The benefits of Microbiology

Sunday, June 27, 2010

Use of bacteria as probiotics and pre-biotics

Probiotics' refers to useful, live bacteria consumed in small amounts for their potential benefits on health. Pre-biotics refer to certain substances which are consumed in order to promote the growth of commensal bacteria in the body with the aim of obtaining increased benefits on human health. Commensal bacteria or friendly bacteria present in out intestines can help in the process of digestion. In the United States, the probiotic bacteria are available in form of conventional dairy foods supplemented with these bacteria or as dietary supplements in the form of capsules, powder or tablets .A special branch of microbiology deals with study and manufacture of such pro-biotic and pre-biotic bacteria.

Vitamin synthesis

Bacteria like E.coli present in human colon are involved in synthesis of vitamins like vitamin B12, folic acid, biotin and K, which may be used by the host. Such bacteria are often used for commercial preparation of vitamins like riboflavin.

Use of bacteria in dairy and food industry

Bacteria, especially the lactic acid producing lactobacillus are specially used in
preparation of food stuffs involving the process of fermentation e.g. yogurts, cheese, breads, fermented soy sauces, pickles, soy sauces etc.

Use of Microbiology in Agriculture and farming
Symbiotically associated bacteria are able to biologically convert nitrogen gas present in the atmosphere into ammonia which helps in enriching the soil and promotes optimal growth of plants. Such bacteria are useful in the areas of agriculture and farming and increasing crop yield without use of chemical fertilizers. Bacteria are also important for production of compost which is decayed ganic matter and serves as a rich source of nutrition for the plants.
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DNA sequencing and genomics

Thursday, January 21, 2010

One of the most fundamental technologies developed to study genetics, DNA sequencing allows researchers to determine the sequence of nucleotides in DNA fragments. Developed in 1977 by Frederick Sanger and coworkers, chain-termination sequencing is now routinely used to sequence DNA fragments. With this technology, researchers have been able to study the molecular sequences associated with many human diseases.

As sequencing has become less expensive, researchers have sequenced the genomes of many organisms, using computational tools to stitch together the sequences of many different fragments (a process called genome assembly). These technologies were used to sequence the human genome, leading to the completion of the Human Genome Project in 2003. New high-throughput sequencing technologies are dramatically lowering the cost of DNA sequencing, with many researchers hoping to bring the cost of resequencing a human genome down to a thousand dollars.

The large amount of sequence data available has created the field of genomics, research that uses computational tools to search for and analyze patterns in the full genomes of organisms. Genomics can also be considered a subfield of bioinformatics, which uses computational approaches to analyze large sets of biological data.
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Molecular models of DNA

Molecular models of DNA structures are representations of the molecular geometry and topology of Deoxyribonucleic acid (DNA) molecules using one of several means, such as: closely packed spheres (CPK models) made of plastic, metal wires for 'skeletal models', graphic computations and animations by computers, artistic rendering, and so on, with the aim of simplifying and presenting the essential, physical and chemical, properties of DNA molecular structures either in vivo or in vitro. Computer molecular models also allow animations and molecular dynamics simulations that are very important for understanding how DNA functions in vivo.
Thus, an old standing dynamic problem is how DNA "self-replication" takes place in living cells that should involve transient uncoiling of supercoiled DNA fibers. Although DNA consists of relatively rigid, very large elongated biopolymer molecules called "fibers" or chains (that are made of repeating nucleotide units of four basic types, attached to deoxyribose and phosphate groups), its molecular structure in vivo undergoes dynamic configuration changes that involve dynamically attached water molecules and ions. Supercoiling, packing with histones in chromosome structures, and other such supramolecular aspects also involve in vivo DNA topology which is even more complex than DNA molecular geometry, thus turning molecular modeling of DNA into an especially challenging problem for both molecular biologists and biotechnologists.
Like other large molecules and biopolymers, DNA often exists in multiple stable geometries (that is, it exhibits conformational isomerism) and configurational, quantum states which are close to each other in energy on the potential energy surface of the DNA molecule. Such geometries can also be computed, at least in principle, by employing ab initio quantum chemistry methods that have high accuracy for small molecules. Such quantum geometries define an important class of ab initio molecular models of DNA whose exploration has barely started.
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DNA Double Helix

DNA is a normally double stranded macromolecule. Two polynucleotide chains, held together by weak thermodynamic forces, form a DNA molecule.

Features of the DNA Double Helix

* Two DNA strands form a helical spiral, winding around a helix axis in a right-handed spiral
* The two polynucleotide chains run in opposite directions
* The sugar-phosphate backbones of the two DNA strands wind around the helix axis like the railing of a sprial staircase
* The bases of the individual nucleotides are on the inside of the helix, stacked on top of each other like the steps of a spiral staircase.

DNA Helix Axis

The helix axis is most apparent from a view directly down the axis. The sugar-phosphate backbone is on the outside of the helix where the polar phosphate groups (red and yellow atoms) can interact with the polar environment. The nitrogen (blue atoms) containing bases are inside, stacking perpendicular to the helix axis.

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Components of DNA

DNA is a polymer. The monomer units of DNA are nucleotides, and the polymer is known as a "polynucleotide." Each nucleotide consists of a 5-carbon sugar (deoxyribose), a nitrogen containing base attached to the sugar, and a phosphate group. There are four different types of nucleotides found in DNA, differing only in the nitrogenous base. The four nucleotides are given one letter abbreviations as shorthand for the four bases.
• A is for adenine
• G is for guanine
• C is for cytosine
• T is for thymine

DNA Backbone
The DNA backbone is a polymer with an alternating sugar-phosphate sequence. The deoxyribose sugars are joined at both the 3'-hydroxyl and 5'-hydroxyl groups to phosphate groups in ester links, also known as "phosphodiester" bonds.
DNA Double Helix

DNA is a normally double stranded macromolecule. Two polynucleotide chains, held together by weak thermodynamic forces, form a DNA molecule.
Features of the DNA Double Helix
• Two DNA strands form a helical spiral, winding around a helix axis in a right-handed spiral
• The two polynucleotide chains run in opposite directions
• The sugar-phosphate backbones of the two DNA strands wind around the helix axis like the railing of a sprial staircase
• The bases of the individual nucleotides are on the inside of the helix, stacked on top of each other like the steps of a spiral staircase.
READ MORE - Components of DNA

Mesothelioma and Early Lung Cancer Identified by Screening

Exposure to asbestos fibers is a known risk factor for lung cancer and the cause of mesothelioma. Although asbestos is still not completely banned in the U.S., it was phased out of American industry to a large degree beginning in the 1970s. However because asbestos-related diseases can take 20 to 40 years to emerge after people have been exposed, former asbestos workers and those exposed to products containing this carcinogen continue to be diagnosed with asbestos caused cancers.

As researchers search for better treatments and even a cure for these diseases, they are also focusing on new diagnostic methods that might identify the cancers earlier. Early diagnosis is particularly crucial with mesothelioma, because many patients survive only one year after they first start to show signs, and symptoms are often difficult to distinguish from those of other lung diseases.

One potential screening method uses low-dose computed tomography (LDCT) to evaluate the lungs and their lining (pleura). LDCT can locate plaques in the lungs, which are a sign of asbestos exposure and have been linked to an increased cancer risk.

Currently, there are no recommendations about using LDCT or any other method to screen people who have been exposed to asbestos, and screening isn’t routinely done. “There are currently no methods for the early detection of mesothelioma available,” says lead author Heidi Roberts, MD, Associate Professor of Radiology at the University of Toronto. “This is why we are doing the research.”

To determine the effectiveness of LDCT as a screening tool for asbestos-related lung cancers, Dr. Roberts and her colleagues recruited 516 people (most of them men) who had been exposed to asbestos at least 20 years before, or who had known plaques. Participants were given LDCT scans of the chest. Patients who had abnormal scans were given follow-up tests. Those with normal test results were invited to have an annual LDCT scan.

Of the 516 participants, 357 had evidence of plaques. Based on the results of the first scan and annual scans, six of the patients were diagnosed with lung cancers and four were diagnosed with mesothelioma.

