CHAPTER 1
Nucleic Acid Structure and Basic Analysis
Ralph Rapley
University of Hertfordshire, School of Life and Medical Sciences, College Lane, Hatfield, AL10 9AB, UK
1.1 Introduction
Major advances have been made in gene analysis and genomics in recent years and this has been accelerated by the continued development and refinement of methods and techniques for studying nucleic acids. One major area of current research is the identification and diagnosis of diseases that are multifactorial in nature. There are numerous diseases where analysis of genomes has provided insights into the disease and one particularly notable example is oncology. Molecular genetic analysis of this area has allowed a discrete set of cellular genes, termed oncogenes and tumour suppressor genes, to be identified and characterised. These genes and the proteins and enzymes they encode are major components of cell signalling and the cell cycle and are intimately involved in many aspects cell regulation. The disruption of oncogenes and tumour suppressor genes contributes to the early changes required for cancers to develop. Identification and analysis of these genes and genomes has already provided information for use in diagnostics and prognostics and a number have been shown to be biomarkers of a particular cancer type. In a number of cancers well-defined molecular events have been correlated with mutations in oncogenes and therefore in the corresponding protein. It is already possible to screen and predict the outcome of some disease processes at an early stage, a point which itself raises significant ethical dilemmas. The application of molecular biology has allowed understanding of cellular processes both in normal and disease states. The advent of this type of analysis has given rise to the development of personalised or precision medicine and there is now great promise in further developments in drug discovery and molecular gene therapy. A number of genetically engineered therapeutic proteins and enzymes have been developed and are already having an effect on disease management. In addition the correction of disorders at the gene level using gene therapy is also under way. Perhaps one of the most startling applications of molecular biology to date is indeed gene editing and the development of gene modifications methods such as the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated system 9 (cas9) system, which may have a profound effect on treating genetic-based diseases. 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 function in vivo and in 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 intended to provide an overview of the general features of nucleic acid structure and function and describe some of the basic methods used in their isolation and analysis.
1.2 Structure of Nucleic Acids
1.2.1 Primary Structure of Nucleic Acids
DNA and RNA are macromolecular structures composed of regular repeating polymers formed from nucleotides. 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 (Figure 1.1). The carbon atoms of the sugar are designated 'prime' (l', 2', 3', etc.). To distinguish them from the carbons of the 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' position of a nucleoside by an ester linkage. 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' hydroxyl of another, thus generating a 5' to 3' phosphodiester bond between adjacent sugars; this process can be repeated indefinitely to give long polynucleotide molecules. DNA has two such polynucleotide strands. However, since each strand has both a free 5' hydroxyl group at one end, and a free 3' hydroxyl at the other end, each strand has a polarity or directionality. The polarities of the two strands of the molecule are in opposite directions, and thus DNA is described as an 'anti-parallel' structure.
The purine 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 other pyrimidines are 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 the structure, which distinguishes one DNA (or RNA) from another.
1.2.2 Secondary Structure of Nucleic Acids
The two polynucleotide chains in DNA are usually found in the shape of a right-handed double helix, in which the bases of the two strands lie in the centre of the molecule, with the sugar–phosphate backbones on the outside.A crucial feature of this double-stranded structure is that it depends on the sequence of bases in one strand being complementary to those in the other strand. A purine base attached to a sugar residue on one strand is always hydrogen bonded to a pyrimidine base attached to a sugar residue on the other strand. Moreover, adenine (A) always pairs with thymine (T) or uracil (U) in RNA, via two hydrogen bonds, and guanine (G) always pairs with cytosine (C) by three hydrogen bonds (Figure 1.2). When these conditions are met a stable double-helical structure results in which the backbones of the two strands are, on average, a constant distance apart. Thus, if the sequence of one strand is known, that of the other strand can be deduced. The strands are designated as plus (+) and minus (-) and an RNA molecule complementary to the minus (-) strand is synthesised during transcription. The base sequence may cause significant local variations in the shape of the DNA molecule, and these variations are vital for specific interactions between the DNA and various proteins to take place. Although the three-dimensional structure of DNA may vary it generally adopts a double helical structure termed the b form or b-DNA in vivo.
It has been well recognized for some time that DNA as a structure may be chemically modified without the underlying DNA sequence being altered. One of the most important modifications is the addition of a methyl (CH3) group to cytosine, termed DNA methylation and catalysed by DNA methyltransferases. This results in what some describe as the fifth base in DNA. Approximately 1.5% of human DNA is methylated (termed the epigenome) and the methylation status appears to have a profound effect on gene expression, where hypomethylation appears to promote gene expression. This feature of gene expression control is termed epigenetics and is a complex process which is also extended to the modification of histone proteins and some small RNA molecules involved in gene expression control. Importantly, epigenetics appears to play a role in disease states, such as cancers and certain neurological diseases, which may lead to a new means of future treatment.
