A Visible Spectrophotometric Assay for Submicrogram Quantities of DNA, Including PCR-Amplified DNA

The use of the cationic thiazine dye toluidine blue for solution measurements of DNA in the submicrogram range was studied with particular regard to the measurement of the small quantities of DNA synthesized in a typical polymerase chain reaction (PCR). Tolouidine blue shows a decrease in absorption at its wavelength of maximal absorption, 628 nm, on binding DNA in low concentrations. Using toluidine blue at 2 × 10⁻⁵ M, there was a linear response to increasing DNA concentrations up to 2000 ng of DNA in a 500 μl assay. The lower limit of detection was 24 ng of DNA. The assay was found to be sufficiently sensitive to measure the DNA produced in a PCR, and to distinguish PCRs that amplified a product from those in which amplification did not occur. The effects of selected metal ions and nonmetals on the assay were assessed. Of the compounds tested only sodium dodecyl sulfate was found to be incompatible with the assay. Compared to other colorimetric DNA assays, the toluidine blue assay is 10- to 100-fold more sensitive, approaching the sensitivity achieved by fluorometric DNA assays. The color change on binding DNA is immediate, and takes place at room temperature, thus allowing for rapid measurements to be made. The toluidine blue assay extends the useful range of visible spectrophotometric DNA assays well into the submicrogram range and is suitable for detection and measurement of the microgram quantities of DNA produced in a typical PCR.     

Development of a multiplex system to assess DNA persistence in taphonomic studies

In this study, we have developed a PCR multiplex that can be used to assess DNA degradation and at the same time monitor for inhibition: primers have been designed to amplify human, pig, and rabbit DNA, allowing pig and rabbit to be used as experimental models for taphonomic research, but also enabling studies on human DNA persistence in forensic evidence. Internal amplified controls have been added to monitor for inhibition, allowing the effects of degradation and inhibition to be differentiated. Sequence data for single‐copy nuclear recombination activation gene (RAG‐1) from human, pig, and rabbit were aligned to identify conserved regions and primers were designed that targeted amplicons of 70, 194, 305, and 384 bp. Robust amplification in all three species was possible using as little as 0.3 ng of template DNA. These have been combined with primers that will amplify a bacterial DNA template within the PCR. The multiplex has been evaluated in a series of experiments to gain more knowledge of DNA persistence in soft tissues, which can be important when assessing what material to collect following events such as mass disasters or conflict, when muscle or bone material can be used to aid with the identification of human remains. The experiments used pigs as a model species. When whole pig bodies were exposed to the environment in Northwest England, DNA in muscle tissue persisted for over 24 days in the summer and over 77 days in the winter, with full profiles generated from these samples. In addition to time, accumulated degree days (ADD) were also used as a measure that combines both time and temperature—24 days was in summer equivalent to 295 ADD whereas 77 days in winter was equivalent to 494 ADD.     

One-PCR-tube approach for in situ DNA isolation and detection

Traditional real-time polymerase chain reaction (PCR) requires a purified DNA sample for PCR amplification and detection. This requires PCR tests be conducted in clean laboratories, and limits its applications for field tests. This work developed a method that can carry out DNA purification, amplification and detection in a single PCR tube. The polypropylene PCR tube was first treated with chromic acid and peptide nucleic acids (PNA) as DNA-capturer were immobilized on the internal surface of the tube. Cauliflower mosaic virus 35S (CaMV-35S) promoter in the crude extract was hybridized with the PNA on the tube surface, and the inhibitors, interfering agents and irrelevant DNA in the crude extract were effectively removed by rinsing with buffer solutions. The tube that has captured the target DNA can be used for the following real-time PCR (RT-PCR). By using this approach, the detection of less than 2500 copies of 35S plasmids in a complex sample could be completed within 3 hours. Chocolate samples were tested for real sample analysis, and 35S plasmids in genetically modified chocolate samples have been successfully identified with this method in situ. The novel One-PCR-tube method is competitive for commercial kits with the same time and simpler operation procedure. This method may be widely used for identifying food that contains modified DNA and specific pathogens in the field.     