Although LDCT was able to detect advanced mesothelioma, as well as early- and late-stage lung cancers, it was not able to diagnose early mesothelioma. The study authors say they need to continue screening patients to help them get a better idea of what early mesothelioma looks like. Also, they say adding biomarkers (substances in the blood that indicate the presence of cancer) to the screening process may provide greater sensitivity to help diagnose those at very high risk for mesothelioma.

Even as techniques are fine-tuned, screening is just one step of a three-tiered effort to combat these cancers, according to Dr. Roberts. “The second step is the parallel development of biomarkers, and the third step is the parallel development of treatment strategies,” she says. “These have to be developed hand-in-hand in order to make this a useful and meaningful tool.”

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Mesothelium

The mesothelium is a membrane that forms the lining of several body cavities: the pleura (thoracal cavity), peritoneum (abdominal cavity including the mesentery) and pericardium (heart sac). Mesothelial tissue also surrounds the male internal reproductive organs (the tunica vaginalis testis) and covers the internal reproductive organs of women (the tunica serosa uteri). Mesothelium that covers the internal organs is called visceral mesothelium, while the layer that covers the body walls is called the parietal mesothelium.

The mesothelium is composed of an extensive monolayer of specialized cells (mesothelial cells) that line the body's serous cavities and internal organs. The main purpose of these cells is to produce a lubricating fluid that is released between layers, providing a slippery, non-adhesive and protective surface to facilitate intracoelomic movement.

The mesothelium is also implicated in the transport and movement of fluid and particulate matter across the serosal cavities, leukocyte migration in response to inflammatory mediators, synthesis of pro-inflammatory cytokines, growth factors and extracellular matrix proteins to aid in serosal repair, and the release of factors to promote the disposition and clearance of fibrin (such as plasminogen). It is an antigen presenting cell. Furthermore, the secretion of glycosaminoglycans and lubricants may protect the body against infection and tumor dissemination.

Role in disease
• Mesothelioma: (cancer of the mesothelium) is a disease in which cells of the mesothelium become abnormal and divide without control or order. They can invade and damage nearby tissues and organs. Cancer cells can also metastasize (spread) from their original site to other parts of the body. Most cases of mesothelioma begin in the pleura or peritoneum. More than 90% of mesothelioma cases are linked to asbestos exposure.

• Intra-abdominal adhesions: Normally, the mesothelium secretes plasminogen, which removes fibrin deposits. During surgical procedures, the mesothelium may be damaged. Its fibrinolytic capacity becomes insufficient and fibrin accumulates, causing fibrous adhesions between opposing surfaces. These adhesions cause intestinal obstruction and female infertility if it occurs in the abdomen, and may impair cardiac and lung function in the thorax.

• Ultrafiltration failure: The peritoneal mesothelium is implicated in the long-term development of ultrafiltration failure in peritoneal dialysis patients. The presence of supra-physiological glucose concentrations, acidity, and glucose degradation products in peritoneal dialysis fluids contribute to the fibrosis of the peritoneal mesothelium, either by epithelial-mesenchymal transition or increased proliferation of existing fibroblasts. A fibrosed peritoneum results in the increased passage of solutes across the peritoneum and ultrafiltration failure.
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Mesothelioma

Mesothelioma is a form of cancer that is almost always caused by exposure to asbestos. In this disease, malignant cells develop in the mesothelium, a protective lining that covers most of the body's internal organs. Its most common site is the pleura (outer lining of the lungs and internal chest wall), but it may also occur in the peritoneum (the lining of the abdominal cavity), the heart, the pericardium (a sac that surrounds the heart) or tunica vaginalis.

Most people who develop mesothelioma have worked on jobs where they inhaled asbestos particles, or they have been exposed to asbestos dust and fiber in other ways. Washing the clothes of a family member who worked with asbestos can also put a person at risk for developing mesothelioma. Unlike lung cancer, there is no association between mesothelioma and smoking, but smoking greatly increases risk of other asbestos-induced cancer. Compensation via asbestos funds or lawsuits is an important issue in mesothelioma (see asbestos and the law).

The symptoms of mesothelioma include shortness of breath due to pleural effusion (fluid between the lung and the chest wall) or chest wall pain, and general symptoms such as weight loss. The diagnosis may be suspected with chest X-ray and CT scan, and is confirmed with a biopsy (tissue sample) and microscopic examination. A thoracoscopy (inserting a tube with a camera into the chest) can be used to take biopsies. It allows the introduction of substances such as talc to obliterate the pleural space (called pleurodesis), which prevents more fluid from accumulating and pressing on the lung. Despite treatment with chemotherapy, radiation therapy or sometimes surgery, the disease carries a poor prognosis. Research about screening tests for the early detection of mesothelioma is ongoing.

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DNA Extraction From Fresh Bone

Extract nucleic acids, such as DNA, from bone samples in order to analyze gene expressions, to look for somatic mutations of tumors or other pathological tissue, or for genotyping archive material when other sources of DNA are not available. You can use several kits that have already provided by biotech companies. But, if you are extracting DNA from large number samples, you can use a homemade method as described here to be effective in cost.


There are four procedures that ascertain the successful extraction of nucleic acids from tissue:
1. disrupting the tissue so that extraction reagents can reach the cells.

2. disrupting the cell membranes so that nucleic acids are liberated.

3. separation of the nucleic acid from other cellular components.

4. precipitation and solubilization of the nucleic acid.

Materials
1. DNA extraction buffer: Add 17.6 mL of 0.75 M sodium citrate, pH 7.0, 26.4 mL of 10% sodium lauryl sarkosyl, and 250 g of guanidinium isothiocyanate to 293 mL of distilled water and mix well. Add 7.2 microliter of beta-mercaptoethanol/mL of lysis buffer on the day of use
All chemicals should be of molecular biology grade. The solutions can be stored at 4oC for up to 3 months.

2. 0.5 M ETDA: Add 93.05 g of EDTA to 300 mL of distilled water and add 10 N NaOH to pH 8.0. Make up to 500 mL. Autoclave.

Tris–EDTA: Add 1 mL of 1 M Tris to 200 microliters of 0.5 M EDTA. Make up to 100 mL with distilled water.

3. 3 M Sodium acetate, pH 5.2: Add 401.8 g of sodium acetate to 800 mL of distilled water. Adjust pH to 5.1 with glacial acetic acid. Make up to 1 L with distilled water. Autoclave.

4. General Reagents: Tris-saturated phenol pH 7.8–8.0 (Sigma), Chloroform, Isopropanol 100%, Ethanol.

Methods
1. Collect the bone sample in a sterile container containing phosphate-buffered saline (PBS) and transport to the laboratory within 1–2 h.
If the DNA extraction is not initiated immediately, freeze the sample at –20oC or below for later use.

2. Place the bone tissue in a clean glass Petri dish. Using bone cutters or a strong sharp pair of scissors, isolate a piece of bone measuring about 1 cm3 and transfer to a clean 5-mL bijoux container.

3. Add 1 mL of DNA extraction buffer and homogenize the tissue with the scissors until a slurry solution is obtained.

4. Transfer 500 microliters aliquots of slurry into screw-capped conical-bottomed 1.5-mL Eppendorf tubes.

5. Add one volume of Tris-saturated phenol, followed by one volume of chloroform per tube. Mix well by inverting the tubes a few times or by shaking. Do not vortex, because vortex-mixing causes long strands of DNA to shear.

6. Centrifuge the tubes at 10,000 g for 20 min to separate the phases.

7. Transfer the upper layer to a fresh centrifuge tube (taking note of the volume), being careful not to disturb the milky layer at the interface. Repeat steps 5–7 if the interface is disturbed.

8. Add one volume of ice-cold isopropanol and 0.1 volumes of 3 M sodium acetate to the supernatant. Mix well and allow to stand for 15 min on ice.

9. Centrifuge the tubes at 10,000g for 20 min to pellet the DNA.
Orientate the Eppendorf tube so that you can identify where the DNA pellet lies. A pellet should be visible the bottom of the tube.

10. Aspirate and discard the supernatant, taking care not to disturb the pellet. Wash the sample with 1.75 mL of ice-cold ethanol and centrifuge at 10,000g for 5 min. Aspirate and discard the supernatant and then repeat the wash.

