Preparation of radioiodinated simian virus 40 DNA for use in DNA - DNA reassociation kinetics experiments.

A method is described for the preparation of 125I-labelled SV40 DNA. Using this method, SV40 DNA can be routinely labelled to 15 - 10(6) dpm per mug; much higher specific activities are easily obtained by minor modifications of the method. Once incorporated, the radioactive label dissociates from DNA exceedingly slowly at 4 degrees C or at 68 degrees C. Iodinated SV40 DNA is shown to be useful in the quantitation of viral nucleic acid sequences in SV40-transformed 3T3 cells by DNA - DNA reassociation kinetics.     

Detection of total DNA with single-stranded DNA binding protein conjugates

We have developed a rapid and sensitive method for total DNA measurement using single-stranded DNA binding protein from E coli conjugated with horseradish peroxidase or urease. To detect DNA, the sample is heated or alkali treated to denature the DNA and then filtered through nylon or nitrocellulose membranes. After the single-stranded DNA is bound to the membrane, single-stranded DNA binding protein enzyme-conjugate is incubated with the membrane. Next, the unbound conjugate is washed off the membrane and the bound conjugate detected colorimetrically. The assay can detect 10 pg of DNA in less than 3 hr. This method can be applied to the detection of DNA contamination in therapeutic proteins produced by recombinant DNA or hybridoma techniques.     

DNA contents of replication without DNA density labeling

A new method for determining the timing of DNA replication in specific regions of the mammalian genome without the use of DNA density labeling and DNA density centrifugation is described. The method is based on determination of average relative DNA copy numbers in specific genomic regions as cells progress through S phase, and "time of replication" for a specific region is described in terms of the cell's DNA content when the region is replicated. DNA is isolated from synchronized populations of G1 and S phase cells, it is slot-blotted at the same DNA concentration(s) for each population, and it is hybridized with 32P-labeled DNA probes that are specific to the regions of interest. Quantitation of the slot blot autoradiograms and flow cytometric analysis allows determination of (a) average relative DNA copy numbers for the regions of interest in synchronized cell populations, and (b) the average total DNA content in each population of synchronized cells. This information and the flow cytometry histograms are then used to calculate the cellular DNA content at which each region of interest is replicated. The results have a precision of less than or equal to +/- 10% of S phase for Chinese hamster (line CHO) rhodopsin, metallothionein II, the 5'-end of dihydrofolate reductase, the telomeric repeated sequence, pHuR-093 (also located near the centromeres in CHO chromosomes), and the c-Ki-ras family.     

Chemiluminescent detection of DNA: application for DNA sequencing and hybridization.

A non-radioactive DNA detection chemistry is described and its application is shown for DNA hybridization and standard dideoxy DNA sequencing. The method employes a biotin-streptavidin system which binds an enzyme specifically to a target DNA and upon exposure to substrate, the enzyme catalyzes a chemiluminescent reaction. The image is captured within seconds by a Polaroid or X-ray film. The method is capable of detecting DNA in the hundred attomol range.     

High-resolution mapping of satellite DNA using biotin-labeled DNA probes

We have developed a novel method for high resolution mapping of specific DNA sequences after in situ hybridization. DNA probes, labeled with biotin-nucleotides in conventional nick-translation reactions, are hybridized to cytological preparations and detected with affinity- purified rabbit antibiotin antibodies followed by antibodies to rabbit IgG that are conjugated to fluorescent or enzymatic reagents. Using peroxidase labeled anti-rabbit IgG, we are able to detect and localize specific sequences at both the light and electron microscopic levels. Initial studies were done with repeated DNA sequences previously mapped by light microscope autoradiography to assess the fidelity and resolution of this method. An analysis using biotin-labeled mouse satellite DNA is presented here.     

DNA extraction techniques for DNA barcoding of minute gall-inhabiting wasps

DNA extraction from minute hymenopterans and their larvae is difficult and challenging because of their small size indicating a low amount of starting material. Hence, 11 DNA extraction methods were compared to determine their efficacy in isolating DNA. Success of each method was scored on a 2% agarose gel after PCR of the cox 1 mitochondrial locus. A silica-membrane-based approach was the most successful, followed by a method using a combination of incubation buffers and a method using magnetic beads. The method using buffers was the most cost- and time effective. Using this method, larvae from Eucalyptus seed capsule galls could be assigned a role (parasitoid, gall former or inquiline) in the gall-inhabiting complex.     