Silica-coated La0.75Sr0.25MnO3 nanoparticles for magnetically driven DNA isolation

Magnetic La_0.75Sr_0.25MnO₃ nanoparticles possessing an approximately 20‐nm‐thick silica shell (LSMO(0.25)@SiO₂) were characterised and tested for the isolation of PCR‐ready bacterial DNA. The results presented here show that the nanoparticles do not interfere in PCR. DNA was apparently reversibly adsorbed on their silica shell from the aqueous phase system (16% PEG 6000–2 M NaCl). The method proposed was used for DNA isolation from complex food samples (dairy products and probiotic food supplements). The isolated DNA was compatible with PCR. The main advantages of the nanoparticles tested for routine use were their high colloidal stability allowing a more precise dosage and therefore high reproducibility of DNA isolation.     

Quantification of DNA in forensic samples

Quantification of DNA in a forensic sample is of major importance for proper DNA amplification and STR profiling. Several methods have been developed to quantify DNA, from basic UV spectrometry, through gel-based techniques, to dye staining, blotting techniques, and, very recently, DNA amplification methods (polymerase chain reaction, PCR). Early techniques simply measured total DNA, but newer techniques can specifically measure human DNA while excluding non-human DNA (foodstuff, animal, or bacterial contamination). These newer assays can be faster and less expensive than traditional methods, making them ideal for the busy forensic laboratory. This paper reviews classic and newer quantification techniques and presents methods recently developed by the authors on the basis of PCR of Alu sequences.     

Microfluidic DNA amplification—A review

The application of microfluidic devices for DNA amplification has recently been extensively studied. Here, we review the important development of microfluidic polymerase chain reaction (PCR) devices and discuss the underlying physical principles for the optimal design and operation of the device. In particular, we focus on continuous-flow microfluidic PCR on-chip, which can be readily implemented as an integrated function of a micro-total-analysis system. To overcome sample carryover contamination and surface adsorption associated with microfluidic PCR, microdroplet technology has recently been utilized to perform PCR in droplets, which can eliminate the synthesis of short chimeric products, shorten thermal-cycling time, and offers great potential for single DNA molecule and single-cell amplification. The work on chip-based PCR in droplets is highlighted.     

DNA共价结合在化学修饰云母片上的AFM研究

原子力显微镜(AFM)自1986年发明以来,已经成为生物学研究领域中的一个有效工具,尤其在核酸及其它生物大分子结构方面的应用已成为普遍关注的热点.原子力显微镜要求基底达到原子级平整,硅片和玻璃表面的起伏很大,因而原子级平整的云母具有重要的价值.     

Label-free selective DNA detection with high mismatch recognition by PNA beacons and ion exchange HPLC

Two 11mer peptide nucleic acid (PNA) beacons were synthesized and tested for the detection of full-matched or single mismatched DNA. Fluorescent measurements carried out in solution showed only partial discrimination of the mismatched sequence, while using anion-exchange HPLC, in combination with fluorimetric detection, allowed DNA analysis to be performed with high sensitivity and extremely high sequence selectivity. Up to >90 : 1 signal discrimination in the presence of one single mismatched base was observed. The analysis was tested on both short and long DNA oligomers. Detection of DNA obtained from PCR amplification was also performed allowing the selective detection of the target sequence in complex mixtures. Label free detection of the DNA with high sequence selectivity is therefore possible using the present approach.     

Disposable on‐chip microfluidic system for buccal cell lysis,DNA purification,and polymerase chain reaction

This paper reports the development of a disposable, integrated biochip for DNA sample preparation and PCR. The hybrid biochip (25 × 45 mm) is composed of a disposable PDMS layer with a microchannel chamber and reusable glass substrate integrated with a microheater and thermal microsensor. Lysis, purification, and PCR can be performed sequentially on this microfluidic device. Cell lysis is achieved by heat and purification is performed by mechanical filtration. Passive check valves are integrated to enable sample preparation and PCR in a fixed sequence. Reactor temperature is needed to lysis and PCR reaction is controlled within ±1°C by PID controller of LabVIEW software. Buccal epithelial cell lysis, DNA purification, and SY158 gene PCR amplification were successfully performed on this novel chip. Our experiments confirm that the entire process, except the off‐chip gel electrophoresis, requires only approximately 1 h for completion. This disposable microfluidic chip for sample preparation and PCR can be easily united with other technologies to realize a fully integrated DNA chip.     

