Settlement of Planulae of the Moon Jellyfish Aurelia aurita onto Hydrophilic Polycarbonate Plates Modified by Atmospheric Plasma Treatment

It has been reported that planula larvae of some jellyfish prefer artificial substrates for settlement. This research focused on the relationship between the settlement of planulae and the wettability of artificial substrate surfaces. We used atmospheric plasmas to change the wettability of the surfaces of polycarbonate (PC) plates because plasma treatment has no chemical side effects. The treatment made the surfaces hydrophilic, as evidenced by the decrease of contact angle from 85° to 35°. X-ray photoelectron spectroscopy revealed that the change of wettability of the PC plates could be attributed to N₂, which was probably ionized in the air above the plates. Scanning electron microscopy revealed no difference in the surface morphology of the plates before and after plasma treatment. Results of bioassays using treated PC plates showed that planulae tended to preferentially settle on hydrophobic surfaces.     

Inactivation of bacterial opportunistic skin pathogens by nonthermal DC-operated afterglow atmospheric plasma

Aims: Multidrug‐resistant opportunistic pathogens are clinically significant and require the development of new antimicrobial methods. In this study, Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus cells were exposed to atmospheric plasma on agar plates and in vitro on porcine skin for the purpose of testing bacterial inactivation. Methods and Results: Microbial inactivation at varying exposure durations was tested using a nonthermal plasma jet generated with a DC voltage from ambient air. The observed reduction in colony forming units was quantified as log₁₀ reductions. Conclusions: Direct plasma exposure significantly inactivated seeded bacterial cells by approx. 6 log₁₀ on agar plates and 2–3 log₁₀ on porcine skin. On agar plates, an indirect ‘bystander’ inactivation outside the plasma delivery area was also observed. The reduced inactivation observed on the skin surface was most likely due to cell protection by the variable surface architecture. Significance and Impact of Study: Atmospheric plasma has potential for clinical application as a disinfectant of patient skin and medically relevant surfaces.     

A 5× genome coverage bovine BAC library: production, characterization, and distribution

A bovine large-insert DNA library has been constructed in a Bacterial Artificial Chromosome (BAC) vector. The source DNA was derived from lymphocytes of a Jersey male. High-molecular-weight DNA fragments were produced by treatment with EcoRI/EcoRI methylase and cloned into the EcoRI site of pBACe3.6. In total, 157,240 individual BACs have been picked into 384-well plates. Approximately 190 randomly chosen clones have been characterized by Pulsed Field Gel Electrophoresis (PFGE) and have an average insert size of 105 kb, suggesting library coverage representing 5–6 genome equivalents. The frequency of clones without inserts is 4%. The chromosomal location of 51 BACs was studied by FISH; 3 showed more than one signal, indicating a chimerism frequency of roughly 6%. Approximately 50% of the clones in the library contain Simple Repeat Sequences (microsatellites), and 4% of the clones contain centromeric repeats. Insert stability was assessed by restriction digestion of DNA prepared from 20 clones after serial culture for one and three nights. Only one clone showed any evidence of an altered restriction pattern. Clones from 360 × 384-well plates (138,240 colonies) were gridded onto high-density membranes, and PCR superpools were produced from the same set of clones. Both membranes and superpools are available from the RZPD, Berlin (http://www.rzpd.de). PCR 4-D superpools have been prepared from an additional 23,000 clones. The library has been screened for a total of 24 single-copy sequences; positive clones have been obtained in all cases. Received: 14 October 1998 / Accepted: 9 March 1999     

Characterization of DNA polymerase from the hyperthermophilic archaeon Thermococcus marinus and its application to PCR

