Once an interesting piece of DNA has been isolated or recognized, there is requirement to find out if the sequence of nucleotides in the fragment is associated to known genes and to find out what type of protein it might produce. DNA sequencing signifies to sequencing methods for finding out the order of the nucleotides bases - adenine, guanine, cytosine and thymine - in the molecule of DNA
DNA sequencing makes it probable to find out the correct order, or sequence of nucleotide bases in a fragment of DNA. In DNA sequencing, most of the copies of a single-stranded DNA fragment which will be employed to synthesize a new DNA strand are made. Through DNA sequencing a gene can be characterized in terms of a linear sequence of AGCT bases which in turn, can be employed to predict the amino acid sequence of the corresponding protein employing the genetic code. There are three processes for finding out DNA sequences.
Chain-Termination Method (Sanger Method):
This method needs a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively labeled nucleotides and modified nucleotides which terminate DNA strand elongation.
The DNA sample is categorized into four separate sequencing reactions, having all four of the stranded deoxynucleotides and the DNA polymerase. To each and every reaction is added just one of the four dideoxynucleotides that are the chain-terminating nucleotides which terminate DNA strand extension and outcome in the DNA fragments of varying lengths.
The newly synthesized and labeled DNA fragments are heat denatured, and separated by size, through gel electrophoresis on a denaturing polyacrylamide-urea gel having each of the four reactions run in one of four individual lanes (lanes A, T, G, C) the DNA bands are then visualized through autoradiography or UV light and the DNA sequence can be directly read off the x-ray film.
Maxam and Gilbert's Chemical (that is, Degradation Method) the method is mainly based on chemical modification of DNA and subsequent clearage at specific bases; it needs potassium labeling at one end and purification of the RNA fragment to be sequenced. The chemical treatment with **** RNAs produces breaks at each and every nucleotide base. Therefore a series of labeled fragments is produced from the potassium labeled end to the first cut site in each molecule. The fragments in the four reactions are arranged or ordered side by side in gel electrophoresis for size separation.
The fragments are visualized if the gel is exposed to hydrolysis enzymes for autoradiography, yielding a sequence of cubes each corresponding to a RNA fragment from which the sequence might be determined.
Introduction to Metabolic pathways:
Metabolic pathways are a sequence of chemical reactions occurring in a cell. In each and every pathway or chemical reaction, a principal chemical is modified by a sequence of chemical reactions.
Enzymes catalyze these reactions, and often need dietary minerals, vitamins and other cofactors in order to function appropriately. Because of the many chemicals (that is, metabolites) that might be comprised metabolic pathways can be elaborate. Moreover, a number of distinct pathways co-exist in a cell; collectively termed as the metabolic network. Metabolic pathways are significant to the maintenance of homeostatis in an organism.
Meaning of Metabolism:
Metabolism: The sum of enzyme-mediated chemical reactions occurring in a cell or a whole organism. These chemical reactions are categorized into two: catabolism and anabolism.
Catabolism (catabolic pathway) is a metabolic pathway which discharges energy by breaking down complex molecules into the simpler compounds.
Anabolism (anabolic pathway) is a metabolic pathway which comprises the synthesis or assembling of organic molecules and new protoplasm.
Before the energy is stored in proteins, lipids and carbohydrates can be employed by the cell to do work, the molecules should be broken down in a sequence of chemical reactions and the energy produced utilized to synthesize adenosine triphosphate (ATP) - an adenine - having nucleoside triphosphate which discharges free energy when its phosphate bonds are hydrolyzed. ATP is considered as the universal energy currency of living organisms. The energy is employed to drive endergonic reactions in the cells.
The complete degradation of an energy-rich compound like glucose to carbon-dioxide and water comprises numerous enzymatically controlled reactions. The complete break-down of glucose comprises four phases:
a) Glycolysis (Phase I)
b) Fermentation or oxidation of Pyruvic acid to acetyl-coenzyme A (Phase II).
c) The Kreb's Critic Acid Cycle (Phase III).
d) Oxidative phosphorylation (Phase IV).