1.2.3 Denaturation of Double-stranded DNA
The two anti-parallel strands of DNA are held together only by the weak forces of hydrogen bonding between complementary bases, and partly by hydrophobic interactions between adjacent, stacked base pairs, termed base-stacking. Little energy is needed to separate a few base pairs, and so, at any instant, a few short stretches of DNA will be opened up to the single-stranded conformation. However, such stretches immediately pair up again at room temperature, so the molecule as a whole remains predominantly double-stranded.
If, however, a DNA solution is heated to approximately 90 °C or above there will be enough kinetic energy to denature the DNA completely, causing it to separate into single strands. The temperature at which 50% of the DNA is melted is termed the melting temperature or Tm, and this depends on the nature of the DNA. If several different samples of DNA are melted, it is found that the Tm is highest for those DNA molecules that contain the highest proportion of cytosine and guanine, and Tm can actually be used to estimate the percentage (C + G) in a DNA sample. This relationship between Tm and (C + G) content arises because cytosine and guanine form three hydrogen bonds when base-paired, whereas thymine and adenine form only two. Because of the differential numbers of hydrogen bonds between A–T and C–G pairs those sequences with a predominance of C–G pairs will require greater energy to separate or denature them. The conditions required to separate a particular nucleotide sequence are also dependent on environmental conditions, such as salt concentration. If denatured DNA is cooled it is possible for the separated strands to re-associate, a process known as renaturation.
Strands of RNA and DNA will associate with each other, if their sequences are complementary, to give double-stranded, hybrid molecules. Similarly, strands of labelled RNA or DNA, when added to a denatured DNA preparation, will act as probes for DNA molecules to which they are complementary. This hybridisation of complementary strands of nucleic acids is a cornerstone for many molecular biology techniques and is very useful for isolating a specific fragment of DNA from a complex mixture. It is also possible for small single-stranded fragments of DNA (up to 40 bases in length), termed oligonucleotides, to hybridise to a denatured sample of DNA. This type of hybridisation is termed annealing and again is dependent on the base sequence of the oligonucleotide and the salt concentration of the sample.
1.3 Isolation and Separation of Nucleic Acids
1.3.1 Isolation of DNA
The use of DNA for analysis or manipulation usually requires that it is isolated and purified to a certain extent. DNA is recovered from cells by the gentlest possible method of cell rupture to prevent the DNA from fragmenting by mechanical shearing. This is usually in the presence of ethylenediaminetetraacetic acid (EDTA) which chelates the Mg2+ ions needed for enzymes that degrade DNA, termed DNAse. Ideally, cell walls, if present, should be digested enzymatically (e.g. by lysozyme treatment of bacteria), and the cell membrane should be solubilised using detergent. If physical disruption is necessary it should be kept to a minimum and should involve cutting or squashing of cells, rather than the use of shear forces. Cell disruption (and most subsequent steps) should be performed at 4 °C, using glassware and solutions which have been autoclaved to destroy DNAse activity (Figure 1.3).
After release of nucleic acids from the cells, RNA can be removed by treatment with ribonuclease (RNAse) that has been heat-treated to inactivate any DNAse contaminants; RNAse is relatively stable to heat as a result of its disulphide bonds, which ensure rapid renaturation of the molecule on cooling. The other major contaminant, protein, is removed by shaking the solution gently with water-saturated phenol, or with a phenol–chloroform mixture, either of which will denature proteins but not nucleic acids. Centrifugation of the emulsion formed by this mixing produces a lower, organic phase, separated from the upper, aqueous phase by an interface of denatured protein. The aqueous solution is recovered and deproteinised repeatedly, until no more material is seen at the interface. Finally, the deproteinised DNA preparation is mixed with two volumes of absolute ethanol and the DNA is allowed to precipitate out of solution in a freezer. After centrifugation, the DNA pellet is redissolved in a buffer containing EDTA to inactivate any DNAses present. This solution can be stored at 4 °C for at least a month. DNA solutions can be stored frozen, although repeated freezing and thawing tends to damage long DNA molecules by shearing.