The family B DNA polymerase gene from the archaeon Thermococcus marinus (Tma) contains a long open reading frame of 3,939 bp that encodes 1,312 amino acid residues. The gene is split by one intervening sequence that forms a continuous open reading frame with the two polymerase exteins. In this study, the Tma DNA polymerase gene both with (precursor form) and without (mature form) its intein was expressed in Escherichia coli, purified by heat treatment and HiTrap™ Heparin HP column chromatography and characterized. Primary sequence analysis of the mature Tma polymerase showed high sequence identity with DNA polymerases in the genus Thermococcus. The expressed precursor form was easily spliced during purification steps. The molecular mass of the purified Tma DNA polymerases is about 90 kDa, as estimated by SDS-PAGE. Both Tma DNA polymerases showed the same properties. PCR performed with this enzyme was found to be optimal in the presence of 50 mM Tris–HCl (pH 8.4), 40 mM KCl, 12.5 mM (NH₄)₂SO_4, 2 mM MgCl_2, 0.05% Triton X-100 and 0.0075% BSA. Furthermore, long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tma DNA polymerases (Tma plus DNA polymerase).     

DNA persistence after treatment of Lyme borreliosis

One hundred twenty-four patients—53 with neuroborreliosis, 48 with erythema migrans, and 23 with Lyme arthritis—were tested in a prospective study for the presence of the DNA of Borrelia burgdorferi sensu lato in plasma, cerebrospinal fluid (CSF), urine, and synovial fluid by nested polymerase chain reaction (PCR). Specific DNA was detected using five amplification systems simultaneously: three targeted chromosomal genes encoding 16S rDNA, flagellin, and p66; and two plasmid sequences of OspA and OspC. Patients were examined clinically and by PCR before and after treatment and again after 3 and 6 months. Before treatment, the specific DNA was detected in 78 patients (62.9 %). Forty-one neuroborreliosis patients were DNA-positive (77.4 %), with CSF positivity in 26 patients, urine in 25, and plasma in 16. Twenty-six erythema migrans patients were DNA-positive (54.2 %), with plasma positivity in 18 cases and urine in 14. Eleven Lyme arthritis cases (47.8 %) were DNA positive (six in urine, five in plasma, and four in synovial fluid). The frequency of PCR positives was comparable in CSF and urine, and it was lower by approximately 50 % in plasma. Specific DNA was also found in a significant number of patients in later testing periods: 48 patients after treatment, 29 patients after 3 months, and 6 patients after 6 months. The prolonged PCR positivity was not explainable by persistent infection according to the clinical manifestations of the disease. Possible explanations of the problem are discussed.     

Analysis of the separation of gadolinium isotopes by the ICR method

The problem of separating gadolinium isotopes is discussed. The parameters of isotope separation in a plasma by the ICR method are estimated for a device with given operating parameters. The mean transverse energy 〈E_⊥〉 of the heated ions of the ¹⁵⁷Gd isotope and the heating efficiency η are calculated as functions of the frequency of the RF electric field in the plasma. The dependence of the heating efficiency η on the longitudinal temperature of the plasma flow is investigated. The issue of how the concentration of the target isotope ions at the collector plate depends on the longitudinal coordinate measured from the front edge of the plate is analyzed for different values of the frequency detuning parameter. The extraction coefficient Г for the target isotope is calculated as a function of the distance b between the collector plates for different frequency detunings.     

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.     

Genetic Variation in a Human Immunodeficiency Virus Type 2 Live-Virus Macaca nemestrina Vaccine Model