The first sequence of reactions in the degradation of glucose is known as glycolysis and the most significant features of glycolysis are:
1) Each and every molecule of glucose (C6H12O6) is broken down to two molecules of Pyruvic acid (C3H4O3).
2) Two molecules of ATP are employed to initiate the procedure; afterward four new ATP molecules are synthesized by a total gain of two molecules of ATP and two molecules of NADH (that is, an energy or electron carrier molecule).
3) ATP molecules are generated by substrate level phosphorylation and NAD+ (that is, nicotinamide adenine dinucleotide) is decreased to NADH by oxidation of the food.
4) The total energy yield from glycolysis, per glucose molecule is 2 ATP and 2 NADH (Phase I).
5) No molecular oxygen is employed; glycolysis can take place by aerobic metabolism or with no oxygen (that is, anaerobic metabolism).
6) The reactions of glycolysis take place in the Cytosol of the cell in the cytoplasm exterior the mitochondria. While the Krebs cycle enzymes and intermediates are dissolved in the fluid in the mitochondria.
1) The fate the Pyruvic acid generated all through glycolysis based on oxygen supply. Fermentation facilitates a cell to continue the reactions of glycolysis in the absence of oxygen.
2) In the absence of O2, the Pyruvic acid might be decreased by NADH to CO2 and ethyl alcohol or lactic acid, in a process termed as fermentation. In the process, the glycolytic pathway leads to the making of alcohol or lactic acid and it let the cell to continue synthesizing ATP molecules through the breakdown of nutrients under anaerobic conditions.
3) Beneath aerobic conditions the Pyruvic acid can be further oxidized, by accompanying the synthesis of ATP. The method starts when Pyruvic acid moves from the cytoplasm to the inner compartment of the mitochondrion to form two each of acetyl-COA, CO2 and NADH (Phase II).
3) The acetyl-CoA made is fed to a complex circular sequence of reactions.
The Krebs Citric Acid Cycle:
The cycle functions as a metabolic 'furnace' which oxidizes organic fuel derived from pyruvate, the product of glycolysis.
1) In the course of cycle, two carbon atoms are lost as CO2, a molecule of ATP is synthesized, and 8 electrons and 8 hydrogen are picked up through carrier compounds, making three molecules of NADH and one of FADH2.
2) The cycle produces 1 ATP per turn by substrate phosphorylation.
3) As one molecule of glucose gives increase to two molecules of acetyl-CoA, two turns of the cycle take place for each molecule of glucose oxidized.
Oxidative Phosphorylation (Electron Transport Chain):
A sequence of oxidation - reduction reactions which passes electrons from higher energy levels to lower energy levels. As an outcome, ADP is phosphorylated to make ATP. The Exergonic reaction of hydrogen having oxygen to form water discharges a huge amount of energy in the form of light and heat. In cellular respiration, an electron transport chain breaks the 'fall' of electrons in this reaction to a sequence of small steps and stores some of the discharged energy in a form which can be employed to make ATP.
The electron transport chain molecules are mainly found in the inner membrane of the mitochondria. The transfer of electrons all along the electron-transport chain outcomes in the pumping of H+ ions from the inner compartment of the mitochondria to the outer compartment.
As an outcome, the H+ concentration rises in the outer compartment and an electrostatic and osmotic concentration gradient is made up across the inner membrane. Special enzyme complexes, termed as ATP synthetases act as H+ ion channels in the inner mitochondrial membrane. As H+ ions move down the electrochemical gradient via the complex, energy is discharged and can be employed to synthesize ATP. The net number of new ATP molecules generated by the complete metabolic breakdown of glucose is generally 36, two from glycolysis, two from Krebs cycle and 32 from electron-transport phosphorylation in the mitochondria. This yield represents around 39% of the energy of glucose; the rest of the energy is discharged as heat.
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