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Modern systematics and phylogenetics


Microbial Systematics:
This paper focuses on past, present & future of microbial systematic considering different technical & theoretical advancement in the field. Based on molecular & genomics tools, understanding of microbial ecology has been expanded greatly. New prospects of metagenomes astonish taxonomists greatly in the field of microbial systematic. Molecular biology has changed the concept of species & their classification. Conclusively, the articles urges to microbiology, taxonomists, systematists, molecular biologists and all the researchers for the theoretical renaissance on microbial systematic based on advancement of bioinformatics, metagenomics & molecular genetics & focusing on the important of depositing newly described species in culture collection centre for maintaining updated database.

Prokaryotic systematic in the genomics era:
Microbial systematics is the scientific study of the kinds & diversity of microorganisms & of relationships between them. It is a basic scientific discipline that encompasses classification, nomenclature & identification & includes studies on genetics mechanism which underpin evolutionary process & phylogeny. Classification, Nomenclature, & Identification are basic steps of microbial systematic. There are several taxonomic marker used for the basis of identification. Morphology, growth requirements, pathogenic potential, serological traits, physiological characters are conventional techniques used in identification. Study of different chemotaxonomic apparatus, chemical composition of genomic DNA, and DNA-DNA hybridization are the significant approaches in the microbial systematic.
Genomic information, like whole genome sequencing, 16S rRNA sequencing, and  Gene duplication are recent advances in prokaryotic systematics. Prokaryotes have evolved other mechanism for rapid adaptation to newly environmental niches which may leads to speciation. The horizontal gene transfer, chromosomal rearrangement, genomics plasticity, single nucleotides polymorphism, insertion/deletion, may make organism differ from each other.
Prokaryotic speciation can be understood with genomics information. The phylophenetic species concept which is based on these independent approaches DNA-DNA hybridization, phenotype descriptions, & relationship based on the phylogeny of 16S rRNA gene has been considered to be the most universally applicable in the delineation of prokaryotic species.
As a whole, the genomics era has changed the pattern of prokaryotic systematic. The advancement in the techniques challenges systematists to reanalyze the molecular mechanisms underlying the taxonomic characteristics of prokaryotes by drawing the knowledge from studies of genomics & bioinformatics tools.

Molecular Phylogenetics  :
Molecular phylogenetics is the study in which molecular and statistical techniques are combined to infer evolutionary relationship among organisms or genes. Advances in computer tools help molecular phylogenetics to study more effectively. The primary objective of molecular phylogenetics studies is to recover the order of evolutionary events & represent them in evolutionary trees that graphically depict relationships among species or genes overtime.
Darwin’s explain the evolution based on change in traits and molecular techniques explain evolution as a molecular process based on genetics information. This evolutionary mechanism creates basis for the phylogeny which explains all organisms have descended from a common ancestor & the graphical representation of this phylogeny is termed as phylogenetics tree which consists of edge, node & root. Its branches can be grouped into monophyletic, paraphyletic, & polyphyletic group. All members within the group are derived from a common ancestor & have inherited a set of unique common traits is a monophyletic group. A paraphyletic group excludes some of its descendents & a polyphyletic group can be a collection of distantly related operational taxonomic unit (May not directly descendent from a common ancestor. Phylogenetic trees are defined by homologous relationship which can be either paralogs or orthologs. Paralogs are homologous sequences separated by gene duplication event. Orthologs are homologous sequences separated by speciation event.




Phylogenetic tree can be estimated from molecular data which may include biomolecular sequence alignments of DNA, RNA or amino acids. The basic steps in phylogenetic analysis include: assemble & align dataset, building trees from sequences using computational methods & statistically test & assess the estimated tress. Sequence can be align using different software among which ClustalW is widely used. There are several computational methods for building trees after alignment. It includes: Distance Matrix methods & discrete Data methods such as Maximum Parsimony & maximum likelihood. Distance-matrix methods compute pairwise distances between sequences that approximate evolutionary distance. There are several different distance-matrix methods among which Neighbour-Joining method is common. Discrete data methods examine each column of a multiple sequence alignment dataset separately & search for the tree that best represents all this information. Commonly used discrete data methods include Maximum Parsimony & Maximum Likelihood. There are several online phylogenetic tools. These include PANTER, P-Pod PFan, Treefan & the PhyloFacts. Molecular phylogenetics has much diverse application. It can be used to trace the evolution of man, origin of SARS & even required in biological research papers.

In sum up, molecular phylogenetics is a broad, diverse field with many applications, supported by multiple computational & statistical methods. It has become an integral part of biological research, pharmaceutical drug design & bioinformatics techniques.


