The Chemical Synthesis of Oligonucleotides and the Phosphoramidite Method
By Klaus D. Linse
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...
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...oligomer, possible truncated failure sequences with free 5’hydroxyl ends, byproducts of deprotection, and silicates from hydrolysis of the glass support. Different purification methods can be used to separate the product oligonucleotide from the contaminating species.
Figure 18: summary of Phosphoamidite Oligonucleotide Synthesis Method.
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the gel electrophorese, 2.5ul of the 10x loading dye is added to each PCR reaction tube. The gel
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These six samples (crude -/+, broken -/+, and whole -/+) were spun at 5000 rpm, and the resulting pellets were isolated and resuspended in DNase buffer. The set of suspensions labeled with a (+) was incubated in DNase enzyme for 15 minutes, and afterwards incubated in 15 uL of STOP solution. All six samples were lysed for DNA extraction with DNA extraction buffer, and micro-centrifuged at maximum speed. To precipitate the extracted DNA, the supernatants from each of the six samples were added to their correspondingly labeled micro-centrifuge tubes containing 7% ethanol (Parent et. al, 2008To bind the DNA, the ethanol lysate mixtures were transferred to labeled spin columns and spun for one minute in the micro-centrifuge at maximum speed. To wash the bound DNA, the spin columns were washed and spun three times at maximum speed. In order to elute the bound DNA, the samples were washed in 80 uL of distilled water and spun again for 2 minutes at maximum speed (Parent et. al,
DNA is made up of nucleotides, and a strand of DNA is known as a polynucleotide. A nucleotide is made up of three parts: A phosphate (phosphoric acid), a sugar (Deoxyribose in the case of DNA), and an organic nitrogenous base2 of which there are four. The four bases are as followed: Adenine (A), Cytosine
Polymerase Chain reaction (PCR) is used to isolate a predetermined strand of DNA on the double helix. Once the desired DNA is isolated it is able to be copied as much as needed (2). In this experiment PCR was used to isoloate Vangl2 from Zebra fish embryos. In a PCR experiment, a primer is used to find and isolate the desired nucleotide sequence of DNA (2). In this experiment two primers were used as follows:
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For example, for the domestic horse, all the proteins had around 379 amino acids. The proteins needed to have the same number of amino acids to construct the cladogram. This process was repeated four more times since there were five-mammalian
Amino acids are the building blocks of all proteins, which are complex molecules containing elements such as nitrogen, oxygen, carbon and hydrogen. Amino acids linked by peptide bonds create polypeptides. When there are many polypeptides linked together in a chain, and it is folded to fit a particular function, it creates a protein. However if it was not folded into a certain shape, it would not become a protein, rather just a Polypeptide.
1% agarose gel was made and allowed to solidify, and put in electrophoresis. 2. DNA was incubated with restriction endonuclease. 3. 20 µl of DNA was loaded into each well according to instructions.
Figure 3 shows the structure of the prepared protein along with the ligand depicted in green colour.
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There is no surprise that food is important in all aspects of our lives—it is shared amongst families, celebrated as a major part of our culture, and crucial to our daily routine that keeps us fit, healthy, and active. Today’s western culture glorifies a skewed perspective on how food is supposed to fit into our lives. Somehow this perception has led us to believe we no longer have the time or money it takes to prepare a wholesome, healthy meal that is shared at the dinner table with family. Instead, we are trained to want a meal that is fast, cheap, and easy. This meal is usually highly processed and filled with sugars and fats. This has led us to a problem of epidemic proportions characterized by the rapid increase in obesity and diabetes.
A polypeptide chain is a series of amino acids that are joined by the peptide bonds. Each amino acid in a polypeptide chain is called a residue. It also has polarity because its ends are different. The backbone or main chain is the part of the polypeptide chain that is made up of a regularly repeating part and is rich with the potential for hydrogen-bonding. There is also a variable part, which comprises the distinct side chain. Each residue of the chain has a carbonyl group, which is good hydrogen-bond acceptor, and an NH group, which is a good hydrogen-bond donor. The groups interact with the functional groups of the side chains and each other to stabilize structures. Proteins are polypeptide chains that have 500 to 2,000 amino acid residues. Oligopeptides, or peptides, are made up of small numbers of amino acids. Each protein has a precisely defined, unique amino acid sequence, referred to as its primary structure. The amino acid sequences of proteins are determined by the nucleotide sequences of genes because nucleotides in DNA specify a complimentary sequence in RNA, which specifies the amino acid sequence. Amino acid sequences determine the 3D structures of proteins. An alteration in the amino acid sequence can produce disease and abnormal function. All of the different ways
One of the most important uses of organic compounds is in medicine. All living things have four organic molecules - carbohydrates, proteins, lipids and nucleic acids. Without carbon chains and nucleic acids, DNA would not exist. Enzymes which produce chemica...