Read an Excerpt
Molecular Biology and Biotechnology
By Ralph Rapley, David Whitehouse The Royal Society of Chemistry
Copyright © 2015 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84973-795-1
CHAPTER 1
Molecular Biology Techniques and Bioinformatics
RALPH RAPLEY AND NATALIA HUPERT
1.1 INTRODUCTION
The development of methods and techniques for studying processes at the molecular level has led to new and powerful ways of isolating, analysing, manipulating and exploiting nucleic acids. The completion of numerous genome projects has allowed the continued 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 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.
1.1.1 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' (1', 2', 3', 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' 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 a molecule that distinguishes one DNA (or RNA) from another.
1.1.2 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 that in the other. 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 BDNA in vivo.
1.1.3 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 DNAs which 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 is also dependent on environmental conditions such as salt concentration. If melted DNA is cooled, it is possible for the separated strands to reassociate, 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.
1.2 ISOLATION AND SEPARATION OF NUCLEIC ACIDS
1.2.1 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 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. 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) which has been heat treated to inactivate any DNase contaminants; RNase is relatively stable to heat as a result of its disulfide 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 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 of 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 equates to 50 µg/mL of DNA and thus:
50×A260=concentration of DNA sample (µg/mL) (1.1)
Contaminants may also be identified in the sample by employing scanning UV-spectrophotometry from 200 nm to 300 nm. A ratio of 260 nm:280 nm of approximately 1.8 indicates that the sample is free of protein contamination, which absorbs strongly at 280 nm.
1.2.2 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, very 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 deproteinisation 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 that 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 indicate that the other RNA components, such as mRNA, are likely to be intact. This 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 of RNA and therefore:
40×A260=concentration of RNA sample (µg/mL) (1.2)
Contaminants may also be identified in the same way as for DNA by scanning UV-spectrophotometry, however in the case of RNA a 260 nm:280 nm ratio of approximately 2 would be expected for a sample containing no protein.
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.
1.2.3 Enzymes Used In Molecular Biology
The discovery and characterisation of a number of key enzymes has enabled the development of various techniques for the analysis and manipulation of DNA. In particular, the enzymes termed type II restriction endonucleases have come to play a key role in all aspects of molecular biology. These enzymes recognise certain DNA sequences, usually 4–6 bp (base-pairs) in length, and cleave them in a defined manner. The sequences recognised are palindromic or of an inverted repeat nature (Figure 1.4). That is, they read the same in both directions on each strand. When cleaved they leave a flush-ended or staggered (also termed a cohesive-ended) fragment depending on the particular enzyme used. An important property of staggered ends is that those produced from different molecules by the same enzyme are complementary (or 'sticky') and so will anneal to each other. The annealed strands are held together only by hydrogen bonding between complementary bases on opposite strands. Covalent joining of the ends of each of the two strands may be carried out using the enzyme DNA ligase. This is widely exploited in molecular biology to enable the construction of recombinant DNA, i.e. the joining of DNA fragments from different sources. Approximately 500 restriction enzymes have been characterised that recognise over 100 different target sequences. A number of these, termed isoschizomers, recognise different target sequences but produce the same staggered ends or overhangs. A number of other enzymes have proved to be of value in the manipulation of DNA, as summarised in Table 1.1, and are indicated at appropriate points within the text (Figure 1.5).
1.2.4 Electrophoresis of Nucleic Acids
Electrophoresis in agarose or polyacrylamide gels is the most usual way to separate DNA molecules according to size. The technique can be used analytically or preparatively, and can be qualitative or quantitative. Large fragments of DNA such as chromosomes may also be separated by a modification of electrophoresis termed pulsed field gel electrophoresis (PFGE). The easiest and most widely applicable method is electrophoresis in horizontal agarose gels, followed by staining with ethidium bromide. This dye binds to DNA by insertion between stacked base pairs (intercalation), and it exhibits a strong orange/red fluorescence when illuminated with ultraviolet light. Very often electrophoresis is used to check the purity and intactness of a DNA preparation or to assess the extent of an enzymatic reaction during, for example, the steps involved in the cloning of DNA (Figure 1.6). For such checks 'mini-gels' are particularly convenient, since they need little preparation use small samples and provide results quickly. Agarose gels can be used to separate molecules larger than about 100 bp. For higher resolution or for the effective separation of shorter DNA molecules polyacrylamide gels are the preferred method.
When electrophoresis is used preparatively, the piece of gel containing the desired DNA fragment is physically removed with a scalpel. The DNA may be recovered from the gel fragment in various ways. This may include crushing with a glass rod in a small volume of buffer, using agarase to digest the agarose leaving the DNA, or by the process of electroelution. In this method, the piece of gel is sealed in a length of dialysis tubing containing buffer, and is then placed between two electrodes in a tank containing more buffer. Passage of an electrical current between the electrodes causes DNA to migrate out of the gel piece, but it remains trapped within the dialysis tubing, and can therefore be recovered easily. An alternative to conventional analysis of nucleic acids by electrophoresis is through the use of microfluidic systems such as an Agilent Bioanalyser. These are automated machine based systems utilising carefully manufactured chip based units, frequently termed 'lab on a chip', where microlitre samples volumes may be used. Using complex miniaturised channels coupled with flow pumps and detection systems, nucleic acids can be separated and assayed rapidly. With the aid of advanced software, the analysis may provide much of the data required for nucleic acid and protein analysis such as sizing and quantitation.
(Continues...)
Excerpted from Molecular Biology and Biotechnology by Ralph Rapley, David Whitehouse. Copyright © 2015 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.
