Nucleotide Units

A number of factors combine to lower the susceptibility of phosphoric mono-, di-, and tri-esters to the type of nucleophilic attack required for intemucleotide-bond formation.8 Although a P=0 bond is present, the polarization of this bond is not so great as with an analogous C=0 bond, due to ptt - dn back-bonding into empty, phosphorus d-orbitals.150 Furthermore, the pKa values for the first dissociations of both phosphoric mono- and di-esters are approximately unity,8 which means that, in reactions in pyridine, the anions are involved. Partial delocalization of the formal, negative charge into phosphorus 

The types of activated-phosphate intermediate-species that could arise during reactions catalyzed by DCC or arenesulfonyl chlorides 

Yields from Condensation Employing Various Reactants for 100 h 

One variable that seems to influence the yields in the nucleophilic attack of hydroxyl groups on activated phosphate groups is the relative proportion of these two components in reaction mixtures. A 1 1 ratio of reactants may not be ideal54 if a considerable proportion of the nu-cleotidic component is converted into pyrophosphate, or if, as with activation by 2,4,6-triisopropylbenzenesulfonyl chloride (TPS) further reaction occurs after formation of one phosphoric diester linkage to 

Another variable is the relative amounts of nucleotide component and condensing agent in joining-reactions. The occurrence of side reactions catalyzed by DCC is minor, so a large (5-10-fold) excess of the reagent is routinely used to optimize yields and minimize reaction times. However, after prolonged reaction in pyridine, byproducts 

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DNA from a gene contains hundreds to thousands of nucleotide units for which the sequence is needed in order to interpret its code. Sequencing methods require only small amounts (5 (tg) of purified DNA, which can be produced by cloning. Automated sequencers are available that can daily sequence DNA containing hundreds of nucleotide units. 

In the human cell there are 23 pairs of chromosomes containing approximately 3000 million base pairs of DNA. Short sequences of DNA, perhaps with as few as 20 nucleotide units and sometimes radiolabeled, can be obtained either by chemical synthesis (gene machine) or from cloning. These short sequences can be used to probe for a complementary sequence by looking for the position to which they bind to any DNA sample under investigation, from blood for example. Such probes can detect as little as 100 fg of DNA and are the basis of forensic genetic fingerprinting tests. 

The primary stmcture of DNA is based on repeating nucleotide units, where each nucleotide is made up of the sugar, ie, 2 -deoxyribose, a phosphate, and a heterocycHc base, N. The most common DNA bases are the purines, adenine (A) and guanine (G), and the pyrimidines, thymine (T) and cytosine (C) (Fig. 1). The base, N, is bound at the I -position of the ribose unit through a heterocycHc nitrogen. 

The first one consists of 11-12 water molecules per nucleotide unit, which are coordinated directly to sites of the DNA double helix. Two of these water molecules are bound very tightly to the ionic phosphate residue and cannot be removed without completely destroying the structure of DNA. There are four other water molecules... 

Molecules of DNA and RNA are polynucleotides, polymeric species built from nucleotide units. Polymerization takes place when the phosphate group of one nucleotide (which is the conjugate base of an organic phosphoric acid) condenses... 

Examples polyethylene nylon. See also copolymer. polynucleotide A polymer built from nucleotide units. Examples DNA RNA. 

A complete molecule of DNA consists of two complementary strands. These strands are held together hy hydrogen bonds between the bases on the nucleotide units, as shown in Figure 2.21. 

Final question how big are the nucleic acids How many nucleotide units do we typically find in DNA or RNA The answer is enormously variable. There are small RNA molecules that contain 25 or fewer nucleotides there are also RNA molecules that contain thousands of nucleotides. But for really, really big molecules, we turn to DNA, which may have tens of thousands of nucleotides linked together Specific examples follow below but we need some additional insights first. 

The initial VNTRs were several hundred nucleotide units long requiring extended laboratory periods for the various segments to separate on the gel. Today, most tests employ shorter, 3 5 nucleotides long, VNTRs that allow for more rapid movement on the gel, resulting in faster and less costly results. It also allows for the production of a greater number of sequences that are looked at, and hence a greater ability to match or not match the results. These shorter sequences are called short tandem repeats (STRs). 