Four pigtailed macaques were inoculated with an infectious, apathogenic human immunodeficiency virus type 2 (HIV-2) molecular clone (HIV-2_KR) and subsequently challenged with a highly pathogenic strain, HIV-2₂₈₇, together with two naive control animals. After challenge, two animals inoculated with a high dose of the immunizing strain were protected from CD4 decline and immunodeficiency. To examine the role of genetic heterogeneity in protection, fragments of the env gene were amplified from peripheral blood mononuclear cell DNA and plasma RNA of challenged animals by PCR, examined by using a heteroduplex tracking assay (HTA), and sequenced. By HTA, variation was detected principally within the V1 and V2 regions of envelope. Extent of variation in viral DNA clones as assessed by HTA correlated with inoculum size, as did the degree of variation in sequences of clones derived from viral DNA. Conversely, a rapid reduction in the number of plasma viral RNA variants was noted by HTA at 8 weeks postinfection in protected animals; this reduction was not present in naive or unprotected macaques. Sequences derived from plasma viral RNA were found to be more closely related than corresponding viral DNA sequences, and protection correlated with a significant reduction in variation in plasma RNA sequences in animals given the identical inocula of HIV-2₂₈₇. Nonsynonymous mutations were significantly less prevalent in the protected animals. An additional potential glycosylation site was predicted to be present in the V2 region in all but one clone, and amino acid signatures related to protection were identified in viral DNA and RNA clones within both the V1 and V2 regions. Examination of the role of viral variation in this HIV-2 live-virus vaccine model may provide valuable insights into immunopathogenesis.     

Strong association between polymorphisms in an intronic microsatellite and in the coding sequence of the BoLA-DKB3 gene: implications for micro-satellite stability and PCR-based DRB3 typing

A highly polymorphic microsatellite in the bovine DRB3 gene was characterized by polymerase chain reaction (PCR) analysis and DNA sequencing. A very strong association between expressed DRB3 polymorphism and microsatellite alleles was revealed by PCR analysis of genomic DNA from 116 animals representing three breeds of cattle. The results indicated a low frequency of microsatellite length mutations as the association was consistent over breeds. The DRB3 microsatellite may be utilized in a PCR-based typing method of bovine class II alleles. The microsatellite polymorphism did not distinguish all known DRB3 alleles, but it was shown that this method may be complemented by the use of allele-specific PCR based on the extensive polymorphism in the DRB3 exon 2. The DNA sequences of seven microsatellite alleles, associated with different class II hap-lotypes, were determined. The DRB3 microsatellite is composed of three repeat motifs, a stretch of at least 10 uninterrupted (TG)_n dinucleotides, a long but interrupted stretch of (GA)_n dinucleotides, and a few (CAGA)_n tetranucleotides. There were pronounced sequence differences beween alleles and the results indicated that the evolution of this microsatellite has involved length mutations of the dinucleotide repeats as well as point mutations causing interruptions in the dinucleotide repeats.     

A Simple and Non-Invasive Method for Nuclear Transformation of Intact-walled Chlamydomonas reinhardtii

Genetic engineering in microalgae is gaining attraction but nuclear transformation methods available so far are either inefficient or require special equipment. In this study, we employ positively charged nanoparticles, 3-aminopropyl-functionalized magnesium phyllosilicate (aminoclay, approximate unit cell composition of [H₂N(CH₂)₃]₈Si₈Mg₆O₁₂(OH)₄), for nuclear transformation into eukaryotic microalgae. TEM and EDX analysis of the process of transformation reveals that aminoclay coats negatively-charged DNA biomolecules and forms a self-assembled hybrid nanostructure. Subsequently, when this nanostructure is mixed with microalgal cells and plated onto selective agar plates with high friction force, cell wall is disrupted facilitating delivery of plasmid DNA into the cell and ultimately to the nucleus. This method is not only simple, inexpensive, and non-toxic to cells but also provides efficient transformation (5.03×10² transformants/µg DNA), second only to electroporation which needs advanced instrumentation. We present optimized parameters for efficient transformation including pre-treatment, friction force, concentration of foreign DNA/aminoclay, and plasticity of agar plates. It is also confirmed the successful integration and stable expression of foreign gene in Chlamydomonas reinhardtii through molecular methods.     

