Introduction
There is a number of different methods that can be used to sequence the whole genome of an organism, some of this methods are the first generation sequencing, next generation sequencing, shotgun sequencing and the third
First Generation Sequencing
The first step under this method is identifying and labelling the desired DNA molecule that is to be sequenced.
In the next step there is introduction of the four base destruction chemical reactions which are carried out. These are C+T, G, A+G and C. Each of the four base destruction chemical reactions destroys only one base of the sequenced desired DNA molecule. After several reactions are made there is a formation or creation of populations of same sized molecules
In the last step the created populations introduced to agel and then separated according to the size they possess.
Under the first generation sequencing we also have another method well known as the Sanger chain
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The first thing to be done to accomplish the sequencing through this method is preparing the DNA library for the desired genome. The DNA library is prepared under three phases, the first phase is cutting the genomic DNA into small pieces. This is done by physical shearing of the desired DNA molecule or by restricting it either using certain enzymes. The shearing of the DNA is only done if its pieces or fragments are very long. The adaptors, primers and barcodes are introduced to all the clones in our DNA library, reason being that there are no vectors in next generation DNA library. The adapters are used to bind cloned fragments onto the beads and the primers are essential for the sequencing of the clone by polymerase. The barcodes are used for amplicon sequencing. The last step under DNA library preparation is the size selection, only fragments of assured size are sequenced. Now the DNA can be sequenced using methods such as the Emulsion PCR and the Polony
Figure 2 shows the results of the electrophoresis. Lanes 5 and 7 indicate the fragments obtained when the plasmids are digested with both restriction enzymes, indicating the approximate fragment size for the hlyA gene, the pK184 plasmid and the pBluescript plasmid. This is useful for identifying the recombinant DNA needed for this experiment
Living organisms undergo chemical reactions with the help of unique proteins known as enzymes. Enzymes significantly assist in these processes by accelerating the rate of reaction in order to maintain life in the organism. Without enzymes, an organism would not be able to survive as long, because its chemical reactions would be too slow to prolong life. The properties and functions of enzymes during chemical reactions can help analyze the activity of the specific enzyme catalase, which can be found in bovine liver and yeast. Our hypothesis regarding enzyme activity is that the aspects of biology and environmental factors contribute to the different enzyme activities between bovine liver and yeast.
As the solution pH can influence the stability of NaClO-NH3 blend and the elimination of SO2, NOx, the impact of the pH of NaClO-NH3 blend solution on the instantaneous removal as well as the duration time was investigated, and the final pH after reaction was also detected and shown in Fig. 5. It can be seen that the variation of solution pH has a negligible effect on the desulfurization, but the elevated pH has a great promotion on the NOx removal, the efficiencies are significantly increased from 36% to 99% for NO2 in the pH range of 5–12 and from 19% to 65% for NO when the pH is between 5 and 10, after where, both of them are constant. Hence, the optimal pH of the NaClO-NH3 solution for the
Using PCR and Gel Electrophoresis to Determine Genotype. In certain situations, it is necessary to identify DNA retrieved from a sample. When there is a small sample in need of identification, Polymerase Chain Reactions are used to multiply the DNA. in the sample in many identical samples.
I would suggest to students performing the nitration to make sure their benzoic acid product is very fine and broken up before reacting it, as it has a tendency to clump together when it dries and thus proves very difficult to react in solution. I would also suggest keeping a very close eye on the temperature when adding the sulfuric/nitric acid mixture dropwise, as the reaction has a tendency to spike in temperature
The study of nucleic acids has now become a fruitful and dynamic scientific enterprise. Nucleic acids are of unique importance in biological systems. Genes are made up of deoxyribonucleic acid or DNA, and each gene is a linear segment, or polymer, of a long DNA molecule. A DNA polymer, or DNA oligonucleotide, contains a linear arrangement of subunits called nucleotides. There are four types of nucleotides. Each nucleotide has three components; a phosphate group, a sugar and a base that contains nitrogen within its structure. The sugar moiety in DNA oligonucleotides is always dexoyribose, and there are four alternative bases: adenine (A), thymine (T), guanine (G), and cytosine (C). The phosphate groups and the deoxyribose sugars form the backbone of each DNA stand. The bases are joined to the deoxyribose sugar and stick out to the side. Both oligomers, DNA and RNA, consist of 5’->3’ phosphodiester-linked nucleotide units that are composed of a 2’-deoxy-D-ribose (DNA) or D-ribose (RNA) in their furanose forms and a heteroaromatic nucleobase (A, T, G, and C; A, U, G, C), and the resulting oligonucleotide chain is composed of a polar, negatively charged sugar-phosphate backbone and an array of hydrophobic nucleobases. The amphiphilic nature of these polymers dictates the assembly and maintenance of secondary and tertiary structures the oligonucleotides can form. In the DNA duplex structure, genetic information is stored as a linear nucleotide code. This code can be accessed and replicated. RNA, or ribonucleic acid, is another structurally related essential biopolymer. RNA differs from DNA in having the sugar ribose in place of the deoxyribos...