The procedure described is suitable for total cellular DNA. If the DNA from a specific organelle or viral particle is needed, it is best to isolate the organelle or virus before extracting its DNA, since the recovery of a particular type of DNA from a mixture is usually rather difficult. Where a high degree of purity is required DNA may be subjected to density gradient ultracentrifugation through caesium chloride which is particularly useful for the preparation plasmid DNA. It is possible to check the integrity of the DNA by agarose gel electrophoresis and determine the concentration of the DNA by using the fact that 1 absorbance unit at a wavelength of 260 nm (A260) equates to 50 µg ml-1 of DNA and thus:
50 × A260 = concentration of DNA sample (µg ml-1)
Contaminants may also be identified in the sample by employing scanning UV-spectrophotometry from 200 nm to 300 nm. A ratio of A260: A280 of approximately 1.8 indicates that the sample is free of protein contamination, which absorbs strongly at 280 nm.
1.3.2 Isolation of RNA
The methods used for RNA isolation are very similar to those described above for DNA; however, RNA molecules are relatively short and therefore less easily damaged by shearing, so cell disruption can be rather more vigorous. RNA is, however, vulnerable to digestion by RNAses, which are present endogenously in various concentrations in certain cell types and exogenously on fingers. Gloves should therefore be worn, and a strong detergent should be included in the isolation medium to immediately denature any RNAses. Subsequent deproteinization should be particularly rigorous, since RNA is often tightly associated with proteins. DNAse treatment can be used to remove DNA, and RNA can be precipitated by ethanol. One reagent which is commonly used in RNA extraction is guanidinium thiocyanate (GTC) which is both a strong inhibitor of RNAse and a protein denaturant. It is possible to check the integrity of an RNA extract by analysing it by agarose gel electrophoresis. The most abundant RNA species are the rRNA molecules. For prokaryotes these are 16S and 23S and for eukaryotes the molecules are slightly heavier at 18S and 28S. These appear as discrete bands following agarose gel electrophoresis and, importantly, if intact indicate that the other RNA components, such as mRNA, are likely to be intact also. Electrophoresis is usually carried out under denaturing conditions to prevent secondary structure formation in the RNA. The concentration of the RNA may be estimated by using UV-spectrophotometry. At 260 nm 1 absorbance unit equates to 40 µg ml-1 of RNA and therefore:
40 × A260 = concentration of RNA sample (µg ml-1)
Contaminants may also be identified in the same way as for DNA by scanning UV-spectrophotometry, however in the case of RNA an A260: A280 ratio of approximately 2 would be expected for a sample containing no contaminants.
In many cases it is desirable to isolate eukaryotic mRNA which constitutes only 2–5% of cellular RNA from a mixture of total RNA molecules. This may be carried out by affinity chromatography on oligo(dT)-cellulose columns. At high salt concentrations, the mRNA containing poly(A) tails binds to the complementary oligo(dT) molecules of the affinity column, and so mRNA will be retained; all other RNA molecules can be washed through the column by further high-salt solution. Finally, the bound mRNA can be eluted using a low concentration of salt. Nucleic acid species may also be subfractionated by more physical means such as electrophoretic or chromatographic separations based on differences in nucleic acid fragment sizes or physicochemical characteristics.
1.3.3 Automated Nucleic Acid Extraction
Many current molecular biology methods and their reagents can now be found in the form of a kit from a manufacturer, such as Sigma, or can be automated. The extraction of nucleic acids by these means is no exception. The advantage of their use lies in the fact that the reagents are standardised and quality control tested, providing a high degree of reliability. For example, glass bead preparations for DNA purification have been used increasingly and with reliable results. Small compact column-type preparations, such as Qiagen spin columns, are also used extensively in research and in routine DNA extraction. Essentially the same reagents for nucleic acid extraction may be used in a format that allows reliable and automated extraction. The process can also be automated with a low-throughput Qiacube system. Further methods are also available using kit-based extraction methods for RNA; these in particular have overcome some of the problems of RNA extraction, such as RNAse contamination. A number of fully automated nucleic acid extraction machines, such as the Qiasymphony system are now employed in areas where high throughput is required, e.g. clinical diagnostic laboratories. Here the raw samples, such as blood specimens, are placed in 96- or 384-well microtitre plates and these undergo a set computer-controlled processing pattern carried out robotically. Thus the samples are rapidly manipulated and extracted in approximately 45 min without any manual operations being undertaken. Nucleic acids can be extracted from a variety of samples, including blood, serum, frozen tissue sections, formalin-fixed paraffin-embedded (FFPE) tissue sections and biopsies among others, and all have their own unique challenges in the extraction process. Reliable and effective methods are a crucial element for further analysis, such as DNA sequencing and long-term storage in DNA banks.