11. Dissolve the DNA pellets in 10–50 microliters of water or Tris–EDTA buffer (you can pool DNA from the same sample at this stage) and quantitate by spectrophotometry or with Hoechst 33258.
Hoechst 33258 is a DNA-specific dye that can be used to quantitate DNA.

12. Store the sample frozen at –20oC or below.
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Benefits of Ayurvedic Medicines

• By using ayurvedic and herbal medicines you ensure physical and mental health without side effects. The natural ingredients of herbs help bring “arogya” to human body and mind. ("Arogya" means free from diseases). The chemicals used in preparing allopathy medicines have impact on mind as well. One should have allopathy medicine only when it is very necessary.

• According to the original texts, the goal of Ayurveda is prevention as well as promotion of the body’s own capacity for maintenance and balance.

• Ayurvedic treatment is non-invasive and non-toxic, so it can be used safely as an alternative therapy or alongside conventional therapies.

• Ayurvedic physicians claim that their methods can also help stress-related, metabolic, and chronic conditions.

• Ayurveda has been used to treat acne, allergies, asthma, anxiety, arthritis, chronic fatigue syndrome, colds, colitis, constipation, depression, diabetes, flu, heart disease, hypertension, immune problems, inflammation, insomnia, nervous disorders, obesity, skin problems, and ulcers.

Ayurvedic Terms Explained
Dosha: In Ayurvedic philosophy, the five elements combine in pairs to form three dynamic forces or interactions called doshas. It is also known as the governing principles as every living things in nature is characterized by the dosha.
Ayurvedic Facial: Purportedly, a "therapeutic skin care experience" that involves the use of "dosha-specific" products and a facial massage focusing on "marma points."

Ayurvedic Nutrition (Ayurvedic Diet): Nutritional phase of Ayurveda. It involves eating according to (a) one's "body type" and (b) the "season." The alleged activity of the doshas--three "bodily humors," "dynamic forces," or "spirits that possess"--determines one's "body type." In Ayurveda, "body types" number seven, eight, or ten, and "seasons" traditionally number six. Each two-month season corresponds to a dosha; for example, the two seasons that correspond to the dosha named "Pitta" (see "Raktamoksha") constitute the period of mid-March through mid-July. But some proponents enumerate three seasons: summer (when pitta predominates), autumn, and winter (the season of kapha); or Vata season (fall and winter), Kapha season (spring), and Pitta season (summer). According to Ayurvedic theory, one should lessen one's intake of foods that increase ("aggravate") the ascendant dosha.
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Serial analysis of gene expression

Serial analysis of gene expression (SAGE) is a technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those transcripts. The original technique was developed by Dr. Victor Velculescu at the Oncology Center of Johns Hopkins University and published in 1995. Several variants have been developed since, most notably a more robust version, LongSAGE, RL-SAGEand the most recent SuperSAGE that enables very precise annotation of existing genes and discovery of new genes within genomes because of an increased tag-length of 25–27 bp.

Overview
SAGE experiments proceed as follows:
1. Isolate the mRNA of an input sample (e.g. a tumour).
2. Extract a small chunk of sequence from a defined position of each mRNA molecule.
3. Link these small pieces of sequence together to form a long chain (or concatemer).
4. Clone these chains into a vector which can be taken up by bacteria.
5. Sequence these chains using modern high-throughput DNA sequencers.
6. Process this data with a computer to count the small sequence tags.
Applications
Although SAGE was originally conceived for use in cancer studies, it has been successfully used to describe the transcriptome of other diseases and in a wide variety of organisms.

Comparison to DNA microarrays
The general goal of the technique is similar to the DNA microarray. However, SAGE is a sequence-based sampling technique. Observations are not based on hybridization, which result in more qualitative, digital values. In addition, the mRNA sequences do not need to be known a priori, so genes or gene variants which are not known can be discovered. Microarray experiments are much cheaper to perform, so large-scale studies do not typically use SAGE. Quantifying gene expressions is more exact in SAGE because it involves directly counting the number of transcripts whereas spot intensities in microarrays fall in non-discrete gradients and are prone to background noise.
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Ayurveda

Ayurveda is a system of traditional medicine native to India, and practiced in other parts of the world as a form of alternative medicine. In Sanskrit, the word Ayurvedacomprises the words āyus, meaning 'life' and veda, meaning 'science'. Evolving throughout its history, Ayurveda remains an influential system of medicine in South Asia. The earliest literature of Ayurveda appeared during the Vedic period in India. The Sushruta Samhita and the Charaka Samhita were influential works on traditional medicine during this era. Ayurvedic practitioners also claim to have identified a number of medicinal preparations and surgical procedures for curing various ailments and diseases.
Ayurveda is considered to be a form of complementary and alternative medicine (CAM) within the western world, where several of its methods—such as herbs, massage, and Yoga as exercise or alternative medicine—are applied on their own as a form of CAM treatment.

Ayurveda stresses the use of vegetable drugs Fats are used both for consumption and for external use. Hundreds of vegetable drugs are employed, including cardamom and cinnamon. Some animal products may also be used, for example milk, bones, and gallstones etc. Minerals—including sulfur, arsenic, lead, copper sulfate, gold—are also consumed as prescribed.. This practice of adding minerals to herbal medicine is known as Rasa Shastra.

In some cases alcohol is used as a narcotic for the patient undergoing an operation. The advent of Islam introduced opium as a narcotic. Both oil and tar are used to stop bleeding. Oils may be used in a number of ways including regular consumption as a part of food, anointing, smearing, head massage, and prescribed application to infected areas.

The proper function of channels—tubes that exist within the body and transport fluids from one point to another—is seen as vital, and the lack of healthy channels may lead to disease and insanity. Sushruta identifies that blockages of these channels may lead to rheumatism, epilepsy, paralysis, and convulsions as fluids and channels are diverted from their ideal locations. Sweating is favored as a manner in which to open up the channels and dilute the Doshas causing the blockages and harming a patient—a number of ways to take steam bathing and other steam related cures are recommended so that these toxins are released.
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Gene expression

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is a functional RNA.

Several steps in the gene expression process may be modulated, including the transcription, RNA splicing, translation, and post-translational modification of a protein. Gene regulation gives the cell control over structure and function, and is the basis for cellular differentiation, morphogenesis and the versatility and adaptability of any organism. Gene regulation may also serve as a substrate for evolutionary change, since control of the timing, location, and amount of gene expression can have a profound effect on the functions (actions) of the gene in the organism.

Transcription

The gene itself is typically a long stretch of DNA and does not perform an active role. It is a blueprint for the production of RNA. The production of RNA copies of the DNA is called transcription, and is performed by RNA polymerase, which adds one RNA nucleotide at a time to a growing RNA strand. This RNA is complementary to the DNA nucleotide being transcribed; i.e. a T on the DNA means an A is added to the RNA. However, in RNA the nitrogen-containing base Uracil is inserted instead of Thymine wherever there is an Adenine on the DNA strand. Therefore, the mRNA complement of a DNA strand reading "TAC" would be transcribed as "AUG".

RNA processing

Transcription creates a primary transcript of RNA at the place where the gene was located. This transcript is often altered before being translated. RNA processing, also known as post-transcriptional modification, can start during transcription, as is the case for splicing, where the spliceosome removes introns from newly formed RNA. Introns are RNA segments which are not found in the mature RNA, although they can function as precursors, e.g. for snoRNAs, which are RNAs that direct modification of nucleotides in other RNAs.
In some cases large aggregates of RNA and RNA processing factors are formed, notably in the nucleolus where ribosomal RNA is processed by snoRNAs and their partner enzymes. These cleave the primary ribosomal RNA transcripts into the correct segments and alter some of its nucleosides, for instance into pseudouridine.

RNA export

While some RNAs function in the nucleus, many other RNAs in eukaryotes are transported through the nuclear pores and into the cytosol, including all the RNA types involved in protein synthesis. In some cases RNAs are additionally transported to a specific part of the cytoplasm, such as a synapse; they are then towed by motor proteins that bind through linker proteins to specific sequences (called "zipcodes") on the RNA.