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
This experiment synthesized luminol (5-Amino-2,3-dihydro-1,4-phthalazinedione) and used the product to observe how chemiluminescence would work. The starting material was 5-nitro-2,3-dihydrophthalazine-1,4-dione, which was, after addition of reaction agents, refluxed and vacuum filtered to retrieve luminol. Using two stock solutions, we missed our precipitated luminol with sodium hydroxide, potassium ferricyanide, and hydrogen peroxide, in their respective solutions, in a dark room, to observe the blue light
Genetics and the study of heredity began with Gregor Mendel, a monk that experimented with peas to show the passing of traits from “parent” to “child.” About 40 years later Thomas Hunt Morgan discovered the gene itself using fruit flies. This began the search for DNA, which was concluded in 1944 by Oswald Avery, Colin McLeod and Maclyn McCarty when they proved DNA was genetic material. Their discovery kicked-off the beginning of the DNA era in which all scientists were scrambling to find out more about this mysterious microscopic molecule. In addition, during this time the Human Genome Project was started. The project was dedicated to finding, identifying and sequencing DNA. The purpose, as listed by the United Stated Department of Energy Office of Science, was to identify all the approximately 20,000-25,000 genes in human DNA, determine the sequences of the 3 billion chemical base pairs that make up human DNA, store this information in databases, improve tools for data analysis, transfer related technologies to the private sector, and address the ethical, legal, and social issu...
Then the sequence was loaded into Velvet where it was trimmed to the desired k-mer length for alignment and contig formation. Mitos and MEGA alignment Explorer were also used in order to get the DNA sequence to a
Gene cloning works by first isolating the desired gene and ‘cutting’ it from the original chromosome using restriction enzymes. The piece of DNA is ‘pasted’ into a vector and the ends of the DNA are joined to the vector DNA by ligation. The vector is introduced into a host cell, often a bacteria or yeast, by a process called transformation. The host cells copy the vector DNA along with their own DNA, creating multiple copies of the inserted DNA. The vector DNA is separated from the host cells’ DNA and purified.
The discovery of genome sequencing by Fredrick Sanger and his team of researchers in the early 70’s gave rise to one of the most empirical research methods that was ever to exist. This revolutionary research technique has allowed scientists to finally encode organisms down to their most basic properties; helping massively in our understanding of pathways, reactions and functions of organisms. The technique involves analysing the DNA of an organism’s genome and therefore all the genes that compose it. The DNA from an organism is run through an electrophoresis gel and the sequence produced is taken up and interpreted by a computer program to then present the nucleotide sequence of the organism. Genome sequencing of pathogenic organisms has lead to huge advancements in the fight against infectious diseases within human and veterinary medicine; three notably virulent infectious diseases of the veterinary world are bluetongue virus, equine strangles and bovine tuberculosis (Goodhead, 2012).
Another way to extract DNA is by solution based methods. There are also different ways of solution base methods. The salting out method was used back around 1979. They used glass powder stuff to purify DNA. Similar steps are used through most of the solution extracts so they can contribute the DNA cells evenly. They usually combine nuclear acid. These solutions also use buffering with pacific Ph. to make a chemical change. After many steps through columns and buffering the DNA is later placed in distilled water after it has been cleaned.
The main purpose of DNA analysis is to get a visual representation of DNA that is left at the crime scene (Harris, 2013). DNA evidence is an instruction handbook and blueprint for everything in your body (Harris 2013). DNA seems very complex, but it's only made up of four nucleotides, which are Adenine, Cytosine, Guanine, and Thymine (Harris 2013). These nucleotides are base pairs and they join together. Adenine and thymine always join together as a set, and cytosine and guanine join together as a set (Harris 2013). In human cells, DNA is firmly bound into 23 pairs of chro...
Secondly the gene has to be cut from its DNA chain. Controlling this process are many restriction endonucleases (restriction enzymes). Each of these enzymes cut DNA at a different base sequence called a recognition sequence. The recognition sequence is 6 base pairs long. The restriction enzymes PstI cuts DNA horizontally and vertically to produce sticky ends.