2.4 Analysis of nucleic acids by electrophoresis and hybridisation
Nucleic acids can be separated according to size by gel electrophoresis, most commonly performed using a horizontal gel (Figure 7a). This is in contrast to the vertical gel electrophoresis set-up, which is generally used for analysis of proteins.
The size of DNA molecules is usually expressed in terms of the number of base pairs. The phosphate backbone, containing one phosphate per nucleotide, gives the molecules a uniform negative charge per base pair. The secondary structure of DNA is such that these negatively charged phosphates lie on the outside of the molecule, meaning that, unlike proteins, no denaturation or pretreatment with SDS is required. Thus the degree of migration in the gel is directly related to the length of the DNA chain.
DNA electrophoresis is usually performed using an agarose (polymerised galactose) gel matrix for molecules larger than 500 bp, and the size of the molecule is determined by comparison with a calibration set of DNA fragments, as shown in Figure 7b. DNA fragments smaller than 500 bp are usually studied using a polyacrylamide gel matrix, which has a smaller pore size and so allows greater length discrimination of small DNA molecules. After electrophoresis, DNA is detected by ‘staining’ with ethidium bromide, the dye that intercalates between stacked bases (Figure 6d) and in doing so becomes fluorescent when exposed to UV irradiation.
RNA can also be examined by electrophoresis, but due to the physical characteristics of RNA molecules there are two important differences compared with DNA electrophoresis. The first is that RNA must be pretreated to disrupt any internal base pairing; that is, its secondary structure is effectively destroyed. This pretreatment is necessary because the extensive base pairing in RNA molecules means that they have very diverse conformations, which can affect the relative mobility of the molecules. RNA samples are therefore pretreated by heating or by the addition of agents such as formamide, which disrupts the hydrogen bonds and denatures the RNA. The second important difference between electrophoresis of DNA and that of RNA is that the latter must be performed under conditions that buffer against alkalinity, since RNA is vulnerable to hydrolysis in alkaline conditions.
Measuring the mobility of nucleic acids through gels allows their size to be estimated, but a second technique, called hybridisation, further extends the utility of electrophoresis. Nucleic acids in solution or attached to solid-phase matrices can be dissociated into single strands by denaturing at 100 °C. In the case of DNA, denaturation can also be achieved by treatment with alkali.
Why does heat or alkali treatment lead to the denaturation of duplex DNA?
Heat provides energy to the system so that base pairing, favoured at lower temperatures, is disrupted. Alkali leads to the deprotonation of individual atoms in bases leading to loss of polarity and, as a consequence, loss of hydrogen bonding.
When two such denatured polynucleotide strands, containing complementary stretches of bases, are mixed together under conditions that allow base-base hydrogen bonding to occur, these stretches associate with each other in a process called strand annealing. This property of two polynucleotide strands is exploited in many techniques used in molecular biology and is called hybridisation. It can occur between DNA–DNA, RNA-RNA and RNA–DNA strands.
Hybridisation has become a standard laboratory analytical tool for detecting specific nucleotide sequences in intact chromosomes or in nucleic acid mixtures fixed to a solid support. Short lengths of single-stranded DNA or RNA are labelled with radioactive or fluorescent tags, and then allowed to interact with denatured target strands. The labelled nucleic acid, also known as aprobe, will then hybridise to any section of the target that has complementary stretches of bases and this hybridisation can be visualised after detection of the radioactive or fluorescent tag.
One application of hybridisation techniques is in the analysis of nucleic acids after electrophoresis, as shown in Figure 7c and d. After electrophoresis, nucleic acid molecules are transferred onto solid membranes, usually by capillary action in a process called blotting. Blotting of nucleic acids is analogous to the transfer of proteins from a polyacrylamide gel, though in the latter, transfer is achieved by applying an electrical current across the gel. Transfer of DNA molecules is called Southern blotting and transfer of RNA molecules is called Northern blotting. (If you are interested, the box that follows describes these blotting techniques.) A complex mixture of nucleic acids, separated according to their length by electrophoresis, can be transferred to a membrane by blotting and individual chains identified using a complementary labelled nucleic acid probe. Examples of applications of these techniques include the identification of DNA fragments generated by digestion of genomic DNA with restriction enzymes (which cleave the backbone at specific sites; see Figure 7d) or of mRNA species in whole cellular RNA extracts.