Molecular-glue-triggered DNA assembly to form a robust and photoresponsive nano-network

A robust and photoresponsive DNA network has been designed and constructed from branched DNA and molecular glue. The molecular glue is photoswitchable and can specifically bind to G-G mismatched double-stranded DNA. The assembly process can be reversibly controlled by manipulating the wavelength of light. The approach is flexible, allowing tuning of the size, morphology as well as the cavity of the network by variation of the molar ratio and the isotropic/anisotropic character of the branched building blocks. The assembled architectures are versatile and heat tolerant. These properties should allow the use of the network in further applications.     

Quantitative DNA methylation analysis by fluorescent polymerase chain reaction single-strand conformation polymorphism using an automated DNA sequencer

A novel DNA methylation assay technique, termed bisulfite single-strand conformation polymorphism (bisulfite-SSCP), is a combination of sodium-bisulfite modification and fluorescence-based polymerase chain reaction (PCR)-SSCP. After bisulfite treatment followed by PCR amplification, methylated and unmethylated alleles can be simultaneously separated in a nondenaturing gel using an automated DNA sequencer. Using bisulfite-SSCP, methylation of hMLH1 was detected in a quantitative manner. This method is not only simple, quick, accurate, and quantitative, but detailed information about methylation is also available with less work. Methylation analysis of large numbers of samples for multiple loci will be facilitated by bisulfite-SSCP.     

Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase

Coupling nucleic acid processing enzymes to nanoscale pores allows controlled movement of individual DNA or RNA strands that is reported as an ionic current/time series. Hundreds of individual enzyme complexes can be examined in single-file order at high bandwidth and spatial resolution. The bacteriophage phi29 DNA polymerase (phi29 DNAP) is an attractive candidate for this technology, due to its remarkable processivity and high affinity for DNA substrates. Here we show that phi29 DNAP-DNA complexes are stable when captured in an electric field across the α-hemolysin nanopore. DNA substrates were activated for replication at the nanopore orifice by exploiting the 3'-5' exonuclease activity of wild-type phi29 DNAP to excise a 3'-H terminal residue, yielding a primer strand 3'-OH. In the presence of deoxynucleoside triphosphates, DNA synthesis was initiated, allowing real-time detection of numerous sequential nucleotide additions that was limited only by DNA template length. Translocation of phi29 DNAP along DNA substrates was observed in real time at ?ngstrom-scale precision as the template strand was drawn through the nanopore lumen during replication.     

DNA packing in chromatine, a manifestation of the Bonnet transformation

The packing of DNA is described using the formalism of differential geometry. Winding of the DNA double helix around the histone 2-5 octamer forming a nucleosome and the condensation of the so-formed bead-on-a-string chromatine aided by histone 1 is interpreted as two consecutive isometric, i.e. Bonnet, transformations. The DNA double helix can be approximated to a helicoid which can be transformed isometrically to a catenoid, an approximation of the nucleosome. Owing to the organization of the histone octamer the extended chromatine takes a helicoidal shape allowing a second Bonnet transformation to consummate the condensation into a chromatine fibre.     

Monitoring viral DNA release with capillary electrophoresis

Viral DNA injection into host cells is one of the primary mechanisms of viral propagation. Drug development that targets viral propagation requires fast and sensitive methods for monitoring the release of viral DNA in vitro. Here we demonstrate the use of capillary electrophoresis (CE) for monitoring DNA release from virus particles. As a model for this study, we used T5 bacteriophages that infect the bacterium Escherichia coli K-12 by binding to the outer membrane FhuA receptor and then injecting DNA. DNA release from the T5 phages in vitro was induced by either elevated temperature or by interaction with the purified FhuA receptor. After DNA release, the viral samples were stained with the high affinity fluorescent dye YOYO-1, injected into the capillary and subjected to electrophoresis. YOYO-1-stained DNA generated a well-defined peak, allowing reliable detection of viral DNA from as few as 10(5) viral particles. The staining to track T5 phage DNA release exemplifies the great versatility that CE offers in studying viral systems. This CE-based method can be used to study molecular mechanisms of viral infections and to evaluate anti-viral drug candidates.     

