DNA isolation

The cacti family is a morphologically heterogeneous group comprising 100 genera and about 1500 species (Hernandez and Barcenas, 1996). With the exception of one genus, all members of this family are native to America (Hernandez and Barcenas, 1996). There are three subfamilies, Opuntioideae, Cactoideae, and Pereskioideae (Gibson and Nobel, 1986). DNA isolation from cacti is notoriously difficult because they contain high amounts of polysaccharides and secondary metabolites which form insoluble complexes with nucleic acids during extraction (Guillemaut and Marechal-Drouard, 1992). Like in other groups of plants, the secondary metabolites and polysaccharides in cacti inhibit enzyme action (Porebski et al., 1997). The polysaccharides are visually evident by their viscous, glue-like texture and they make the DNA unmanageable when pipeting and hard to amplify by the polymerase chain reaction (PCR) (Poresbski et al., 1997). We report an easy and inexpensive protocol to isolate DNA from cacti. We used this method to isolate DNA from 85 species (170 individuals) of 39 genera of the subfamilies Pereskioideae, Opuntioidea, and Cactoideae. This procedure is a modification of a protocol described by De la Cruz et al. (1995) for the Cacti family. It requires only a few grams of tissue and does not require destruction of the whole plant to produce high molecular weight genomic DNA. The DNA from this procedure can be amplified consistently by PCR and used for RAPD analysis.

Broad-range amplification of bacterial DNA from clinical specimens has proved useful for the diagnosis of various bacterial infections, especially during antimicrobial treatment of the patient. Optimal sample processing protocols for diagnostic broad-range bacterial PCR should release DNA from an array of target organisms with equal efficiencies and wash out inhibitory factors from various sample types without introducing bacterial DNA contamination to the amplification reaction. In the present study, two physical cell wall disintegration methods, bead beating and sonication, for enhanced detection of organisms with difficult-to-lyse cell walls were studied. The analytical sensitivities of several commercially available DNA purification kits, which were used with and without additional cell disintegration steps, were compared by using dilution series of model bacteria. Selected purification methods were used to process routine clinical specimens in parallel with the standard phenol-...

A thorough understanding of the mechanisms of R-, C-and G-banding will come only from studies of the binding of Giemsa dyes to isolated and characterized preparations of heterochromatin and euchromatin. Since such studies require an exact knowledge of the optical characteristics of Giemsa, the spectral adsorption curves and extinction coefficients of Giemsa and its component dyes at various concentrations in the presence and absence of DNA were determined. — Although Giemsa is a complex mixture of thiazin dyes plus eosin; methylene blue, and azure A, B or C alone gave good banding. Thionin, with no methyl groups, gave poor or no banding. Eosin was not a necessary component for banding. — The most striking characteristic of the thiazin dyes is that they are strongly metachromatic, i.e., their adsorption spectra and extinction coefficients change as the concentration of the dye increases or as they bind to positively charged compounds (chromotropes). These changes, especially for methylene blue, are described in detail and allow a distinction between concentration dependent binding to DNA by intercalation and binding by side stacking.

The major objective of our cucumber breeding program involves breeding for CMV disease resistance to reduce losses in quality and yield. Seven half diallel cross hybrids resistant to CMV were developed at the Vegetables Breeding Dept., Hort. Res. Inst., Egypt during March, 2006. Sixteen genetically different pure inbred lines of Cucumis sativus were used to develop the hybrids obtained from different sources and selected for their CMV disease-resistance trait. The inbred lines and one commercial (Beit alpha) cultivar as a ...

Molecular genetic markers have numerous potential applications in environmental forensics if DNA can be isolated from 'difficult' non-human biological material such as hairs, feathers, or wood. The identification of the origin of wood is particularly important in order to identify illegally harvested and traded timber and wood products. We describe success rates of DNA isolation from wood based on a simple, previously published extraction protocol. The protocol was used to isolate DNA from a total of 406 wood samples, mainly of the important tropical tree family Dipterocarpaceae. The reliability of the extraction method was confirmed by comparing fragment sizes and sequences after isolation of DNA from leaves and wood of the same trees. We observed the success of amplification of chloroplast DNA (cpDNA) fragments of different lengths by means of PCR, investigated key factors influencing PCR, and conducted inhibitor tests for a subset of the samples. The average rate of succe...

