31
CONTENTS
(T = theory, X = experiment) Page
------
Lecturers ...... 3
(T) Protein electrophoresis (J. Mork)...... 4
(T) Genetic interpretation of banding patterns on gels (J. Mork)..... 9
(X) Isoelectric focusing of LDH in gadoids (J. Mork) ...... 13
(T) Analysis of genetic differentiation and structure (J. Mork)...... 14
(X) Starch gel electrophoresis of fish tissue enzymes (M-A. Østensen) 18
(T/X) Bruk av isozyner for å studere hybridsoner
(diploid-tetraploid hybridsone hos orkidéer) (S. Såstad) ...... 23
(T/X) RFLP markers (the cDNA RFLP SypI*) (S. Karlsson) ...... 28
(T/X) DNA markers (mini/micro-satellites,PCR reaction) (T. Ryan) .... 32
APPENDICES ...... 40
(T) Hints on software for statistical tests and genetics (J. Mork).... 41
(T) Measurements of similarities and distances (B.I. Honne) ...... 45
(T) Analysis of data from Avena sterilis RAPDs (B.I. Honne) ...... 47
(T) DNA analysis techniques (P. Galvin)...... 56
(T) Plant DNA markers (M. Heun)...... 78
BI 315
POPULATION GENETICS METHODOLOGY COURSE
AT TBS AUTUMN 2000 (WEEK 47 & 48)
Prof. Mork and Prof. Fenster are responsible for the course.
Personnel involved:
Name / Telephone / Telefax / E-mailProf. Jarle Mork, TBS / 47 73 59 15 89 / 47 73 59 15 97 /
Prof. Charles Fenster, Bot.Inst., KB-Fak. / 47 73 55 0337 / 47 73 59 61 00 /
Prof. Manfred Heun, NLH Ås / 47 64 94 76 91 / 47 64 94 76 79 /
Prof. Bjørn Ivar Honne, Planteforsk / 47 74 82 62 11 / 47 74 82 88 11 /
Dr. Tony Ryan, Max Planck, Leipzig / 49 341 9952 593 / 49 341 9952 555 /
Dr. Sigurd Såstad, Bot. Avd. VM / 47 73 59 22 51 / 47 73 59 22 49 /
Cand. Scient. Sten Karlsson, TBS / 47 73 59 15 80 / 47 73 59 15 97 /
Leading Eng. Mari-Ann Østensen, TBS / 47 73 59 67 99 / 47 73 59 15 97 /
Web address for course information: http://www.ntnu.no/~jmork/jmork/courses/315H00/AGEN00.html
Lecture
Protein electrophoresis
(J. Mork, TBS)
Principle
In an electric field (DC), charged particles like molecules in aquous solution migrate towards the electrode of opposite charge. Amphotheric molecules (e.g., proteins and peptides) may have a large number of charged groups, and their net charge will depend on the pK value (the dissociation constant) of their charged groups which depends on the pH of the aquous medium. Due to differences in charge, different molecules in a mixture will migrate with different velocities and thereby be separated in single fractions. In addition to pI (the isoelectric point) of a protein/peptide, its electrophoretic migration velocity is influenced by the type, concentration and pH of the buffer, by the temperature and field strength (the voltage between the electrodes), as well as of type and pore size of the stabilizing medium (paper, agar, starch etc).
Allelic variation (substitution of amino acids) in proteins usually does not affect molecular size appreciably. However many such substitutions result in a change of net charge which alters the electrophoretic mobility and makes the different genotypes detectable by electrophoresis. Of special value for population genetics is that such ’biochemical’ variation is co-dominant and allows the scoring of both allels at a locus (i.e. no dominance or recessivity).
Electrophoretic separations can take place in free solution (e.g. in capillars) or in stabilizing media such as silica plates, variuos paper types, or gels. The development of stabilizing media during the last 50 years has been from paper via agar, cellolose-acetat, agarose, starch and to synthetic polymers of acrylamid. At the same time there has been a development of new techniques from the ‘continuous’ separation based on charge, via separation based on molecule size, to disc electrophoresis, immuno-electrophoresis and isoelectric focusing.
No other biochemical technique has shown such a rich diversification and played such a central role in modern biochemistry. By electrophoresis it is possible to obtain very efficient separations with relatively simple equipment. Application areas range from biological and biochemical research to protein chemistry, pharmacology, forensic medicin, veterinary science, food quality control, molecular biology and genetics. Samples may be as diverse as whole cells or particles, proteins, peptides, amino acids , organic acids and bases, nucleic acid, drugs, and pesticides - in short, all substances that can carry electric charges.
In biological research it will probably become increasingly important to choose the most adequate separation technique for a specific purpose, and to be able to carry out the practical procedures involved in electrophoresis. An thorough guide to the techniques is Westermeier (1993).