Structural effect of synthetic zwitterionic cosolutes on the stability of DNA duplexes

The molecular design of useful cosolutes for polymerase chain reaction (PCR), which is one of the most important techniques in molecular biology, plays a significant role in amplification of highly stable genome sequences because during PCR, strand dissociation sometimes fails due to high melting temperature. Here, we designed and synthesized eight new zwitterionic cosolutes derived from glycine betaine, a destabilizing reagent for GC-rich DNA duplexes, and systematically compared their ability to destabilize DNA duplexes and to amplify genome DNA by PCR. We found that introduction of n-butyl groups rather than methyl groups into the ammonium group reduced the melting temperature of DNA duplexes 11-fold more than what was observed for the scaffold cosolute, glycine betaine, and furthermore, the cosolute can amplify the stable genome sequence by PCR.     

Single-stranded DNA (ssDNA) production in DNA aptamer generation

The discovery that synthetic short chain nucleic acids are capable of selective binding to biological targets has made them to be widely used as molecular recognition elements. These nucleic acids, called aptamers, are comprised of two types, DNA and RNA aptamers, where the DNA aptamer is preferred over the latter due to its stability, making it widely used in a number of applications. However, the success of the DNA selection process through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments is very much dependent on its most critical step, which is the conversion of the dsDNA to ssDNA. There is a plethora of methods available in generating ssDNA from the corresponding dsDNA. These include asymmetric PCR, biotin-streptavidin separation, lambda exonuclease digestion and size separation on denaturing-urea PAGE. Herein, different methods of ssDNA generation following the PCR amplification step in SELEX are reviewed.     

DNA sequencing

Determination of the sequence of DNA is one of the most important aspects of modern molecular biology. New sequencing methods currently being developed enable DNA sequence to be determined increasingly faster and more efficiently. One of the major advances in sequencing technology is the development of automated DNA sequencers. These utilize fluorescent rather than radioactive labels. A laser beam excites the fluorescent dyes, the emitted fluorescence is collected by detectors, and the information analyzed by computer. Robotic work stations are being developed to perform template preparation and purification, and the sequencing reactions themselves. Research is currently in progress to develop the technology of mass spectrometry for DNA sequencing. Success in this endeavor would mean that the gel electrophoresis step in DNA sequencing could be eliminated. A major innovation has been the application of polymerase chain reaction (PCR) technology to DNA sequence determination, which has led to the development of linear amplification sequencing (cycle sequencing). This very powerful yet technically simple method of sequencing has many advantages over conventional techniques, and may be used in manual or automated methods. Other recent innovations proposed recently to increase speed and efficiency include multiplex sequencing. This consists of pooling a number of samples and processing them as pools. After electrophoresis, the DNA is transferred to a membrane, and sequence images of the individual samples are obtained by sequential hybridizations with specific labeled oligonucleotides. Multiplex DNA sequencing has been used in conjunction with direct blotting electrophoresis to facilitate transfer of the DNA to a membrane. Chemiluminescent detection can also be used in conjunction with multiplex DNA sequencing to visualize the image on the membrane.     

Quantitative and qualitative profiling of mitochondrial DNA length heteroplasmy

Quantitative and qualitative analysis of mitochondrial DNA length heteroplasmy for the first hypervariable segment (HV1) and second hypervariable segment (HV2) regions were performed using size-based separation of fluorescently-labeled polymerase chain reaction (PCR) products by capillary electrophoresis. In this report, the relative proportions of length heteroplasmies in individuals were determined, and each length variant in the heteroplasmic mtDNA mixture was identified. The study demonstrated that 36% and 69% of Koreans show length heteroplasmy in the HV1 and HV2 regions, respectively. Electropherograms revealed that length heteroplasmy in the HV1 region resulted in over 5 length variants in an individual. The peak patterns of length heteroplasmy in the HV1 region were classified into five major types. In the HV2 region, length heteroplasmy resulted in 3-6 length variants in an individual, and showed seven variant peak patterns. The increased knowledge concerning mtDNA length heteroplasmy is believed to not only offer a useful means of determining genetic identity due to increased mitochondrial DNA haplotype diversity by allowing mtDNAs to be classified into several peak patterns, but also represent a promising tool for the diagnosis of several common diseases which are etiologically or prognostically associated with mtDNA polymorphisms.