Development and Application of a Real-time PCR Method for Pharmacokinetic and Biodistribution Studies of Recombinant Adenovirus

A replication-deficient recombinant adenovirus (Ad5-LFA-3/IgG₁) that encodes secreted LFA-3/IgG₁ was constructed for gene therapy treatment of psoriasis. The purpose of this study was to develop a real-time PCR method for pharmacokinetic and biodistribution studies of Ad5-LFA-3/IgG₁ within the circulation and organs. This method showed good specificity, sensitivity and reproducibility over a wide dynamic range of concentrations. Quantitative measurement of recombinant adenoviral DNA suggested that the level of Ad5-LFA-3/IgG₁ DNA in circulating blood peaked within 10 min following intravenous injection in rhesus macaques. Following this peak, the adenoviral DNA level dropped significantly to a very low level. Real-time PCR revealed that Ad5-LFA-3/IgG₁ DNA was enriched in the spleen, lung and liver after injection of the adenovirus into rats through the tail vein. The adenoviral DNA was barely detected in other tissues. These data provide important information for clinical trials of Ad5-LFA-3/IgG₁ and confirm the utility of the real-time PCR assay for monitoring gene therapy trials.     

Optimized Quantification of Fragmented,Free Circulating DNA in Human Blood Plasma Using a Calibrated Duplex Real-Time PCR

Background

Duplex real-time PCR assays have been widely used to determine amounts and concentrations of free circulating DNA in human blood plasma samples. Circulatory plasma DNA is highly fragmented and hence a PCR-based determination of DNA concentration may be affected by the limited availability of full-length targets in the DNA sample. This leads to inaccuracies when counting PCR target copy numbers as whole genome equivalents.

Methodology/Principal Findings

A model system was designed allowing for assessment of bias in a duplex real-time PCR research assay. We collected blood plasma samples from male donors in pools of 6 to 8 individuals. Circulatory plasma DNA was extracted and separated by agarose gel electrophoresis. Separated DNA was recovered from the gel in discrete size fractions and analyzed with different duplex real-time PCR Taqman assays detecting a Y chromosome-specific target and an autosomal target. The real-time PCR research assays used differed significantly in their ability to determine the correct copy number ratio of 0.5 between Y chromosome and autosome targets in DNA of male origin. Longer PCR targets did not amplify quantitatively in circulatory DNA, due to limited presence of full-length target sequence in the sample.

Conclusions

PCR targets of the same small size are preferred over longer targets when comparing fractional circulatory DNA concentrations by real-time PCR. As an example, a DYS14/18S duplex real-time PCR research assay is presented that correctly measures the fractional concentration of male DNA in a male/female mixture of circulatory, fragmented DNA.     

Variable (CA/GT)n simple sequence repeat DNA in the alga Chlamydomonas

A partial genomic DNA library of Chlamydomonas reinhardtii was screened with an (AC)₁₁ probe for the presence of (CA/GT)_n simple sequence repeats (SSRs). Based on the frequency of these repeats in the partial genomic library, we estimate that (CA/GT)_n repeats occur at a rate of about one every 17.7 kb in the C. reinhardtii genome. Ten positive clones were sequenced and four polymerase chain reaction (PCR) primer sets flanking (CA/GT)_n sequences were constructed for four loci. The PCR was used to specifically amplify these regions from multiple isolates of C. reinhardtii. All four loci were highly polymorphic in the C. reinhardtii isolates. A simple Mendelian inheritance pattern was found for all four loci, which showed 2:2 segregation in the tetrads resulting from a cross between C. reinhardtii and C. smithii. Our results suggest that these simple sequence repeat DNA loci will be useful for identity testing, population studies, linkage analysis, and genome mapping in Chlamydomonas.     

甲肝病毒减毒株(H2)RNA的全长RT-PCR扩增

通过RT-PCR技术扩增了甲肝病毒减毒株(H₂)全长RNA,并对长片段RT-PCR扩增进行了方法学上的探讨.采用抗血清特异沉淀病毒;盐酸胍-酸性酚、氯仿一步法分离纯化病毒RNA,可得到高质量的RNA样品;以此RNA为模板,在无RNA酶的逆转录酶作用下,合成单链cDNA;继续以此cDNA为模板,利用32 mer寡核苷酸引物, 在Taq和Deep Vent DNA多聚酶的作用下进行PCR扩增,得到7.4 kb的扩增产物.     