Translation
For most RNA, the mature RNA is the gene product (see non-coding RNA). In the case of messenger RNA however, the RNA is but an information carrier for the synthesis of a protein. Each triplet of nucleotides of the coding region of a messenger RNA corresponds to a binding site for a transfer RNA. Transfer RNAs carry amino acids, and these are chained together by the ribosome. The ribosome helps transfer RNA bind to messenger RNA and takes the amino acid from each transfer RNA and makes a structure-less protein out of it.
Some proteins have parts that should be within a membrane, these parts are moved into the membrane by the signal recognition particle which binds to the ribosome when it finds a signal sequence on the nascent amino acid chain.

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Cleave DNA Using Restriction Endonulease:

In order to manipulate DNA you have to posses the ability to cleave DNA at specific sites by using bacterial enzyme, which is restriction endonulease. Restriction endonucleases are bacterial enzymes that cleave duplex DNA at specific target sequences with the production of defined fragments. The name of the enzyme (such as BamHl, EcoRl, AluI, and so on) tells us about the origin of the enzyme but does not give us any information about the specificity of cleavage. The recognition site for most of the commonly used enzymes is a short palindromic sequence, usually either 4, 5, or 6 bp in length, such as AGCT (for AZul),GAATTC (for EcoRl), and so on. Each enzyme cuts the palindrome at a particular site, and two different enzymes may have the same recognition sequence but cleave the DNA at different points within that sequence.


Materials

1. 10X stock of the appropriate restriction enzyme buffer.

2. DNA to be digested in either water or TE (10 mM Tris-HCl pH 8.3, 1 mM ethylenediaminetetraacetic acid [EDTA]).

3. Bovine serum albumin (BSA) at a concentration of 1 mg/mL.
BSA is routinely included in restriction digests to stabilize low protein concentrations and to protect against factors that cause denaturation.

4. Sterile distilled water.
Good-quality sterile distilled water should be used in restriction digests. Water should be free of ions and organic compounds, and must be detergent free.

5. The correct enzyme for the digest.

6. 5X Loading buffer: 50% (v/v) glycerol, 100 mM Na2EDTA, pH 8, 0.125% (w/v) bromophenol blue, 0.125% (w/v) xylene cyanol.

7. 100 mM Spermidine.
Digests of genomic DNA are dramatically improved by the inclusion of spermidine in the digest mixture to a final concentration of 1 mM since the polycationic spermidine binds negatively charged contaminants.
Spermidine can cause precipitation of DNA at low temperatures, so it should not be added while the reaction is kept on ice.


Methods

1. Thaw all solutions, with the exception of the enzyme, and then place on ice.

2. Decide on a final volume for the digest, usually between 10 and 50 microliters, and then into a sterile Eppendorf tube, add 1/10 vol of reaction buffer, 1/10 vol BSA, between 0.5 and 1 micrograms of the DNA to be digested, and sterile distilled water to the final volume.
The amount of DNA to be digested depends on subsequent steps. A reasonable amount for a plasmid digestion to confirm the presence of an insertion would be 500 ng-l microgram, depending on the size of the insert. The smaller the insert, the more DNA should be digested to enable visualization of the insert after agarose gel analysis.

3. Take the restriction enzyme stock directly from the -20oC freezer, and remove the desired units of enzyme with a clean sterile pipet tip. Immediately add the enzyme to the reaction and mix.
Stock restriction enzymes are very heat labile and so should be removed from -20oC storage for as short a time as possible and placed on ice.

4. Incubate the tube at the correct temperature (see Note 12) for approx 1 h. Genomic DNA can be digested overnight.
Note that the incubation temperature for the vast majority of restriction endonucleases is 37oC but that this is not true for all enzymes.

5. An aliquot of the reaction (usually 1-2microliter) may be mixed with a 5X concentrated loading buffer and analyzed by gel electrophoresis.


Hopefully this method can help your work.

READ MORE - Cleave DNA Using Restriction Endonulease:

Cloning

Cloning
Cloning in biology is the process of producing populations of genetically-identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. More generally, the term refers to the production of multiple copies of a product such as digital media or software.

Cellular cloning
Cloning a cell means to derive a population of cells from a single cell. In the case of unicellular organisms such as bacteria and yeast, this process is remarkably simple and essentially only requires the inoculation of the appropriate medium. However, in the case of cell cultures from multi-cellular organisms, cell cloning is an arduous task as these cells will not readily grow in standard media.
A useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings (cylinders). According to this technique, a single-cell suspension of cells which have been exposed to a mutagenic agent or drug used to drive selection is plated at high dilution to create isolated colonies; each arising from a single and potentially clonally distinct cell. At an early growth stage when colonies consist of only a few of cells, sterile polystyrene rings (cloning rings), which have been dipped in grease are placed over an individual colony and a small amount of trypsin is added. Cloned cells are collected from inside the ring and transferred to a new vessel for further growth.

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Cell culture

Cell culture is the process by which prokaryotic or eukaryotic cells are grown under controlled conditions. In practice the term "cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture.


Applications of cell culture

Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and many products of biotechnology. Biological products produced by recombinant DNA (rDNA) technology in animal cell cultures include enzymes, synthetic hormones, immunobiologicals (monoclonal antibodies, interleukins, lymphokines), and anticancer agents. Although many simpler proteins can be produced using rDNA in bacterial cultures, more complex proteins that are glycosylated (carbohydrate-modified) currently must be made in animal cells. An important example of such a complex protein is the hormone erythropoietin. The cost of growing mammalian cell cultures is high, so research is underway to produce such complex proteins in insect cells or in higher plants.

Tissue culture and engineering
Cell culture is a fundamental component of tissue culture and tissue engineering, as it establishes the basics of growing and maintaining cells ex vivo.

Vaccines
Vaccines for polio, measles, mumps, rubella, and chickenpox are currently made in cell cultures. Due to the H5N1 pandemic threat, research into using cell culture for influenza vaccines is being funded by the United States government. Novel ideas in the field include recombinant DNA-based vaccines, such as one made using human adenovirus (a common cold virus) as a vector,or the use of adjuvants.

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Types of cells

Cells are often categorized by their source:

* Autologous cells are obtained from the same individual to which they will be reimplanted. Autologous cells have the fewest problems with rejection and pathogen transmission, however in some cases might not be available. For example in genetic disease suitable autologous cells are not available. Also very ill or elderly persons, as well as patients suffering from severe burns, may not have sufficient quantities of autologous cells to establish useful cell lines. Moreover since this category of cells needs to be harvested from the patient, there are also some concerns related to the necessity of performing such surgical operations that might lead to donor site infection or chronic pain. Autologous cells also must be cultured from samples before they can be used: this takes time, so autologous solutions may not be very quick. Recently there has been a trend towards the use of mesenchymal stem cells from bone marrow and fat. These cells can differentiate into a variety of tissue types, including bone, cartilage, fat, and nerve. A large number of cells can be easily and quickly isolated from fat, thus opening the potential for large numbers of cells to be quickly and easily obtained. Several companies have been founded to capitalize on this technology, the most successful at this time being Cytori Therapeutics.



* Allogeneic cells come from the body of a donor of the same species. While there are some ethical constraints to the use of human cells for in vitro studies, the employment of dermal fibroblasts from human foreskin has been demonstrated to be immunologically safe and thus a viable choice for tissue engineering of skin.

* Xenogenic cells are these isolated from individuals of another species. In particular animal cells have been used quite extensively in experiments aimed at the construction of cardiovascular implants.

* Syngenic or isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

* Primary cells are from an organism.

* Secondary cells are from a cell bank.

* Stem cells (see main article: stem cell) are undifferentiated cells with the ability to divide in culture and give rise to different forms of specialized cells. According to their source stem cells are divided into "adult" and "embryonic" stem cells, the first class being multipotent and the latter mostly pluripotent; some cells are totipotent, in the earliest stages of the embryo. While there is still a large ethical debate related with the use of embryonic stem cells, it is thought that stem cells may be useful for the repair of diseased or damaged tissues, or may be used to grow new organs.

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Tissue engineering

Tissue engineering is the use of a combination of cells, engineering and materials methods, and suitable biochemical and physio-chemical factors to improve or replace biological functions. While most definitions of tissue engineering cover a broad range of applications, in practice the term is closely associated with applications that repair or replace portions of or whole tissues (i.e., bone, cartilage, blood vessels, bladder, etc.). Often, the tissues involved require certain mechanical and structural properties for proper functioning. The term has also been applied to efforts to perform specific biochemical functions using cells within an artificially-created support system (e.g. an artificial pancreas, or a bioartificial liver). The term regenerative medicine is often used synonymously with tissue engineering, although those involved in regenerative medicine place more emphasis on the use of stem cells to produce tissues.