Addition of new nucleotide units to the growing chain occurs in the 5 to C direction, and is catalysed by the enzyme DNA polymerase. The most important step is the addition of a 5 -mononucleoside triphosphate to the free 3 -hydroxyl group of the growing chain as the 3 -hydroxyl attacks the triphosphate and expels a diphosphate leaving group. 

FIGURE 8-4 Deoxyribonucleotides and ribonucleotides of nucleic acids All nucleotides are shown in their free form at pH 7.0. The nucleotide units of DNA (a) are usually symbolized as A, G, T, and C, sometimes as dA, dG, dT, and dC those of RNA (b) as A, G, U, and C. In their free form the deoxyribonucleotides are commonly abbreviated dAMR dGMR dTMR and dCMP the ribonucleotides, AMR... 

The successive nucleotides of both DNA and RNA are covalently linked through phosphate-group bridges, in which the 5 -phosphate group of one nucleotide unit is... 

FIGURE 8-7 Phosphodiester linkages in the covalent backbone of DNA and RNA. The phosphodiester bonds (one of which is shaded in the DNA) link successive nucleotide units. The backbone of alternating pentose and phosphate groups in both types of nucleic acid is highly polar. The 5 end of the macromolecule lacks a nucleotide at the 5 position, and the 3 end lacks a nucleotide at the 3 position. 

The nucleotide sequences of nucleic acids can be represented schematically, as illustrated on the following page by a segment of DNA with five nucleotide units. The phosphate groups are symbolized by (3, and each deoxyribose is symbolized by a vertical line, from C-l at the top to C-5 at the bottom (but keep in mind that... 

The triplets of nucleotide units in DNA determine the amino acids in a protein through the intermediary mRNA. One of the DNA strands serves as a template for synthesis of mRNA, which has nucleotide triplets (codons) complementary to those of the DNA. In some bacterial and many eukaryotic genes, coding sequences are interrupted at intervals by regions of noncoding sequences (called introns). 

In the next step, translation, the sequence of nucleotides in the newly synthesized mRNA strand is used to determine the sequence of amino acids in the protein to be synthesized. This is done by way of a genetic code, which was fully deciphered by 1966 and is shown in Figure 13.34. According to the genetic code, it takes three mRNA nucleotides—each three-nucleotide unit is called a codon—to code for a single amino acid. The mRNA nucleotide sequence AGU, for example, codes for the amino acid serine, and AAG codes for lysine. (Note from Figure 13.34 that more than one codon can call for the same amino acid.) A few codons, such as AUG and UGA, are the signals for protein synthesis to either start or stop. 

RNA codons are three-nucleotide units that either code for specific amino acids in a protein being synthesized or else signal the synthesis to either start or stop. For example, the codon CUA codes for the amino acid leucine. 

The genetic instructions for a cell are found in the DNA molecules. All DNA is derived from four different kinds of monomers, which we call nucleotides. DNA molecules are double-stranded two polymer chains are coiled together, their nucleotide units being associated as nucleotide pairs (see Fig. 5-7). The genetic messages in the DNA are in the form of... 

It has been shown that the code for specifying a particular amino acid in a protein is determined by a sequence of three nucleotides (a codon) in a DNA chain. There are four different kinds of nucleotide units in DNA. How many different codons exist Note that this is larger than the number of different amino acids (20) that are incorporated into proteins plus the three stop (termination) codons (see Tables 5-5 and 5-6 for a list of codons). 

The calibration of the E. coli genetic map in minutes was a temporary expedient. It was followed by physical maps expressed directly as micrometers of DNA length (total length 1.6 mm) or thousands of nucleotide units (kb). A physical map obtained by restriction enzyme mapping is shown in Fig. 26-4B. To obtain this map DNA fragments were prepared using specific restriction endonucleases (Section E, 1). 

Figure 27-14 Schematic representation of DNA polymerase action on a nicked strand of DNA in which the nick has been enlarged. At the catalytic center new nucleotide units are added at the 3 end of a growing strand. At the 3 -5 exonuclease site the 3 terminal nucleotide may be removed hydrolytically. This will happen to the greatest extent if the nucleotide is poorly paired in the duplex. At the 5 -3 exonuclease site nucleotides are hydrolytically removed from the 5 end of a strand in the chain.265,267...

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