This research is focused on the extraction of plant genomis DNA and the order of five commonly used herbal medicines, namely Impatiens Balsamina, Ficus Deltidea, Centella Asiatica, Andrographis Paniculata and Orthosiphon Aristatus. This molecular technique is another reliable alternative to identifying plant species other than phytochemical profiles.
The Three Cetyl Hexadecyltrimethylammonium Bromide (CTAB) method with little modification at the time of incubation, salt content and other additives are used for DNA extraction. The CTAB method of Doyle and Doyle produces a higher concentration of DNA than L Balsamina, most likely because of the existence of ammonium acetate in a washing buffer and longer incubation time (2 hours). CTAB-based methods are suitable for A. Paniculata because the concentration of high DNA with acceptable quality is obtained for all modified methods.
However,. Aristatus is likely to have a lower concentration of DNA (33-87 μg / g) and quality, perhaps because of the high concentration of phenolic compounds, especially rosmarinate acid. DNA genomes extracted effectively reinforced by a chain reaction polymerase using the Primary Universal Spacer Internal Transcript (ITS), specifically AB 101 and AB 102 at optimal annealing temperature of 48 ° C.
DNA sequences are analyzed by phenetic analysis and found that they have high similarities in the order Nucleotides from their territory for similar plant species in the Genbank database of the National Biotechnology Center.
The protocol is fast and cheap for consistent extraction of DNA and high-quality RNA from challenging plant and fungal samples for Genotiping and Snp Throughput sequencing applications.
Modern genotype techniques, such as SNP analysis and genotypes with sequencing (GBS), are hampered by poor quality and purity of DNA, especially in challenging plant species, rich in secondary metabolites. Therefore we investigate the pre-washy step utility using the Sorbitol buffered solution, before the DNA extraction uses high salt CTAB extraction protocols, in high throughput settings or miniprep.
This pre-washing appears to remove disturbing metabolites, such as polyphenols and polysaccharides, from network macerates. We also investigate the ability to adapt the pre-washing sorbitol for RNA extraction using a lithium-based chloride-based protocol. This method is successfully applied to various networks, including leaves, cambiums and fruits from diverse plant species including annual plants, forests and fruit trees, herbarium leaves and lyophilized mushroom mushrooms. We consistently get good DNA or high purity RNA results in all species tested.
The protocol has been validated for thousands of DNA samples by producing high quality data in a solid SNP array. DNA is extracted from Eucalyptus spp. Leaves and cambiums and mycelium from trichoderma spp. Easily digested with restriction enzymes and is carried out consistently in the AFLP test. Enhanced DNA extraction is also suitable for long reading sequencing. Successful RNA quality control and good RNA-SEQ data for Eucalyptus and MEQUEWS confirm the effectiveness of pre-washing buffer sorbitol for high-quality RNA extraction.
Protocol: Method of flexible genomical genomical DNA extraction, cheap, high suitable for genotiping-by-sequencing.
DNA DNA Technology Development Recently, such as genotiping-by-sequencing (GBS), produces thousands of single informative nucleotide polymorphism markers in almost all species, regardless of genome resources. This allows poor resources / species or “orphans” to high density, a high marker platform that has revolutionized the population genetic study and plant breeding. DNA quality supports the success of the GBS method because DNA must be able to accept digestive limits and enzyme sequencing.
A barrier to implement GBS Technologies is access to cheap and high-high extraction methods that produce DNA quality genomes of sequencing (GDNA) of plants. Some DNA-throughput extraction methods are available, but usually produce low results or poor quality, or expensive (US $ 6- $ 9 / samples) for consumables.
We modify non-organic solvent protocols to extract the number of micrograms (1-13 μg) from cheap high-quality molecular weight gdna on a 96-well plate from dry, dry or silica dry plant tissue. The protocol is effective for some forages, plants, horticulture, and general models that are easily extracted including trifolium, Medicago, Loloum, Secale, Festuca, Malus, Oryza, and Arabidopsis.
The extracted DNA is high molecular weight and digested easily with restriction enzymes. Contrast with other extraction protocols we wear, the order of Library GBS Illumina developed from GDNA has a high-quality base call that is very uniform to the end of the order. Furthermore, DNA is extracted using this method has been successfully sorted by a platform on-read pacbio. Protocol can be scaled, easy automatic without requirements for Fume Hoods, requires around three hours to process 192 samples (384-576 samples / day), and are not expensive at US $ 0.62 / samples for consumables.
This versatile, scalable and simple protocol produces a high molecular weight DNA that is suitable for digestion of the restriction enzyme and the next generation sequence application including GBS and a long reading sequencing platform such as Pacbio. Low costs, high throughput, and high-quality GDNA extraction of various fresh and dry plant materials make this method suitable for many sequencing and genotypes including large-scale sample screening that underlies breeding programs.
The protocol is optimized for DNA extraction in plants with a high content of secondary metabolites, based on Mimosa Tenuiflora (Will.) Poir. (Leguminosae).
Some species are characterized by tannins, alkaloids, and phenols that are high in the leaves. This secondary metabolite was released during DNA extraction and might hamper molecular studies based on PCR (polymerase chain reaction). To provide an efficient method to extract DNA, Mimosa Tenuiflora, an important pollery plant from the Brazilian semi-dry area used in popular treatment and as a source of firewood or forage, used. Eight previously reported procedures for plants tested and adapted from the M. Tenuiflora leaf tissue stored at -20 ° C.
The procedure that was optimized in this study included the use of phenol during deproteinization, increased concentration of cetyltrimethylammonium bromide and sodium chloride, and a shorter period and a shorter period lower incubation temperature about other methods.
The extracted DNA does not present degradation, and amplification through PCR managed to use the primary ISSR, Trnl, ITS, and ETS. In addition to M. tenuiflora, this procedure is also tested and proven efficient in genetic studies of other plant species.