A commonly applied definition of tissue engineering, as stated by Langer and Vacanti, is "an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ".Tissue engineering has also been defined as "understanding the principles of tissue growth, and applying this to produce functional replacement tissue for clinical use." A further description goes on to say that an "underlying supposition of tissue engineering is that the employment of natural biology of the system will allow for greater success in developing therapeutic strategies aimed at the replacement, repair, maintenance, and/or enhancement of tissue function."
Powerful developments in the multidisciplinary field of tissue engineering have yielded a novel set of tissue replacement parts and implementation strategies. Scientific advances in biomaterials, stem cells, growth and differentiation factors, and biomimetic environments have created unique opportunities to fabricate tissues in the laboratory from combinations of engineered extracellular matrices ("scaffolds"), cells, and biologically active molecules. Among the major challenges now facing tissue engineering is the need for more complex functionality, as well as both functional and biomechanical stability in laboratory-grown tissues destined for transplantation. The continued success of tissue engineering, and the eventual development of true human replacement parts, will grow from the convergence of engineering and basic research advances in tissue, matrix, growth factor, stem cell, and developmental biology, as well as materials science and bioinformatics.

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Biochemical engineering

Biochemical engineering is a branch of chemical engineering or biological engineering that mainly deals with the design and construction of unit processes that involve biological organisms or molecules, such as bioreactors. Biochemical engineering is often taught as a supplementary option to chemical engineering or biological engineering due to the similarities in both the background subject curriculum and problem-solving techniques used by both professions. Its applications are used in the food, feed, pharmaceutical, biotechnology, and water treatment industries.
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Genetic Version

In a strange genetic version of the Russian doll, scientists have discovered the genome of a bacterial parasite nestled inside the genome of its host. The findings, published today in the journal Science, suggest that organisms might quickly acquire new genes and functions through the large-scale transfer of genes.

The parasite, known as Wolbachia, invades the eggs and sperm of many different types of insects, ensuring that it is passed down to the host's offspring. In this case, scientists discovered the bacterium's genome within the chromosome of its fruit-fly host. While microbiologists have previously seen cases of gene swapping between microbes or between parasites and their hosts, this is the first example of such an extensive exchange.

According to a press release from the University of Rochester,

"This study establishes the widespread occurrence and high frequency of a process that we would have dismissed as science fiction until just a few years ago," says W. Ford Doolittle, Canada Research Chair in Comparative Microbial Genomics at Dalhousie University, who is not connected to the study. "This is stunning evidence for increased frequency of gene transfer."

"It didn't seem possible at first," says [Jack] Werren, professor of biology at the University of Rochester and a world-leading authority on the parasite, called Wolbachia. "This parasite has implanted itself inside the cells of 70 percent of the world's invertebrates, coevolving with them. And now, we've found at least one species where the parasite's entire or nearly entire genome has been absorbed and integrated into the host's. The host's genes actually hold the coding information for a completely separate species."

A similar phenomenon may have happened in our own distant past.

"In our very own cells and those of nearly all plants and animals are mitochondria, special structures responsible for generating most of our cells' supply of chemical energy. These were once bacteria that lived inside cells, much like Wolbachia does today. Mitochondria still retain their own, albeit tiny, DNA, and most of the genes moved into the nucleus in the very distant past. Like Wolbachia, they have passively exchanged DNA with their host cells. It's possible Wolbachia may follow in the path of mitochondria, eventually becoming a necessary and useful part of a cell. In a way, Wolbachia could be the next mitochondria," says Werren. "A hundred million years from now, everyone may have a Wolbachia organelle."

READ MORE - Genetic Version

Gene expression

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is a functional RNA.

Several steps in the gene expression process may be modulated, including the transcription step and translation step and the post-translational modification of a protein. Gene regulation gives the cell control over structure and function, and is the basis for cellular differentiation, morphogenesis and the versatility and adaptability of any organism. Gene regulation may also serve as a substrate for evolutionary change, since control of the timing, location, and amount of gene expression can have a profound effect on the functions (actions) of the gene in the organism.

Transcription

The gene itself is typically a long stretch of DNA and does not perform an active role. It is a blueprint for the production of RNA. The production of RNA copies of the DNA is termed transcription, and is performed by RNA polymerase, which adds one RNA nucleotide at a time to a growing RNA strand. This RNA is complementary to the DNA nucleotide being read, i.e. a T on the DNA means an A is added to the RNA. However, In RNA the nitrogen base Uracil is inserted instead of Thymine. Wherever there is an Adenine on the DNA strand, a Uracil is inserted into the complementary RNA strand. I.e the mRNA complement of a DNA strand reading "TAC" would be transcribed as "AUG", which is translated into the amino acid methionine, which is generally the starting point in a messenger RNA for expressing a protein.

RNA processing

Transcription creates a primary transcript of RNA at the place where the gene was located. This transcript often needs to be altered by enzymes. RNA processing, also known as post-transcriptional modification, can start already during transcription, as is the case for e.g. splicing where the spliceosome removes introns from newly formed parts of the RNA.[ Introns are RNA segments which are not found in the mature RNA, although they can function as precursors for e.g. snoRNA which are a group of RNAs that direct nucleotide modification of other RNAs.

In some cases large aggregates of RNA and RNA processing factors are formed, notably the nucleolus where ribosomal RNA localises to be processed by snoRNAs and their partner enzymes. These chop the primary ribosomal RNA transcripts into the correct segments and alter some of its nucleotides into e.g. pseudouridine.

RNA export
While some RNAs function in the nucleus, many other RNAs in eukaryotes need to be transported through the nuclear pores and into the cytosol, including all the RNA types involved in protein synthesis.[ In some cases the RNA is additionally transported to a specific part of the cytoplasm, such as a synapse, they are then towed by motor proteins that bind through linker proteins to specific sequences (called "zipcodes") on the RNA.

Translation

For most RNA, the mature RNA is the gene product (see non-coding RNA). In the case of messenger RNA however, the RNA is but an information carrier for the synthesis of a protein. Each triplet of nucleotides of the coding region of a messenger RNA corresponds to a bindning site for a transfer RNA. Transfer RNAs carry amino acids, and these are chained together by the ribosome. The ribosome helps transfer RNA bind to messenger RNA and takes the amino acid from each tranfer RNA and makes a structure-less protein out of it.
Some proteins have parts that should be within a membrane, these parts are moved into the membrane by the signal recognition particle which binds to the ribosome when it finds a signal sequence on the nascent amino acid chain.

Folding
Enzymes called chaperones assist the newly formed protein to attain (fold into) the 3-dimensional structure it needs to function. Similarly, RNA chaperones help RNAs attain their functional shapes.
Protein export
Many proteins that are destined for other parts of the cell than the cytosol. A commonly used mechanism for transporting these proteins to where they should be is translocation to the endoplasmatic reticulum, followed by transport via the Golgi apparatus.
READ MORE - Gene expression

Vomitoxin

Vomitoxin, also known as deoxynivalenol (DON), is a type B trichothecene, an epoxy-sesquiterpeneoid. This mycotoxin occurs predominantly in grains such as wheat, barley, oats, rye, and maize, and less often in rice, sorghum, and triticale. The occurrence of deoxynivalenol is associated primarily with Fusarium graminearum (Gibberella zeae) and F. culmorum, both of which are important plant pathogens which cause Fusarium head blight in wheat and Gibberella ear rot in maize. A direct relationship between the incidence of Fusarium head blight and contamination of wheat with deoxynivalenol has been established. The incidence of Fusarium head blight is strongly associated with moisture at the time of flowering (anthesis), and the timing of rainfall, rather than the amount, is the most critical factor. Furthermore, deoxynivalenol contents are significantly affected by the susceptibility of cultivars towards Fusarium species, previous crop, tillage practices, and fungicide use

F. graminearum grows optimally at a temperature of 25 °C and at a water activity above 0.88. F. culmorum grows optimally at 21 °C and at a water activity above 0.87. The geographical distribution of the two species appears to be related to temperature, F. graminearum being the commoner species and occurring in warmer climates. Deoxynivalenol has been implicated in incidents of mycotoxicoses in both humans and farm animals.

