I.

Introduction
Nucleic acids, like proteins, also have levels of structure. The primary structure of nucleic
acids is the order of bases in the polynucleotide sequence, and the secondary structure is the three-
dimensional conformation of the backbone. The tertiary structure is specifically the supercoiling of
the molecule. The monomers of nucleic acids are called nucleotides, which consist of three parts a
nitrogenous base, a sugar, and a phosphoric acid residue all of which are covalently bonded
together (Campbell and Farrell, 2009).
Nucleic acids are the storage of genetic information and they directly participate in gene
replication, transcription, and expression, and the control of nucleic acids can lead to modulation
and regulation of genetic information flow and gene expression. Moreover, nearly all the functions
of nucleic acids are accomplished by interacting with proteins through non-covalent interactions.
Recognition and selectivity are achieved through non-covalent contacts between virtually all
biopolymers that are present in living organisms. The formation and dissociation of these weak
interactions between nucleases and DNA or RNA are central to the recombination, repair, and
replication of these molecules in cells (Zhang, Xi and Chattopadhyaya, 2011).
There are two principal types of nucleic acids, DNA (deoxyribonucleic acid) and RNA
(ribonucleic acid). Important differences between the two appear in their secondary structures. Even
though nothing in nucleic acid is directly analogous to the quaternary structure of proteins, the
interaction of nucleic acids with other classes of macromolecules to form complexes is similar to the
interactions of the subunits in an oligomeric protein. One well-known example is the association of
RNA and proteins in ribosomes, the polypeptide-generating machinery of the cell; another is the
self-assembly of tobacco mosaic virus, in which the nucleic acid strand winds through a cylinder of
coat-protein subunits (Campbell and Farrell, 2009).
The order of bases in the nucleic acids of DNA contains the information necessary to produce
the correct amino acid sequence in the proteins of cells. The nucleic acid bases are of two types
pyrimidines and purines.

Figure 7.1. Structures of the common nucleobases.

Three pyrimidine bases (single-ring aromatic compounds) cytosine, thymine, and uracil commonly
occur. The primary difference between the nucleobases of DNA and RNA is that for the former
thymine is present while for the latter this is replaced by uracil. The common purine bases (double-
ring aromatic compounds) are adenine and guanine, both of which are found in RNA and DNA. In
addition to these five commonly occurring bases, there are unusual bases, with slightly different
features, that are found principally, but not exclusively, in a type of RNA called transfer RNA
(Campbell and Farrell, 2009

Figure 7.2. Structures of some of the less common nucleotides.

The linkage between monomers in nucleic acids involves formation of two ester bonds by
phosphoric acid. The hydroxyl groups to which the phosphoric acid is esterified are those bonded
to the 3 and 5 carbons in adjacent residues. The resulting repeated linkage is a 35-
phosphodiester bond. The nucleotide residues of nucleic acids are numbered from the 5 end, which
normally carries a phosphate group, to the 3 end, which normally has a free hydroxyl group. A
portion of a DNA chain differs from the RNA chain just described only in the fact that the sugar is
2-deoxyribose rather than ribose (Figure 7.3). The three-dimensional structure of DNA (Figure 7.4),
which is relatively simple to the linear polymeric form of the nucleic acid, involves regular double
helices. There are also larger-scale regular structures, but it has been clearly established that the
information content of DNA is held at the level of the base sequence itself. RNA molecules are linear
polymers as well, but are smaller than genomic DNA. RNA molecules also tend to have a less-
regular three-dimensional structure than DNA (Zvelebil and Baum, 2008).
 Figure 7.3. Differences in a DNA (left) and RNA (right) chain.

Figure 7.4. The double helix of DNA.