A catalytic antioxidant metalloporphyrin blocks hydrogen peroxide-induced mitochondrial DNA damage

Reactive oxygen species (ROS) have been implicated as the cause of cumulative damage to DNA, proteins and lipids that can ultimately result in cell death. A common problem when measuring oxidative DNA damage has been the introduction of modifications in the native state of the molecule by many DNA isolation methods. We circumvented this problem by employing direct PCR (DPCR) of whole cell lysates. DPCR of mouse lung fibroblasts performed better than PCRs containing template acquired by phenol/chloroform extraction or a commercially available genomic DNA isolation kit. We investigated the direct use of whole cell preparations in the polymerase chain reaction (PCR) to detect hydrogen peroxide (H₂O₂)-mediated DNA damage. We observed a concentration-dependent decrease in amplification efficiency of a 4.3 kb mitochondrial (mt)DNA target in H₂O₂-treated mouse lung fibroblasts (MLFs). At low doses the efficiency of amplification returns to control levels over 24 h. We detected no change in amplification efficiency of a plasmid control containing our mtDNA target under any of the culture conditions employed in these studies. Treatment of MLFs with the catalytic antioxidant manganese(III) meso-tetrakis(4-benzoic acid)porphyrin (MnTBAP) attenuates the effects of H₂O₂ exposure. When quantitated with an external standard the use of DPCR in tandem with a PCR amplification efficiency assay provides a powerful approach to assess oxidative mtDNA damage.     

Nickel subsulfide is genotoxic in vitro but shows no mutagenic potential in respiratory tract tissues of BigBlue™ rats and Muta™Mouse mice in vivo after inhalation

Carcinogenic nickel compounds are known to induce promutagenic DNA lesions such as DNA strand breaks and DNA adducts in cultured mammalian cells. In standard mutation assays, in contrast, they were found to be either inactive or weakly active. In our in vitro mutation studies in a lacI transgenic embryonic fibroblast cell line, nickel subsulfide (Ni₃S₂) increased mutation frequency up to 4.5-fold. We subsequently applied the comet assay and transgenic rodent mutation assays to investigate the DNA damaging effect and mutagenic potential of nickel subsulfide in target cells of carcinogenesis. A 2-h in vitro treatment of freshly isolated mouse nasal mucosa and lung cells with nickel subsulfide clearly induced DNA fragmentation in a concentration dependent manner. The strong effect was not seen in the same cell types following inhalative treatment of mice and rats, leading only in the mouse nasal mucosa to high DNA damage. When the same inhalative treatment was applied to lacZ and lacI transgenic mice and rats, the spontaneous mutation frequency of these target genes in the respiratory tissues was not increased. These results support a recently proposed non-genotoxic model of nickel carcinogenesis, which acts through gene silencing via DNA methylation and chromatin condensation. This model may also explain our in vitro mutation data in the lacI transgenic cell line, in which nickel subsulfide increased mutation frequency, but in about one-third of the mutants, molecular analysis did not reveal any DNA sequence change in the coding region of the lacI gene despite of the phenotypic loss of its function.     

A review of the correlation of tergites, sternites, and leg pairs in diplopods

Background

Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (λ) can be estimated by determining the rate of decline.

Results

The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean λ_predator = 0.0106 per nucleotide; mean λ_prey = 0.0176 per nucleotide). This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples.

Conclusion

We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will allow researchers to measure template quality in order to evaluate alternate sources of DNA, different methods of sample preservation and different DNA extraction protocols. The technique could also be applied to study the process of DNA decay.     