When compared to other trichothecene mycotoxins which can form in grains and forages, vomitoxin is relatively mild. Reduced feed intake, and the accompanying decrease in performance, are the only symptoms of vomitoxin toxicity livestock producers will likely encounter. This response to vomitoxin appears to occur through the central nervous system. Vomitoxin belongs to a class of mycotoxins (tricothecenes) which are strong protein inhibitors. Inhibition of protein synthesis following exposure to vomitoxin causes the brain to increase its uptake of the amino acid tryptophan and, in turn, its synthesis of serotonin. Increased levels of serotonin are believed to be responsible for the anorexic effects of DON and other tricothecenes. Irritation of the gastrointestinal tract may also play a role in reducing feed intake... This fact may also partially explain the high incidence of pars esaughageal stomach ulcers observed in sows off feed during feed refusal.

• Human foods: Vomitoxin is not a known carcinogen as with aflatoxin. Large amounts of grain with vomitoxin would have to be consumed to pose a health risk to humans. The FDA has established a level of 1 ppm (parts per million) restriction of vomitoxin.

• Companion animals: Dogs and cats are restricted to 5 ppm and of grains and grain byproducts and that the grains not exceed 40% percent of the diet.

• Livestock and farm animals: In animals and livestock, vomitoxin causes a refusal to feed and lack of weight gain when fed above advised levels. Restrictions are set at 10 ppm for poultry and ruminating beef and feedlot cattle older than 4 months. Ingredients may not exceed 50% of the animal's diet. Dairy cow limits are set at 2 ppm.

READ MORE - Vomitoxin

A Genome within a Genome

In a strange genetic version of the Russian doll, scientists have discovered the genome of a bacterial parasite nestled inside the genome of its host. The findings, published today in the journal Science, suggest that organisms might quickly acquire new genes and functions through the large-scale transfer of genes.

The parasite, known as Wolbachia, invades the eggs and sperm of many different types of insects, ensuring that it is passed down to the host's offspring. In this case, scientists discovered the bacterium's genome within the chromosome of its fruit-fly host. While microbiologists have previously seen cases of gene swapping between microbes or between parasites and their hosts, this is the first example of such an extensive exchange.

According to a press release from the University of Rochester,

"This study establishes the widespread occurrence and high frequency of a process that we would have dismissed as science fiction until just a few years ago," says W. Ford Doolittle, Canada Research Chair in Comparative Microbial Genomics at Dalhousie University, who is not connected to the study. "This is stunning evidence for increased frequency of gene transfer."

"It didn't seem possible at first," says [Jack] Werren, professor of biology at the University of Rochester and a world-leading authority on the parasite, called Wolbachia. "This parasite has implanted itself inside the cells of 70 percent of the world's invertebrates, coevolving with them. And now, we've found at least one species where the parasite's entire or nearly entire genome has been absorbed and integrated into the host's. The host's genes actually hold the coding information for a completely separate species."

A similar phenomenon may have happened in our own distant past.

"In our very own cells and those of nearly all plants and animals are mitochondria, special structures responsible for generating most of our cells' supply of chemical energy. These were once bacteria that lived inside cells, much like Wolbachia does today. Mitochondria still retain their own, albeit tiny, DNA, and most of the genes moved into the nucleus in the very distant past. Like Wolbachia, they have passively exchanged DNA with their host cells. It's possible Wolbachia may follow in the path of mitochondria, eventually becoming a necessary and useful part of a cell. In a way, Wolbachia could be the next mitochondria," says Werren. "A hundred million years from now, everyone may have a Wolbachia organelle."

READ MORE - A Genome within a Genome

RNA - Transcription

Introduction:

The differences in the composition of RNA and DNA have already been noted. In addition, RNA is not usually found as a double helix but as a single strand. However, the single polynucleotide strand may fold back on itself to form portions which have a double helix structure like the tertiary structure of proteins.

The biosynthesis of RNA, called transcription, proceeds in much the same fashion as the replication of DNA and also follows the base pairing principle. Again, a section of DNA double helix is uncoiled and only one of the DNA strands serves as a template for RNA polymerase enzyme to guide the synthesis of RNA. After the synthesis is complete, the RNA separates from the DNA and the DNA recoils into its helix.

The transcription of a single RNA strand is illustrated in the graphic on the left. One major difference is that the heterocyclic amine, adenine, on DNA codes for the incorporation of uracil in RNA rather than thymine as in DNA. Remember that thymine is not found in RNA and do not confuse the replacement of uracil in RNA for thymine in DNA in the transcription process. For example, thymine in DNA still codes for adenine on RNA not uracil, while the adenine on DNA codes for uracil in RNA.

Note that the new RNA (red) is identical to non coding DNA with the exception of uracil where thymine was located in DNA.

There are three major types of RNA which will be fully explained in a later section. Although RNA is synthesized in the nucleus, it migrates out of the nucleus into the cytoplasm where it is used in the synthesis of proteins.

RNA Transcription Process:

The RNA transcription process occurs in three stages: initiation, chain elongation, and termination.

The first stage occurs when the RNA Polymerase-Promoter Complex binds to the promoter gene in the DNA. This also allows for the finding of the start sequence for the RNA polymerase. The promoter enzyme will not work unless the sigma protein is present (shown in blue in graphic). Specific sequences on the non coding strand of DNA are recognized as the signal to start the unwinding process.

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Transcription

Transcription is the synthesis of RNA under the direction of DNA. RNA synthesis, or transcription, is the process of transcribing DNA nucleotide sequence information into RNA sequence information. Both nucleic acid sequences use complementary language, and the information is simply transcribed, or copied, from one molecule to the other. DNA sequence is enzymatically copied by RNA polymerase to produce a complementary nucleotide RNA strand, called messenger RNA (mRNA), because it carries a genetic message from the DNA to the protein-synthesizing machinery of the cell.


One significant difference between RNA and DNA sequence is the presence of U, or uracil in RNA instead of the T, or thymine of DNA. In the case of protein-encoding DNA, transcription is the first step that usually leads to the expression of the genes, by the production of the mRNA intermediate, which is a faithful transcript of the gene's protein-building instruction. The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit.

A DNA transcription unit that is translated into protein contains sequences that direct and regulate protein synthesis in addition to coding the sequence that is translated into protein. The regulatory sequence that is before (upstream (-), towards the 5' DNA end) the coding sequence is called 5' untranslated region (5'UTR), and sequence found following (downstream (+), towards the 3' DNA end) the coding sequence is called 3' untranslated region (3'UTR). Transcription has some proofreading mechanisms, but they are fewer and less effective than the controls for copying DNA; therefore, transcription has a lower copying fidelity than DNA replication.

As in DNA replication, RNA is synthesized in the 5' → 3' direction (from the point of view of the growing RNA transcript). Only one of the two DNA strands is transcribed. This strand is called the template strand, because it provides the template for ordering the sequence of nucleotides in an RNA transcript. The other strand is called the coding strand, because its sequence is the same as the newly created RNA transcript (except for uracil being substituted for thymine). The DNA template strand is read 3' → 5' by RNA polymerase and the new RNA strand is synthesized in the 5'→ 3' direction.

A polymerase binds to the 3' end of a gene (promoter) on the DNA template strand and travels toward the 5' end.

READ MORE - Transcription

The Dna Testing Process

DNA testing is becoming increasingly used to determine genetic links between individuals as a highly accurate and individual way of identifying people and their relationships with one another. The process itself is one carried out in advanced laboratories under the strictest of lab conditions to ensure no cross-contamination and improve result accuracy. As such DNA testing can be said to present with a high degree of accuracy any particular biological relationship that may exist, particularly in paternity disputes where samples of both the mother and the father are provided.