Box 4: Study of gene expression
The first stage of gene expression involves transcribing the gene into an RNA transcript. Identification of a transcript of a gene in a cell or tissue is therefore the most direct way of determining whether the gene is activated. The simplest procedures for studying gene expression are based on identifying specific mRNA molecules using blotting and hybridisation techniques. The technique is based on a method devised for DNA identification by the British scientist Ed Southern. He invented the technique of electrophoretic separation and subsequent immobilisation of DNA onto a filter membrane. This procedure is named Southern blotting, after him. Similar methods are used today to separate and identify RNA (named northern blotting to contrast with Southern blotting), and protein molecules (western blotting).
The total RNA content of a cell or tissue is isolated and purified. The purified RNA is then separated by specialised forms of gel electrophoresis, and transferred onto a nylon membrane. Specific mRNA molecules are then identified by hybridisation. In hybridisation, a complementary sequence (called a ‘probe’) to the nucleic acid of interest (in this case, a specific mRNA) is artificially ‘labelled’ and incubated with the filter membrane. Probes are usually synthetic single-stranded DNA oligonucleotides, and can be labelled using radioisotopes such as 32P or 35S, or by coupling to enzymes that can produce a coloured reaction product. If the mRNA of interest is present in the filter membrane, the labelled probe anneals with it, forming a duplex, which is bound to the membrane. The label can then be visualised using autoradiography, or by the addition of a suitable reagent to act as a substrate for the enzyme and to produce a coloured product. This whole process is called northern blotting; the main features of the technique are shown below.
In-situ hybridisation: identification of specific mRNA molecules in intact cells
The presence of specific mRNA molecules in intact cells can be detected by the technique known as hybridisation. This method is based on the same principle of hybridisation of a labelled probe to the mRNA described above. However, the cells are not disrupted, but tissue sections or whole cells (e.g. in culture) are used. The cells are made permeable to facilitate the penetration of the probe, and thus localise the mRNA within the cells.
The total RNA content of cells/ tissues is isolated.
The different RNA molecules are separated on a size basis by gelelectrophoresis. RNA ‘markers’ of known size are included so the size of the RNAspecies in the samples can be estimated.
The gel is blotted onto a filter membrane, which is placed directly on the gel, followed by several layers of absorbent paper; clingfilm is placed around the gel to form a seal. The RNA is blotted onto the filter as the buffer solution soaks into the paper by capillary action, drawn by the dry, absorbent material above. The filter is then baked in a vacuum oven at 90°C in order to ‘fix’ the RNA on the filter.
The blot is then hybridised with a radioactively labelled DNA probe to identify specific mRNA molecules. Finally, the filter membrane is processed by autoradiography to visualise the labelled probe attached to the target RNA molecule.
In a similar manner, DNA and RNA probes can be used to indicate the position of a specific DNA sequence on a chromosome in a technique called in situ hybridisation (ISH). Results of ISH using a fluorescently labelled DNA probe (fluorescence in situ hybridisation, FISH) against a human X chromosome and a Drosophila polytene chromosome are shown in, respectively, Figure 8a and b. ISH can also be used to detect the distribution of specific mRNAs within tissues.
Hybridisation of complementary nucleic acid strands has also been exploited in the recent development of gene chips or microarrays. In these investigations, single-stranded DNA molecules are attached to a solid support (a membrane or a glass slide) and labelled DNA or RNA is used to probe the array, as shown in Figure 8c. Image analysis software can be used to quantify how much hybridisation occurs. By using many thousands of target DNAs, such as copies of genes or markers along a chromosome, this approach can be used to analyse hybridization to many targets simultaneously. One major application of this technique is to use a gene chip that carries target DNAs for every gene within an organism. After hybridisation with fluorescently labelled whole-cell mRNA, the expression of each individual gene can be quantified. This technique can be exploited to detect differences between different cell or tissue types, different developmental stages or to compare physiological and disease states.