Automated DNA Extraction,Quantification, Dilution,and PCR Preparation for Genotyping by High-Resolution Melting

Genotyping by high-resolution amplicon melting uses only two PCR primers per locus and a generic, saturating DNA dye that detects heteroduplexes as well as homoduplexes. Heterozygous genotypes have a characteristic melting curve shape and a broader width than homozygous genotypes, which are usually differentiated by their melting temperature (T_m). The H63D mutation, associated with hemochromatosis, is a single nucleotide polymorphism, which is impossible to genotype based on T_m, as the homozygous WT and mutant amplicons melt at the same temperature. To distinguish such homozygous variants, WT DNA can be added to controls and unknown samples to create artificial heterozygotes with all genotypes distinguished by quantitative heteroduplex analysis. By automating DNA extraction, quantification, and PCR preparation, a hands-off integrated solution for genotyping is possible. A custom Biomek® NX robot with an onboard spectrophotometer and custom programming was used to extract DNA from whole blood, dilute the DNA to appropriate concentrations, and add the sample DNA to preprepared PCR plates. Agencourt® Genfind™ v.2 chemistry was used for DNA extraction. PCR was performed on a plate thermocycler, high-resolution melting data collected on a LightScanner-96, followed by analysis and automatic genotyping using custom software. In a blinded study of 42 H63D samples, 41 of the 42 sample genotypes were concordant with dual hybridization probe genotyping. The incorrectly assigned genotype was a heterozygote that appeared to be a homozygous mutant as a result of a low sample DNA concentration. Automated DNA extraction from whole blood with quantification, dilution, and PCR preparation was demonstrated using quantitative heteroduplex analysis. Accuracy is critically dependent on DNA quantification.     

A specific marker,pat, for studying the fate of introduced bacteria and their DNA in soil using a combination of detection techniques

A specific eucaryotic DNA marker from Solanum tuberosum cv Bintje (688 bp patatin cDNA fragment) was cloned into the unique HindIII-site of plasmid RP4. RP4:: pat was transferred from Escherichia coli to Pseudomonas fluorescens R2f by filter mating.Homology to pat was not detected in the microbial population of Ede loamy sand soil, nor in that of the rhizosphere of wheat growing in this soil, as evidenced by colony filter hybridization. More sensitive molecular detection techniques like most-probable-number recovery/hybridization analysis, and analysis of total community DNA from soil by polymerase chain reaction (PCR) amplification did not reveal the presence of the pat sequence either. P. fluorescens R2f (RP4:: pat), introduced into sterile soil extract microcosms, initially showed poor survival and plasmid loss, after which the introduced populations grew and stabilized at a level of about Log₁₀ 7 cfu per mL. Between 25 and 50% of the population maintained the plasmid, as evidenced by filter hybridization of colonies from non-selective agar plates using the pat fragment as probe.Introduced R2f (RP4:: pat) could be recovered from soil microcosms using selective plating followed by colony hybridization and MPN recovery/hybridization with the pat probe. The presence of the pat marker always coincided with the presence of the resistance genes on RP4:: pat, indicating pat was an adequate marker of the presence of this plasmid. In addition, it adequately described the population dynamics of the introduced strain in soil, since no loss of the plasmid occurred.Hybridization to pat was also useful to show transfer of plasmid RP4:: pat to a recipient strain in soil; transfer to indigenous bacteria was not detected.Analysis by slot-blot hybridization of total community DNA extracted from inoculated soils indicated about Log₁₀ 6 cfu per g of dry soil were still detectable. Application of the PCR on this DNA indicated pat was detectable at least at a level of Log₁₀ 4 immunofluorescence-detectable cells per g of dry soil. Thus extraction of total community DNA followed by PCR permitted the detection of genetically engineered microorganisms present in soil as non-culturable cells.     

Comparison of DNA extraction methods for multiplex polymerase chain reaction

We compared six DNA extraction methods for obtaining DNA from whole blood and saliva for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate saliva sampling as an alternative to blood sampling to obtain DNA for molecular diagnostics, genetic genealogy, and research purposes. The DNA quantity, DNA purity (A_260/280), PCR inhibition ratio, and mitochondrial DNA/genomic DNA ratio were measured to compare the extraction methods. The different extraction methods resulted in variable DNA quantity and purity, but there were no significant differences in the efficiency of multiplex PCR and oligomicroarray signals after single-base extension on the arrayed primer extension 2 (APEX-2).