Scanning electron microscopy
Scanning electron microscope (SEM) serves to image micro-morphological structures, which are smaller than 0.001mm (=1 µm). Instead of light waves electron waves scan the surfaces of objects. . In the vacuum of the sample chamber an electron beam hits the samples and generates secondary electrons, which are detected and converted into electronic signals. These images can be photographed and digitally examined.
Pollen, diatoms and leaves are among the objects whose surfaces are most frequently examined in the SEM for waxes, hairs or conspicuous structures. Small morphological differences are often of help in differentiating between species.
In order to enlarge objects under a microscope as high as possible, light penetration must be optimal. To achieve this, they are cut in very thin slices or squashed. Small and thin objects suchas diatoms are transferred directly onto the glass slide.
The number of chromosomes in a plant can be determined from tissues with a great many nuclear divisions such as root tips, for example. The tissue is then macerated in an acid-reactive solution, stained and squashed under slight pressure between slide and cover slip, until it has spread out in a single-cell layer. In order to analyse the anatomy of plant organs such as flowers or seeds, these are embedded in a suitable medium such as wax, ice or plastic and cut with a microtome. These wafer-thin sections are lined up on slides, stained and glued with a cover slip to a permanent mount.
Isolation of DNA
DNA (Deoxyribo Nucleic Acid) is a primary carrier of genetic information for all living things. It is a double stranded molecule, which takes the form of a screw-shaped spiral ladder or double helix. Besides a sugar-phosphate backbone, DNA consists of four building blocks, the bases Adenine, Cytosine, Guanine and Thymine. The order of the bases – which is known as a sequence – is often characteristic for species or even individuals. Specific DNA fragments, or genes, contain ‘blueprints’ for proteins or for molecules used in protein synthesis. Depending on the species, it is distributed during cell division among a varied number of chromosomes. Apart from the nucleus, shorter DNA molecules are found in the ‘powerplants of the cells’, the mitochondria; in algae and plants, it is also present in the photosynthesising chloroplasts. In order for DNA to be examined, it must be separated from all residual cell and tissue component parts. For this tissue samples are dried and chopped up. The cell contents are then macerated and partly broken down by means of enzymes. Proteins and polysaccharides are precipitated and, together with cell and tissue remains, removed with the help of a centrifuge. After that the raw DNA is bound to a substrate, purified with alcohol and, as pure DNA, dissolved in an aqueous buffer. It can now be directly analysed and is available long term for further investigations.
DNA can be cut using enzymes into pieces of differing length. When these sections are unravelled according to their size, an individual unique pattern is produced: the genetic fingerprint. The more similar the patterns of two living beings are, the more closely they are related to one another.
For the fingerprinting method AFLP genomic DNA samples are first treated with restriction enzymes. Subsequently, some of the DNA sections are amplified by means of Polymerase Chain Reaction (PCR) and labelled with the appropriate dyes. Then this mixture of DNA fragments is separated in the electric field according to their size. This process is called electrophoresis. The resulting pattern can be translated into a numerical table and statistically evaluated.
Genetic fingerprinting is particularly well suited compare closely related organisms. With its help it is possible to distinguish within species different sub-species, varieties or populations.
Polymerase Chain Reaction
The American Kary Mullis invented the Polymerase Chain Reaction (PCR) method in 1983. It allows even the smallest amounts of DNA, even if they come from very old tissue samples or from well preserved fossils, to be multiplied a million times over. For this Mullis was awarded the Nobel Prize for Chemistry in 1993.
Precisely determined DNA sections of a sample are copied using PCR. To this end, the double-stranded source DNA is first separated into single strands. Then the added artificial starter DNA, or primer, anneals to the single strands. In a third stage a special enzyme, DNA polymerase, with the addition of individual DNA building blocks, converts these single strands into identical double strands. By repetition of this procedure, the source DNA can be exponentially multiplied in just a few cycles.
In this way even a small quantity of DNA, as found in a few pollen grains or in a single cell, can be amplified to such an extent that precise examination becomes possible.
For successful analysis of DNA it is important to assess the quality of the DNA samples. Furthermore, at every stage in the process of genetic fingerprinting and DNA sequencing, the deployed molecular methods must be evaluated to determine whether or not they have been successful. Molecular samples are thus tested using agarose gel electrophoresis and absorption spectroscopy.
Agarose gel electrophoresis separates the DNA molecules by size according to their movement in the electric field. A fluorescent dye makes the molecules visible in UV light. The average size of the isolated DNA is a measure of its quality.
Absorption spectroscopy is used to verify the purity of a DNA sample and search for inhibiting molecules. The concentration of DNA in the sample can also indirectly be calculated by this method.
DNA sequence analysis ‘Sanger method’
DNA sequence analysis using the method pioneered by Frederick Sanger is carried out in a largely automated way. Replicated DNA fragments in a sample are thus separated again into single strands. Unlike amplification using PCR, only a single DNA strand is converted with the help of DNA polymerase enzyme. The mixture of individual bases, which are now added, contains chemically altered chain termination bases. According to the base (A, G, C or T), these are labeled with different fluorescent dyes.
This produces several incomplete copies of the DNA fragment, which can be separated by electrophoresis according to length. The fluorescence signals are detected by a detector and represented in a colour-coded chromatogram. They directly reproduce the sequence of the bases of the sequenced DNA section.
Next Generation Sequencing
In recent years scientists have shown an increasing interest in decoding the entire genetic makeup of organisms, with the aim of better understanding the ‘blueprints of life’ and the biological processes dependent on it. At the same time, new techniques have been developed and existing processes greatly advanced. These are often referred to as high-throughput sequencing or next generation sequencing.
The technique used for NGS are is so efficient that, with just one test run up to 520 million bases can be identified. The ascertained sequences are based on short-chain DNA fragments which overlap with one another multiple times. In order to decode a genome, these are synthesised using very powerful computers similar to those used in cryptography until a complete version is reconstructed.