Helicase-dependent isothermal DNA amplification

Polymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to separate two DNA strands. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate duplex DNA. We have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling. In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.     

Synthesis of non-natural DNA using DNA polymerase.

Nonnatural nucleotide modified by glucose or galactose was synthesized to increase functional diversity of DNA library. These compounds were incorporated in a DNA double strand using Klenow Fragment as well as dTTP. These functional group could be ordered sequentially on a DNA double strand at intervals of few angstroms according to the designed template sequence within a few hours. This method must be useful to constructing nonnatural DNA library or designed supramolecular structures.     

DNA chip replication for a personalized DNA chip

We report the replication technology of DNA chip using by sequence specific localization of nucleic acids via hybridization and electric transfer of the nucleic acids onto a new substrate without losing their array information. The denatured DNA fragments are first spotted and UV-cross-linked on a nylon membrane. The membrane is then immersed and hybridized in a DNA mixture solution that contains all complementary sequences of the nucleic acids to be hybridized with the DNA fragments on the membrane. The hybridized DNA fragments are transferred to another membrane at the denatured condition. After separating two membranes, the transferred membrane contains a complementary array of DNA fragments. This method can be used for the replication of the same copy of DNA chip repeatedly and moreover could be applied for a personalized DNA chip fabrication, where specific information of each spot of DNA chip is originated from the genetic information of a personal sample.     

Global DNA methylation measurement by HPLC using low amounts of DNA

Epigenetic changes in chromatin structure can influence gene expression without affecting the DNA sequence. The most commonly studied epigenetic modification, DNA methylation, has been implicated in normal tissue development and disease progression, and can be influenced by diet and other environmental factors. Current HPLC methods of determining DNA methylation may require relatively large amounts of DNA (50 μg); as many tissues have low DNA yields, this can be hard to achieve. We isolated DNA from mouse colon and liver in a study investigating post-natal supplementation with selenium and folic acid. After optimizing the methods to account for lower initial DNA amounts, we digested 3 μg of DNA to deoxynucleotide monophosphates, then purified and quantified it. Samples were analyzed by reversed-phase HPLC to determine global DNA methylation levels using commercial nucleotide standards. The HPLC column was cooled to 6(C (reducing run time), and detection was at 280 nm (UV). We showed that methylated cytosine can be accurately and reproducibly measured in as little as 3 μg of DNA using this HPLC analysis method (within-assay CV <2%). We also used this method to detect reduced DNA methylation in liver (P = 0.009) in response to post-natal supplementation with selenium and folate.     

Recovery of DNA from soils and sediments

Experiments were performed to evaluate the effectiveness of two different methodological approaches for recovering DNA from soil and sediment bacterial communities: cell extraction followed by lysis and DNA recovery (cell extraction method) versus direct cell lysis and alkaline extraction to recover DNA (direct lysis method). Efficiency of DNA recovery by each method was determined by spectrophotometric absorbance and using a tritiated thymidine tracer. With both procedures, the use of polyvinylpolypyrrolidone was important for the removal of humic compounds to improve the purity of the recovered DNA; without extensive purification, various restriction enzymes failed to cut added target DNA. Milligram quantities of high-purity DNA were recovered from 100-g samples of both soils and sediments by the direct lysis method, which was a greater than 1-order-of-magnitude-higher yield than by the cell extraction method. The ratio of labeled thymidine to total DNA, however, was higher in the DNA recovered by the cell extraction method. than by the direct lysis method, suggesting that the DNA recovered by the cell extraction method came primarily from active bacterial cells, whereas that recovered by the direct lysis method may have contained DNA from other sources.     

Modeling DNA shuffling.