2016 May11


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Protocol for Polyamine Analysis in Bacterial Cells:

1.Extraction & Dansylation  (Derivatization):
·         Take approx. 40 mg of lyophilized cells in a tube.
·         Extract (Hydrolysed) with 1 ml of 0.2 M Perchloric Acid (HClO4).
·         Add internal standard 1, 8-diaminooctane (25 µmol/40 mg of cells) (Required Usually for HPLC, for TLC =????)
·         Incubate at 1000C for 30 min. with occasional shaking after 15 min. interval.
·         After extraction the samples were centrifuged, at 8000 rpm for 10 min.
·         Transfer 0.2 ml of supernatant to a tube containing 0.3 ml of Na2CO3solution (100 mg/ml) and 0.8 ml of dansylchloride solution (7.5mg/ml in acetone).
·         The tube was closed tightly & dansylation was performed by incubating the tube for 20 min at 600C (usually in dark condition).
·         Add 0.1 ml of proline solution (50 mg/ml) to bind excessive dansyl chloride. Incubate for 10 min. at 600C.
(Usually the volume can be adjusted as;
Supernatant (Samples) : Na2CO3 : Dansylchloride : Proline = 0.4 ml : 1.2 ml : 3.2 ml : 0.4 ml)
·         Cool in refrigerator (at 4-50C)
·         Add 200-500 µl of toluene and shake or vortex for 30 sec.
·         Centrifuge if required to separate toluene phase from aqueous phase.
Ø  Toluene Extract can be analyzed either by HPLC or TLC.



2.For TLC:

Ø  The solvent cyclohexane/ethylacetate (2:3) (usually 100 ml) must be prepared some hours before chromatography & placed in tank for saturation.
(Chloform/triethylamine , 4:1 v/v may also be use as solvet)
Ø  Take TLC Plate (silica gel 60,20 X 20 cm; Merck Cat. No. 105553).
Ø  Samples & Standards are loaded in the application Zone by using micropipette. Generally 10-40 µl of each toluene extract are applied. Large volume must be loaded by repeated applications of small aliquots with solvent evaporation in between increment.
Ø  After loading the sample, the plates are dried & then placed vertically into the chamber dipping the immersion zone into the developing solvent.
Ø  The chamber is then sealed tightly.
Ø  Plates are developed for 40 min. when used cyclohexane/Ethylacetate solvent & for 1 hr. 15 min. is used chloroform/triethylamine solvents.
Ø  Once developed, the plate is removed from the chamber & dried quickly for 10 min. at 600C .
Ø  Spots corresponding to the different amines are visualized under UV light, and identified by comparison to standards.
Ø  When developed in 2:3 (V/V) cyclohexane/ethylacetate, the migration order of the different polyamines is spermine (firstly separated) followed by spermidine, and finally, putrescine and cadaverine (if present) poorly separated.
Ø  When developed in 4:1 (V/V) chloroform/triethylamine, the migration order of the different polyamines is diaminopropane (firstly separated) followed by putrescine, cadaverine, and finally, spermidine and spermine.
Ø  Once visualized, the spot bounders are marked with pencil.


3.For HPLC analysis:

Ø  After the extraction of the dansylated polyamines, 400 ul of the organic phase (the toluene extract) is removed, dried (e.g., under a stream of ) and re-dissolved in 800ul of acetonitrile (which is compatible with the HPLC column). Finally, the samples are passes through a 0.45 pore size syringe filter before injection in HPLC.

4.Preparation of standard solutions:
Ø    Stock solutions of commercial polyamine standards: diaminopropane, putrescine, cadaverine, spermidine and spermine (in the form of hydrochlorides) are prepared at the concentration of 1 mM in water (or in 0.01 N HCl).
Ø    For the HPLC procedure, a stock solution 1 mM of 1,7- diaminoheptane is also prepared. The unnatural amine 1,7 diaminoheptane is used in HPLC as internal reference compound because it resolves well from derivatives of endogenous amines, elutes near amines of interest, and it is stable under storage conditions (Smith and Davies, 1987).
Ø    Working standard solutions for HPLC (0.05 mM) are prepared by 1:20 dilution of the stock [i.e., 50 1 mM stock + 950 (or 0.01 N HCl)].
Ø    Working standard solutions for TLC will be the 1 mM stock solutions, undiluted.
Ø    Standards are stable for at least 2 months if stored at –20 °C in plastic tubes. Plastic containers (e.g., Eppendorf tubes) must be used for storage because polyamines adsorb to the surface of glass (Smith and Davies, 1987).

Dansylation of standards is carried out in the same way described for samples, but consider:

Ø  Polyamine standards for HPLC are prepared by mixing 40 of each 0.05 mM working solutions (diaminopropane, putrescine, cadaverine, 1,7-diaminoheptane, spermidine and spermine), having a final volume of 240 Moreover, 40 µL 1,7-diaminoheptane 0.05 mM are added to each sample before dansylation, as internal reference, thus having equal experimental volumes for samples and
standards.
Ø  Polyamine standards for TLC are prepared by mixing 20 of each 1 mM stock solutions (diaminopropane, putrescine, cadaverine, spermidine and spermine). Final volume of 200 (equal to sample aliquots) is achieved by adding 100 (or HCl 0.1 N). Alternatively, different concentrations of standards can be prepared (e.g., 25, 20, 15, 10 of each 1mM stock + 75, 100, 125, 150 µL water in order to obtain a standard curve for each amine (i.e.,
concentration vs. fluorescence).
Ø  Dansylation must be performed in the dark since the derivatives are
light sensitive, but dim light in the laboratory is tolerable. Solution of
dansyl chloride in acetone can be stored for 24 h at 4 °C in the dark. After
the dansylation reaction, samples must be kept in the dark or in dark vials.



Fig:HPLC analysis of Putrescine, Spermidine & Spermine
Fig: Polyamine Bands in TLC


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