development of new exciting areas of biological sciences such as biotechnology,
genome mapping, molecular medicine and gene therapy.
In considering the potential utility of molecular biology techniques it is important
to understand the basic structure of nucleic acids and gain an appreciation of how this
dictates the functionin vivoandin vitro. Indeed many techniques used in molecular
biology mimic in some way the natural functions of nucleic acids such as replication
and transcription. This chapter is therefore intended to provide an overview of the
general features of nucleic acid structure and function and describe some of the basic
methods used in its isolation and analysis.5.2 Structure of nucleic acids
5.2.1 Primary structure of nucleic acids
DNA and RNA are macromolecular structures composed of regular repeating polymers
formed fromnucleotides. These are the basic building blocks of nucleic acids and are
derived from nucleosides which are composed of two elements: a five-membered
pentose carbon sugar (2-deoxyribose in DNA and ribose in RNA), and a nitrogenous
base. The carbon atoms of the sugar are designated ‘prime’ (l^0 ,2^0 ,3^0 , etc.) to distinguish
them from the carbons of nitrogenous bases of which there are two types, either a
purine or a pyrimidine. A nucleotide, or nucleoside phosphate, is formed by the
attachment of a phosphate to the 5^0 position of a nucleoside by an ester linkage
(Fig. 5.1). Such nucleotides can be joined together by the formation of a second ester
bond by reaction between the phosphate of one nucleotide and the 3^0 hydroxyl of
another, thus generating a 5^0 to 3^0 phosphodiester bondbetween adjacent sugars; this
process can be repeated indefinitely to give long polynucleotide molecules (Fig. 5.2).
DNA has two such polynucleotide strands; however, since each strand has both a free
50 hydroxyl group at one end, and a free 3^0 hydroxyl at the other end, each strand has
a polarity or directionality. The polarity of the two strands of the molecule is in
opposite directions, and thus DNA is described as anantiparallelstructure (Fig. 5.3).
Thepurine bases(composed of fused five- and six-membered rings), adenine (A)
and guanine (G), are found in both RNA and DNA, as is the pyrimidine (a single six-
membered ring) cytosine (C). The otherpyrimidinesare each restricted to one type of
nucleic acid: uracil (U) occurs exclusively in RNA, whilst thymine (T) is limited to
DNA. Thus it is possible to distinguish between RNA and DNA on the basis of the
presence of ribose and uracil in RNA, and deoxyribose and thymine in DNA. However,
it is the sequence of bases along a molecule that distinguishes one DNA (or RNA) from
another. It is conventional to write a nucleic acid sequence starting at the 5^0 end of the
molecule, using single capital letters to represent each of the bases, e.g. CGGATCT.
Note that there is usually no point in including the sugar or phosphate groups, since
these are identical throughout the length of the molecule. Terminal phosphate groups
can, when necessary, be indicated by use of a ‘p’; thus 5^0 pCGGATCT 3^0 indicates the
presence of a phosphate on the 5^0 end of the molecule.139 5.2 Structure of nucleic acids