Preparing For the DNA Test and Collecting Samples
Normally a DNA testing kit is sent to the person who ordered the test by the company from whom the order was made. The test begins with samples being collected from everyone preparing to undertake the test. In most cases, that will mean the mother, the father (alleged) and the child concerned. Samples are taken by the way of oral swabs, which collect cheek cells which are then dried and passed on for testing. In order to prepare the sample, it is first important to make sure that the cotton of the swab never touches any other surface including your hands, and that you have a number of swabs for each person taking the test to ensure reliability in the end results. Press the swab into the inside of the cheek and behind the lips, as well as the tongue area in order to get as good as possible a sample from the mouth. Having left to dry for around an hour, the swab should be carefully sealed off before the collation and mailing process.

Testing the Samples
After all the samples have been collected and labelled accordingly, they should be sent off to the laboratory for the DNA testing analysis. At this stage, the samples will be individually examined and DNA will be extracted from within the cells present in the sample. The same will be done for both the other two parties to the test and the results of the DNA profiles will be compared.

The person analysing your results will be looking for a 50/50 split between your alleles, contained within the DNA, between those found on your mother and father. As you can only inherit genes already carried by one or both parents, no alleles can be present in the child's DNA that are not present in that of either parent. Naturally, this is where it becomes obvious when there is and is not a genetic link between those taking the DNA test. Further to that, the results are processed through the appropriate systems and a conclusion is reached, having covered 16 of the locus which are used as the template by which samples are matched.

Receiving the DNA Test Results

Once the DNA test is completed, the result will be sent to the participants via email, letter, fax or as otherwise agreed. The DNA test report should show the individual profile of each person that submitted a sample for the paternity test. Also the result should show the percentage probability of the stated relationship, for example in a DNA paternity test this is normally in excess of 99.99%.

There's no doubt about it - DNA testing is here to stay. Whilst most people are not very knowledgeable on how DNA paternity testing works, it is probably a good idea to gain some level of understanding given the way in which DNA testing is likely to continue to affect our lives over the coming decades. With growing calls for more extensive DNA databases and records for crime prevention, DNA testing and analysis looks set to remain at the forefront of the civil liberties/state interests debate.

READ MORE - The Dna Testing Process

Dna Testing to Find Your Ancestors

The use of DNA testing for determining a person’s ancestry is becoming more and common. By linking your maternal DNA (mitochondrial DNA) and your paternal DNA (the y-chromosome), these ancestry databases are effectively able to link you to other people to whom you may be related and thereby determining to some degree your ancestral lineage and where your ancestors came from.

DNA Ancestry Testing – Y-Chromosome and Mitochondial DNA

The first thing that genealogists look for is a father-to-son linkage, tracked down the Y chromosome which only men posses. Therefore, they are able to observe the Y chromosome that appears in other people and compare them, to determine where a paternal link may be present. This comparison, in essence, allows for the genealogist to try and find paternal linkages amongst people. The other thing that can be done is to link maternal DNA. This in particular is a very powerful testing method that allows for accurate tracking back over many generations because of the mitochondria.

Unlike DNA found in the nucleus, which can be altered and changes as environments change, mitochondrial DNA is a direct connection from child to mother that can’t be altered along the way. By taking a sample of the mitochondrial DNA, which is different than the DNA found in the nucleus, the genealogist can determine a maternal linkage. By taking this information, they can, once again, find, perhaps those long lost cousins or celebrity ancestors.

DNA Ancestry Testing – Matching to a DNA Database

However, can this really be effective at tracing family lines? How can they tell you who you’re related to throughout history? Some online ancestry websites create a database of DNA against which your can be matched. By taking a simple mouth swab and run the DNA tests, they then save the DNA profile that is collected. However, the key is for them to continuously compare other people’s DNA profiles to what your profile is. In essence, this will create a massive database that will determine instantly if a piece of code is a direct comparison to yours. So, as the database grows more and more, more and more relatives and ancestors can be discovered for more and more people.

DNA Ancestry Testing – Determine geographical ancestry

Furthermore, these DNA tests are able to help you find out where you come from. It’s argued that 170,000 years ago, humans left Africa and migrated elsewhere across the globe. Some went to Europe, some went to southern Africa, while others went to Asia to settle. By comparing the DNA profile of a person to that of researched ethnic groups, it is possible to provide information about where people are from.

DNA testing has become a very useful method for people to find long lost relatives. Furthermore, the argument of the true nature of one’s ethnic origins can finally be resolved by DNA testing processes. Of course, as the databases grow and more research is conducted on, the usefulness of these types of tests will increase greatly.

READ MORE - Dna Testing to Find Your Ancestors

How Is Dna Testing Done

DNA testing is done for many different reasons. DNA evidence can link an alleged criminal to a crime scene. DNA paternity and maternity testing can identify a child's father or mother. DNA relationship testing can determine if two individuals are full or half siblings. DNA ancestry testing can determine ethnic origins and genealogical roots.

How DNA testing is done depends on the results desired and the samples available. DNA fingerprinting (or profiling as it's also known) is the process of analyzing and comparing two DNA samples. Only identical twins have the exact same DNA sequence, everyone else's DNA is unique. This makes DNA the perfect way to link individuals to each other or to locations where they have been.

The entire DNA chain is incredibly long, much to long to examine all of it. Human DNA is made up of about 3.3 billion base pairs. The differences between DNA samples occur only in small segments of the DNA--the rest of the DNA is pretty much the same. DNA testing focuses on those segments that are known to differ from person to person.

As DNA testing has evolved over time, the testing methods have become more precise and are able to work with much smaller DNA samples. Early DNA testing was done using dime-size drops of blood. Today's tests can extract DNA from the back of a licked stamp. The DNA must be extracted from whatever sample is provided. DNA must be isolated and purified before it can be compared. In essence, it has to be "unlocked" from the cell in which it exists. The cell walls are usually dissolved with a detergent. Proteins in the cell are digested by enzymes. After this process, the DNA is purified, concentrated, and tested.

DNA testing is done most often today using a process called "short tandem repeats," or STR. Human DNA has several regions of repeated sequences. These regions are found in the same place on the DNA chain, but the repeated sequences are different for each individual. The "short" tandem repeats (repeated sequences of two to five base pairs in length) have been proven to provide excellent DNA profiling results. STR is highly accurate--the chance of misidentification being one in several billion.

source:articlebase
READ MORE - How Is Dna Testing Done

Bacteriocin

Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). They are typically considered to be narrow spectrum antibiotics, though this has been debated They are phenomenologically analogous to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse.

Methods of classification

Alternative methods of classification include: method of killing (pore forming, dnase, nuclease, murein production inhibition, etc), genetics (large plasmids, small plasmids, chromosomal), molecular weight and chemistry (large protein, polypeptide, with/without sugar moiety, containing atypical amino acids like lanthionine) and method of production (ribosomal, post ribosomal modifications, non-ribosomal).

Class I bacteriocins

The class I bacteriocins are small peptide inhibitors and include nisin.

Class II bacteriocins

The class II bacteriocins are small heat-stable proteins. The action of Class IIa bacteriocins seems to involve disruption of mannose transport into target cells. Class IIb bacteriocins form pores in the membranes of target cells and disrupt the proton gradient of target cells. Other bacteriocins can be grouped together as Class IIc. These have a wide range of effects on membrane permeability, cell wall formation and pheromone actions of target cells.

Class III bacteriocins

Large, heat-labile protein bacteriocins.

Medical significance

Bacteriocins are of interest in medicine because they are made by non-pathogenic bacteria that normally colonize the human body. Loss of these harmless bacteria following antibiotic use may allow opportunistic pathogenic bacteria to invade the human body.

Bacteriocins have also been suggested as a cancer treatment. They have shown distinct promise as a diagnostic agent for some cancers, , but their status as a form of therapy remains experimental and outside the main thread of cancer research. Partly this is due to questions about their mechanism of action and the presumption that anti-bacterial agents have no obvious connection to killing mammalian tumor cells. Some of these questions have been addressed, at least in part.

In the long quest for medical applications, bacteriocins have also been tested as AIDS drugs.