Application of DNA fingerprinting methods enables the detection of diverse members of soil bacterial consortia, even including those bacteria not yet cultivated. However, extraction and purification of DNA from soil samples without bias is difficult. We compared five different DNA isolation methods and three purification methods for rhizosphere soil samples. Purified DNA extracts were amplified in PCR using universal bacterial primers and the PCR products were analysed with denaturing gradient gel electrophoresis (DGGE) for the visualisation of DNA bands representing dominant bacterial species. Both the isolation and purification methods affected the apparent bacterial community structure of the samples.

The tissue is homogenised to break open the cells and the nuclear pellet is isolated by differential centrifugation. One treatment with high salt concentration, the DNA solubilise and dissociates from proteins on addition of cold 95% ethanol, DNA precipitates out like their long threads which can be spooled on a glass rod.

Erythrina crista-galli L. (Fabaceae), known in Rio Grande do Sul by corticeira-do-banhado, is a natural tree of Brazil, Uruguay, Paraguay, eastern Bolivia and Argentina. It is found in marshy or swampy lowlands, very frequent in formations of galleries forests and is cultivated for ornamental purposes and restoration of natural environments. This species undergoes significant insect damage in their seeds and unaware efficient methodologies for isolating genomic DNA applied to the species. The work was developed in the region of the state campaign, involving the city of Bagé, Aceguá, Dom Pedrito and Hulha Negra, RS. Emergence tests between sand grains obtained from different types of fruit were performed to assess the potential of these types of emergency, the damage level and its effect on the emergence of seeds. The work also tested two protocols for DNA isolation and three sources of material (frozen fresh leaves, dried leaves and vascular cambium frozen) and the RAPD technique was used to evaluate the protocol tested using five primers. Test results showed that seeds from all kinds of fruits can emerge even with marked differences in moisture content or with different levels of insect damage. Young plants from seeds damaged by insects, however, have less vigor and size during the study period. Success was obtained in isolating DNA from dried leaves, however protocols were not very effective in removing samples of polysaccharides work. The volumes of samples of polysaccharides present in higher levels in relation to the standard curve of the spectrophotometer, seem to have influenced the amplification of DNA fragments with molecular markers, which requires optimization of the protocol tested in order to improve the quality of the isolated DNA.

In the last 20 years, the applications of genomics tools have completely transformed the field of microbial research. This has primarily happened due to revolution in sequencing technologies that have become available today. This review therefore, first describes the discoveries, upgradation and automation of sequencing techniques in a chronological order, followed by a brief discussion on microbial genomics. Some of the recently sequenced bacterial genomes are described to explain how complete genome data is now being used to derive interesting findings. Apart from the genomics of individual microbes, the study of unculturable microbiota from different environments is increasingly gaining importance. The second section is thus dedicated to the concept of metagenomics describing environmental DNA isolation, metagenomic library construction and screening methods to look for novel and potentially important genes, enzymes and biomolecules. It also deals with the pioneering studies in the area of metagenomics that are offering new insights into the previously unappreciated microbial world.

Several DNA isolation techniques
have been developed and some ready-to-use
kits are now widely commercialized. To test
the reliability of these products, two standard
methods and two kit-extractions have been
performed on a total number of ten rabbit
blood samples.
The DNA obtained revealed significant
difference between the methods in
concentration, but not in terms of DNA purity.
The highest DNA quantity was obtained by the
method saline (305μg) and the lowest with the
kit Invitrogen ® (7.33μg). The quality of the
DNA, characterized by the A260/A280 ratio of
1.8 is good for all samples, except for
extractions with saline extraction method,
which gives a lower value (1.57) than all the
others.
A comparative study concerning the extraction
costs reveals that the used kits are up to 5
times more expensive than the standard
methods.
Other parameters such as extraction time, the
initial volume of blood required may explain
the use of a method or the other. These are the
specific needs of the experimenter who decide
on the method of DNA isolation used.