In vitro evolution is a new, important laboratory method to evolve molecules with desired properties. It has been used in a variety of biological studies and drug development. In this paper, we study one important mutagenesis method used in in vitro evolution experiments called DNA shuffling. We construct a mathematical model for DNA shuffling and study the properties of molecules after DNA shuffling experiments based on this model. The model for DNA shuffling consists of two parts. First we apply the Lander-Waterman model for physical mapping by fingerprinting random clones to model the distribution of regions that can be reassembled through DNA shuffling. Then we present a model for recombination between different DNA species with different mutations. We compare our theoretical results with experimental data. Finally we propose novel applications of the theoretical results to the optimal design of DNA shuffling experiments and to physical mapping using DNA shuffling.     

DNA replication-dependent formation of joint DNA molecules in Physarum polycephalum

Two-dimensional neutral/neutral agarose gel electrophoresis is used extensively to localize replication origins. This method resolves DNA structures containing replication forks. It also detects X-shaped recombination intermediates in meiotic cells, in the form of a typical vertical spike. Intriguingly, such a spike of joint DNA molecules is often detectable in replicating DNA from mitotic cells. Here, we used naturally synchronous DNA samples from Physarum polycephalum to demonstrate that postreplicative, DNA replication-dependent X-shaped DNA molecules are formed between sister chromatids. These molecules have physical properties reminiscent of Holliday junctions. Our results demonstrate frequent interactions between sister chromatids during a normal cell cycle and suggest a novel phase during DNA replication consisting of transient, joint DNA molecules formed on newly replicated DNA.     

一种利用Taq酶快速标记DNA探针的方法

目的：探索低成本、高效、快速的DNA探针标记方法。方法：以特定引物和16nler的随机引物作为延伸引物,利用Taq酶标记DNA探针。以大肠杆菌Klenow片断随机引物延伸标记法作为对照。点杂交方法检测探针标记效果。结果与结论：Taq酶标记法和大肠杆菌Klenow片段随机引物延伸标记法同样有较好的标记效果,且随机引物或特定引物作为延伸引物均可以合成足够有效的探针。Taq酶标记法是一种低成本、高效、快速的DNA探针标记方法。     

Exoquence DNA sequencing.

We have developed a strategy for DNA sequencing based on exonuclease III digestion followed by double strand specific endonuclease digestion and direct dideoxynucleotide sequencing reaction. This strategy eliminates the need for subcloning, oligonucleotide primers, and prior knowledge of the DNA to be sequenced. All template and primer duplexes needed for sequencing a complete insert can be prepared in one day from uncharacterized starting DNA. Sequence information can be obtained from different regions of the DNA simultaneously. The method uses double-stranded DNA to generate single-stranded template and primer, and thus produces high quality sequence results. Commercially available dideoxy-sequencing kits are well suited for this method. The strategy should be applicable for both automatic and routine laboratory DNA sequencing.     

Purification of mitochondrial DNA from total cellular DNA of small tissue samples

A method is presented for the isolation of highly purified mitochondrial (mt)DNA from a crude DNA extract, making use of the different mobilities of covalently closed circular mtDNA vs. endonuclease-digested nuclear DNA in agarose gels. The preparation is virtually free of any contaminating linear DNA, as judged from its electron microscopic appearance, and can be used for further procedures such as polymerase chain reaction (PCR). Since isolation of mitochondria is not a prerequisite for this method, it can be applied to tissue samples in the mg range. In principle, the method can be applied to every eukaryotic species, provided a molecular hybridization probe is available which permits the position of mtDNA to be located in an agarose gel. This probe can be a cDNA, a DNA fragment generated by PCR, or mtDNA itself, if only the approximate size of the genome is known.     

Construction of DNA Hemicatenanes from Two Small Circular DNA Molecules

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

Application of a novel chemiluminescence-based DNA detection method to single-vector and multiplex DNA sequencing

A chemiluminescent DNA detection method is described and its application shown for both single-vector and multiplex DNA sequencing using the standard dideoxy chain-termination process. This recently developed detection method, which utilizes the light emitted by an enzyme-catalyzed dioxetane reaction, is highly sensitive and affords significant advantages in safety and speed over the traditional radioactive labeling method. When adapted to a multiplex strategy, this chemiluminescent detection method constitutes a safe, simple and rapid method for increasing the throughput of DNA sequencing procedures.