The prokaryotic and eukaryotic cells, which principally distinguishes living organisms, contain
a nuclear region, which houses the genetic material of the cell surrounded by cytoplasm. The genetic
material of a prokaryotic cell is present in a nucleoid, which is a poorly demarcated region of the
cell that lacks a boundary membrane to separate it from the surrounding cytoplasm. In constrast,
eukaryotic cells possess a nucleus: a region bounded by a complex membraneous structure called
the nuclear envelope. In prokaryotic cells, the amounts of DNA are relatively small e.g., the DNA
content of bacteria ranges from about 600,000 base pairs to nearly 8 million base pairs and
encodes between about 500 and several thousand proteins. Both prokaryotic and eukaryotic cells
have DNA-containing chromosomes. Eukaryotic cells possess a number of separate chromosomes,
each containing a single linear molecule of DNA. On the other hand, nearly all prokaryotes that
have been studied contain a single, circular chromosome. More importantly, the chromosomal DNA
of eukaryotes, unlike that of prokaryotes, is tightly associated with proteins to form a complex
nucleoprotein material known as chromatin (Karp, 2009).
To facilitate the analysis of genomic DNA, it is first necessary to isolate genomic DNA in a
useful (purified and high molecular weight) state. The isolation of any macromolecule from cells or
tissue typically involves a sequential process of selective degradations and selective recoveries,
such that biomolecules other than the desired macromolecule (DNA) are eliminated. In this way, the
desired macromolecule is recovered in a pure, non-degraded state. For the isolation of genomic
DNA, this can be done through a series of defined biochemical steps involving the selective
degradation of proteins and lipids followed by the selective recovery of nucleic acids. This is often
followed by the selective degradation of RNA and the selective recovery of DNA. These steps have
classically included digestion with proteinases to degrade proteins in the presence of detergents to
break down membranes and other lipid-containing structures, followed by organic extraction to
separate the degraded proteins and lipids from the nucleic acids. Treatment with RNase can be
used to eliminate RNA selectively followed by ethanol precipitation to selectively recover the
genomic DNA. Alternatively commercially available kits such as the Qiagen Genomic DNA Isolation
kit, can be used to accomplish the DNA genome isolation (Williams, Slatko and McCarrey, 2007).
The choice of method for DNA extraction depends on a number of factors, including the
sample type and quantity; the speed and in some cases ability to automate the extraction
procedure; the success rate which involves extracting the maximum amount of DNA from a sample;
the chemicals that are used in the extraction; and the cost of the procedure. A DNA extraction
procedure is facilitated greatly by the use of Proteinase K, detergents, and chelating agents. The
detergents dissolve cell membranes and denature proteins present in the cell. Proteinase K is a
potent enzyme that degrades proteins. Sources of DNA genome that have low deoxyribonculease
(DNase) activity should be considered. The presence of high DNase activity in some tissues makes it
difficult to obtain an appropriate source for DNA isolation. The use of chelating agents inhibit
DNase that degrade DNA by binding divalent cation ions that are necessary cofactors for the
nucleases. (McClatchey, 2002).
Organic agents such as phenol, chlofororm and isoamylacohol, or mixtures of the three or
the last two are usually used for treating the cell extracts from a biological source containing the
genomic DNA in order to selectively isolate the target substance. Moreover, to assess the DNA
quality and get the amount of DNA in extracted product solution, the method of agarose gel
electrophoresis is often used to separate DNA molecules according to different molecular sizes.
DNA is separated by the electric filed to push the DNA molecules through the agarose gel, because
the main factor that affects its movement through the gel is the DNA molecular length. Shorter DNA
molecules have lower molecular weight so that they move faster through gels compared with
fragments with higher molecular weight (Xu, 2016). After electrophoresis, the molecules in the gel
can be stained to make them visible. The most common dye used to make DNA bands visible for
agarose gel electrophoresis is ethidium bromide (EtBr). By running DNA through an EtBr-treated gel
and visualizing it with UV light, any band containing more than ~20 ng DNA becomes distinctly
visible.
The DNA isolate from extraction can be characterized by obtaining the absorbance at 260
and 280 nm, finding the ratio of the two in order to determine the purity of the isolate and
converting the absorbance at 260 nm into an appropriate value that estimates the concentration of
the genomic DNA isolate (Vogt, Tilley and Edmonds, 2015).
The experiment aims to isolate genomic DNA from young malunggay leaves (Moringa
oleifera L.) using an appropriate extracting organic solvent, to study the theory and principle of
agarose gel electrophoresis and how to use the process and equipment involved as a tool for
checking purity of the genomic DNA isolate and to characterize the DNA isolate using
spectrophotometry.