ cells). The improved plasmid DNA yields in the DO- and pH-based feed-back controlled process were assumed to be a result of a combination of increased cell density, reduced growth rate (μ) from 0.69 h⁻¹ to 0.13 h⁻¹ and the carbon/nitrogen limitation in the fed-batch stage. The DO- and pH-based feed-back control, fed-batch process has proven itself to be advantageous in regulating cell growth rate to achieve both high cell density and plasmid yield without having to use pure oxygen. The process was reproducible in triplicate fermentations at both 7-L and 80-L scales. Received 22 March 1996/ Accepted in revised form 20 September 1996     

A comparative study of PCR product detection and quantitation by electrochemiluminescence and fluorescence

Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)₃²⁺)-end-labelled primers. In this way, biotin for capture and Ru(bpy)₃²⁺ for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)₃²⁺-labelled primers amplified a 1 kilobase region. Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.     

Integration of vector-containing Bacillus subtilis chromosomal DNA by a Campbell-like mechanism

Summary Using plasmid pHV60, which contains a chloramphenicol resistance (Cm^r) gene that is expressed in Bacillus subtilis, a set of transformation-deficient strains of B. subtilis was isolated by insertional mutagenesis. When chromosomal DNA from these mutants was used to transform a transformation-proficient B. subtilis strain, almost all of the Cm^r transformants had the mutant phenotype as expected. However, with a frequency of approximately 3×10^-4 atypical transformants with the wild-type phenotype were produced. Data concerning amplification of the DNA containing the Cm^r marker and duplication of DNA sequences are presented that suggest that these atypical transformants are the result of a Campbell-like integration of the chromosomal DNA containing the integrated plasmid. Transductional mapping showed that in the atypical transformants the vector-containing DNA had a strong tendency to integrate at sites adjacent to the original site of integration, although integration at sites elsewhere on the chromosome was also observed. The production of atypical transformants is explained on the basis of integration of chromosomal DNA by a Campbell-like mechanism. Circularization of vector-containing chromosomal DNA is thought to occur through joining of the extremities of single-stranded DNA molecules by fortuitous base pairing with an independently entered single-stranded DNA molecule.     

One-step isolation of plant DNA suitable for PCR amplification

We report a one-step extraction technique for the isolation of plant DNA, DNA suitable for amplification by PCR can be produced from leaf material smaller than 0.3 mm² in less than 20 min, with no tube changes. The method was tested on several plant specA00AK020ies. The described method was found to extract DNA that could be amplified without any further purification or treatment. The isolated DNA was amplified using a universal chloroplast primer set. The method was validated by comparing size of PCR products generated by the novel method to PCR products generated using standard DNA isolation techniques.