Background: Application of PCR techniques requiring only minute amounts of Genomic DNA. Thus, a less invasive, simpler to perform, and cheaper method to obtain DNA from exfoliated cells is desirable. We aim to develop a method that can obtain high quality of genomic DNA from one sample that allows for numerous application of PCR analysis. Objective: This study describes a simple, inexpensive and non invasive protocol to isolate a high quality of genomic DNA from exfoliated cells by using swish method. Methodology: Twenty two subjects vigorously swished 10 ml of normal saline in their mouth for 60 s and spitted into a collection tube. DNA extraction assay was performed by using saliva DNA isolation kit (Norgen, USA). The washed pellets were suspended in TE buffer and analyzed for the quality and purity of DNA content by using the NanoDrop Spectrophotometer. A ratio of A260/A280 was calculated. The extracted genomic DNA was amplified with primers of p53 intron 6 by using PCR machine. The presence of amplified DNA was then confirmed by electrophoreses analysis, which DNA bands were scanned by Typhoon 9410 variable imager. Results: In this study, the extracted genomic DNA demonstrated an average value of 1.94 O.D. in DNA content purity and 42.9 \ensuremathμg/\ensuremathμl in DNA yields. The electrophoresis images of the DNA products showed visible and detectable bands of higher molecular weight DNA in all the samples. Conclusion: The results showed that the extracted genomic DNA from the exfoliated cells by applying the swish method, that provides substantially larger amounts and higher molecular weight of DNA for down-stream DNA identification application. In addition, all samples were successfully genotyped by PCR-based assays for p53 gene intron 6 regions, which confirmed that the quality of isolated DNA was reliable in supporting the PCR amplification for the molecular studies.

DNA markers are being increasingly used in studies related to population genetics and conservation biology of endangered species. DNA isolation for such studies requires a source of biological material that is easy to collect, non-bulky and reliable. Further, the sampling strategies based on non-invasive procedures are desirable, especially for the endangered fish species. In view of above, a rapid DNA extraction method from fish scales has been developed with the use of a modified lysis buffer that require about 2 hr duration. This methodology is non-invasive, less expensive and reproducible with high efficiency of DNA recovery. The DNA extracted by this technique, have been found suitable for performing restriction enzyme digestion and PCR amplification. Therefore, the present DNA extraction procedure can be used as an alternative technique in population genetic studies pertaining to endangered fish species. The technique was also found equally effective for DNA isolation from fre...

This review revealed a new mechanism for gene regulation through
“gene silencing” at the transcriptional level (TGS) or at the post -transcriptional level (PTGS), which play a key role in many essential
cellular processes. Today dsRNA is used as a powerful tool to
experimentally elucidate the function of essentially any gene in a
cell. The immense impact of the discovery of RNA interference
(RNAi) on biomedical research and its novel medical applications in
the future are reviewed in this article, with particular stress on
therapeutic applications of radio -labeled antisense oligonucleotides
(RASONs) for diagnosis and treatment of various  cancers and
neurodegenerative diseases by “gene silencing”. Antisense
oligonucleotides (ASONs) can also modulate alternative splicing
which 74% of all human genes undergo.  Epigenetic changes affect
chromatin structure and thus regulate processes such as transcription,
X-chromosome inactivation, allele-specific expression of imprinted
genes, and inactivation of tumor suppressor genes. Treatment with
inhibitors of DNA methylation and histone deacetylation can
reactivate epigenetically silenced genes and has been shown to
restore normal gene function. In cancer cells, this results in
expression of tumor suppressor genes and other regulatory functions,
inducing growth arrest and apoptosis.

Optimization of DNA isolation protocol for genetic
characterization of any medicinal plant is the necessary and
primary step. Plant leaves of medicinal plants are rich in
secondary metabolites, polysaccharides and polyphenols that are
problematic during isolation of genomic DNA. Besides,
succulent plants freezed under liquid nitrogen turn to stonelike
tissues that are difficult to grind. Flowers were not considered as
a source of genomic DNA as the case of young leaves, this might
be due to the seasonal nature and short half-life feature. Several
protocols described for plant DNA isolation from Aloe
barbadensis Miller using leaf, found difficult and tedious. This
plant contains exceptionally high amount of secondary
metabolites that interfere with DNA isolation. To address this
problem, young leaf gel was used for DNA isolation that gave
good quality DNA suitable for molecular biological applications.
Cell lyses was carried out in extraction buffer supplemented with
cetyltrimethylammoniumbromide and sodium chloride. Inclusion
of high concentration of CTAB and PVP removed polyphenols.
Chloroform Isoamyl washes followed by precipitation with NaCl
efficiently removed high protein and polysaccharide
contamination. The yield of pure DNA ranges from 25-50μg per
gram of leaf tissues. The RAPD analysis from the isolated DNA
was optimized to produce clear amplification in all the
germplasm tested.