Production

There are many ways to demonstrate bacteriocin production, depending on the sensitivity and labor intensiveness desired. To demonstrate their production, technicians stab inoculate multiple strains on separate multiple nutrient agar Petri dishes, incubate at 30 °C for 24 h., overlay each plate with one of the strains (in soft agar), incubate again at 30 °C for 24 h. After this process, the presence of bacteriocins can be inferred if there are zones of growth inhibition around stabs. This is the simplest and least sensitive way. It will often mistake phage for bacteriocins. Some methods prompt production with UV radiation, Mitomycin C, or heat shock. UV radiation and Mitomycin C are used because the DNA damage they produce stimulates the SOS response. Cross streaking may be substituted for lawns. Similarly, production in broth may be followed by dripping the broth on a nascent bacterial lawn, or even filtering it. Precipitation (ammonium sulfate) and some purification (e.g. column or HPLC) may help exclude lysogenic and lytic phage from the assay.

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The Analysis of DNA or RNA

In order to measure DNA content you can use UV Spectrophotometer with the advantages are nondestructive and allows the sample to be recovered for further analysis or manipulation. Spectrophotometry uses the fact that there is a relationship between the absorption of ultraviolet light by DNA/RNA and its concentration in a sample. In this particular posting, I want to give you facts about the relationship between DNA/RNA with the wavelength in the Spectrophotometer assay.

1. The absorption maximum of DNA/RNA is approx 260 nm. This figure is an average of the absorption of the individual nucleotides that vary between 256 and 281 nm.

2. In the case of RNA, the concentration of a sample containing RNA may be calculated following Equation:

40 x OD260 of the sample = concentration of RNA (microgram/mL)


And this equation for DNA concentration:

50 x OD260 of the sample =concentration of DNA (microgram/mL)


The equation above describe that when the OD-260 of the sample is 1 the concentration of RNA will be approx 40 micrograms/mL (50 micrograms/mL for DNA).

3. We can also assess the degree of purity of the nucleic acids by examining the absorption at other wavelengths in which protein and polysaccharides have known absorption maxima. Proteins are known to absorb strongly at 280 nm and polysaccharides may be identified by their maximum at 230 nm.

4. Therefore, in assessing the purity degree of the nucleic acid sample we use the ratio of measurements of these three wavelengths 230 nm, 260 nm, and 280 nm.

5. For example a sample containing only RNA following an extraction method is judged as being uncontaminated if the ratio is 1 :2 : 1, and for DNA is 1 : 1.8 : 1 (it reflects OD-230 : 260 : 280 ratio). If there is significant deviation from the ratio, then it is evident that contaminants are present and that further purification of the sample is necessary.


In many cases, the purity and the concentration may be further obscured by the presence of reagents that are used in the extraction process itself. Some of these have characteristics that are evident on a spectrophotometric scan that includes the three wavelengths indicated. Therefore, when using spectrophotometry in the analysis of DNA or RNA it is necessary to be aware of the potential problems that may result in misleading ratio. Further, when analyzing ratios and concentrations of DNA or RNA spectrophotometrically it is also necessary not only to derive readings at 280,260, and 230 nm but also to scan throughout the range 200-320 nm. Trace amounts of reagents used in the extraction process can influence adversely and provide misleading data that may affect any subsequent manipulation.

READ MORE - The Analysis of DNA or RNA

Abamectin

INTRODUCTION

Abamectin is a mixture of avermectins containing > 80% avermectin B1a and <>

Use

Abamectin is used to control insect and mite pests of a range of agronomic, fruit, vegetable and ornamental crops, and it is used by homeowners for control of fire ants. Abamectin is also used as a veterinary antihelmintic. Resistance to abamectin based antihelmintics, although a growing problem, is not as common as to other classes of veterinary antihelmintics.

ACUTE TOXICITY

Abamectin is a highly toxic material, however most formulated products containing abamectin are of low toxicity to mammals. Emulsifiable concentrate formulations may cause moderate eye irritation and mild skin irritation. Symptoms of poisoning observed in laboratory animals include pupil dilation, vomiting, convulsions and/or tremors, and coma .

Abamectin acts on insects by interfering with neural and neuromuscular transmission. It acts on a specific type of synapse located only within the brain and is protected by the blood-brain barrier. However, at very high doses, the mammalian blood-brain barrier can be penetrated, causing symptoms of CNS depression such as incoordination, tremors, lethargy, excitation and pupil dilation. Very high doses have caused death from respiratory failure.

Abamectin is not readily absorbed through skin. Tests with monkeys show that less than 1% of dermally applied abamectin was absorbed into the bloodstream through the skin . Abamectin does not cause allergic skin reactions.

The amount of a chemical that is lethal to one-half (50%) of experimental animals fed the material is referred to as its acute oral lethal dose fifty, or LD50. The oral LD50 for abamectin in rats is 11 mg/kg, and in mice range from 14 to > 80 mg/kg. The dermal LD50 for technical abamectin on rats and rabbits is > 330 mg/kg. The oral LD50 for the product Affirm 0.011% Fire Ant Bait in rats is > 5,000 mg/kg, and its dermal LD50 on rabbits is > 2,000 mg/kg. The oral LD50 for the 1.8% w/v Abamectin EC product in rats is 300 mg/kg, and the dermal LD50 for this product on rabbits is > 2,000 mg/kg .

CHRONIC TOXICITY

In a 1-year study with dogs given oral doses of 0, 0.25, 0.5, or 1 mg/kg/day, there were no changes in tissue at any dose level. However, some dogs at the 0.5 and 1 mg/kg/day levels had pupillary dilation, weight loss, lethargy, tremors and recumbency. The NOEL for this study was 0.25 mg/kg/day. Similar results were seen in a 2-year study with rats fed 0, 0.75, 1.5, or 2 mg/kg/day. No changes in the nervous or muscular systems were observed, but rats in all the dosage levels exhibited body weight gains significantly higher than the controls. A few individuals in the high dose group exhibited tremors .

When mice were fed 8 mg/kg/day, the highest dose tested, for 94 weeks, the males developed dermatitis and changes in blood formation in the spleen, while females exhibited tremors and weight loss.
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Characteristics and Properties of Amino Acids

Introduction:

Each amino acid has at least one amine and one acid functional group as the name implies. The different properties result from variations in the structures of different R groups. The R group is often referred to as the amino acid side chain. Amino acids have special common names, however, a three letter abbreviation for the name is used most of the time. A second abbreviation , single letter, is used in long protein structures.Consult the table on the left for structure, names, and abbreviations of 20 amino acids.

There are basically four different classes of amino acids determined by different side chains:

(1) non-polar and neutral,

(2) polar and neutral,

(3) acidic and polar,

(4) basic and polar.

Principles of Polarity:

The greater the electronegativity difference between atoms in a bond, the more polar the bond. Partial negative charges are found on the most electronegative atoms, the others are partially positive

Non-Polar Side Chains:

Side chains which have pure hydrocarbon alkyl groups (alkane branches) or aromatic (benzene rings) are non-polar. Examples include valine, alanine, leucine, isoleucine, phenylalanine.

The number of alkyl groups also influences the polarity. The more alkyl groups present, the more non-polar the amino acid will be. This effect makes valine more non-polar than alanine; leucine is more non-polar than valine.

Acid - Base Properties of Amino Acids:

Acidic Side Chains:

If the side chain contains an acid functional group, the whole amino acid produces an acidic solution. Normally, an amino acid produces a nearly neutral solution since the acid group and the basic amine group on the root amino acid neutralize each other in the zwitterion. If the amino acid structure contains two acid groups and one amine group, there is a net acid producing effect. The two acidic amino acids are aspartic and glutamic.

Basic Side Chains:

If the side chain contains an amine functional group, the amino acid produces a basic solution because the extra amine group is not neutralized by the acid group. Amino acids which have basic side chains include: lysine, arginine, and histidine.

Amino acids with an amide on the side chain do not produce basic solutions i.e. asparagine and glutamine.

Neutral Side Chains:

Since an amino acid has both an amine and acid group which have been neutralized in the zwitterion, the amino acid is neutral unless there is an extra acid or base on the side chain. If neither is present then then the whole amino acid is neutral.

Amino acids with an amide on the side chain do not produce basic solutions i.e. asparagine and glutamine. You need to look at the functional groups carefully because an amide starts out looking like an amine, but has the carbon double bond oxygen which changes the property. Amides are not basic.

Even though tryptophan has an amine group as part of a five member ring, the electron withdrawing effects of the two ring systems do not allow nitrogen to act as a base by attracting hydrogen ions.
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