Basically, there are three different principles for electrophoretic separation:
a) common zone electrophoresis b) isotachophoresis (ITP) c) Isoelectric focusing (IEF)
Similarities and differences between these three is shown in the following figure (mr = relative mobility (to a standard), pK = the dissociation constant, T = trailing ion, L = leading ion, and pI = isoelectric point (i.e., the pH where the amphoteric compound has no net charge).
a) In electrophoresis we use a buffer system that is homogeneous over the entire separation area to ensure equal pH. This is valid also for disc electrophoresis, although there the buffer system is discontinuous in the start of the experiment in order to concentrate the substances in a very narrow start band (i.e., utilizing the isotachophoresis effect).
b) In isotachophoresis (ITP) the separation takes place in a discontinuous buffer system. The ionized compound migrates trapped between a front ‘leading electrolute’ and a tail ‘trailing electrolyte’ which migrates with the same velocity. The various components of the sample distribute themselves according to their respective electrophoretic mobilities and form a ‘stack’ with the front bands closely behind the ‘leading ion’ and the tail bands just in front of the ‘trailing ion’. Isotachophoresis is mostly used in quantitative separations (and as a ‘stacking and concentration’ step in disc electrophoresis).
c) In isoelectric focusing the separation takes place in a pH gradient created by several hundred different ampholytes with different isoelectric points and with buffer capacity at their isoelectric points. As anolyte and catholyte are used e.g. 1 M phosporic acid and 1 M sodium hydroxide. The function of these is to keep the gradient ‘in place’ between the electrodes. IEF is very sensitive to electro-osmosis (see below), and the supporting medium should thus be as electrically inert (usual media are polyacrylamide and specifically pure agarose). IEF is suitable for amphoteric substances in which the net charge depends on pH, e.g. proteins and peptides. The molecules migrate to the position in the pH gradient where their net charge is zero (i.e., their isoelectric point) and the mobility is zero. Should they diffuse away from this position, the buffer effect of the nearby ampholytes will induce a charge which will force them back in position between ampholytes with slightly lower and slightly higher pI. The higher the field strength (voltage drop) is, the more concentrated the bands will be, thereof the name ‘focusing’. IEF is mostly used for qualitative characterization of substances or mixtures of substances and purity control, but also for preparative purposes. The pH gradient gels can easily be made inhouse, but are also commersially available as ready-made gels with different gradients (e.g., 2-10, 3-9, 4-9, 4-6, 5-7, 5-8 etc). Ampholyte mixtures are marketed by many firms (e.g., Pharmacia’s «Pharmalytes» , Serva’ «Serva-Lytes», Bio-Rad’s «Biolytes» (the two latter are identical). Ready-made gels cost much more than home-made (~600 kr vs ~80 kr per gel of size 24,5x12,5x0,1 cm). Both home-made and ready-made gels have fridge shelf lives of up to one year. The chemical composition (ampholyte type etc) as well as the linearity of the pH gradient vary considerably between brands. There is also some variation in prices.
The buffer system in electrophoresis
Common electrophoresis takes place in a buffer with accurate pH and constant ionic strength. The ionic strength should be as low as practically possible in order to achieve high field strength (voltage drop) and thereby rapid migration/separation, but not so low that the proteins are not pH-buffered by the medium, or the buffer capacity is used up before the separation is completed. During electrophoresis, the buffer ions migrate through the gel in the same manner as the sample molecules; anions towards the anode and cations towards the cathode (in vertical electrophoresis the buffer pH is set so that all molecules of interest migrates towards the same electrode; in practice from the upper to the lower part of the gel).
The buffer ions are responsible for most of the conductivity in the supporting medium. The lower the conductivity (i.e. the ionic strength) , the less Joule heat is produced, and the higher field strength can be employed without overloading the cooling capacity of the system. The cooling is usually achieved by a cooling plate connected to a circulating thermostat. The buffer capacity must be large enough to ensure constant pH during the entire experiment. The capacity is regulated by the amount of buffer and/or its concentration.
The problem of electro-osmosis
If the gel support (glass plate, plastic film etc) or the separating medium itself have electrical charges, a phenomenon called electro-osmosis occurs. If the charges are negative, water in the buffer will migrate towards the cathode and carry sample molecules with them (socalled cathodic drfit). This can either counteract or increase the ordinary electrophoretic mobility. The high voltages employed makes IEF particularly sensitive for electro-osmosis, not least by the use of media which are not totally electrically inert (like some brands of «IEF-grade» agaroses). However, the pheomenon is also common in ordinary electrophoresis in agar, paper and cellulose-acetat. Gels made from starch and polyacrylamide have no electro-osmosis.
Joule heat and the cooling system
After separation, the bands should be as distinct and concentrated as possible. Prolonged analysis time will usually lead to unwanted band diffusion. One way to shorten the analysis time (i.e., diffusion time) is to increase the field strength (i.e the applied voltage) over the gel. However, this will also increase the Joule heat produced (cf figure below), and this may lead to problems like protein denaturation, gel artifacts (melting agarose), «smearing» of bands, etc. It is therefore very important to design the experiment so that the separation takes place as quickly as possible, but with no more Joule heat produced than can be carried away by the cooling system. Very basic knowledge about the aparatus and to the relations between voltage, current, conductivity, affect and Joule heat makes it relatively easy to avoid problems of this kind. It is the total applied effect (measured in Watts) that determines the heat production in the system that must be matched by the cooling plate capacity. The current is necessarily the same at any point between the electrodes. In places where the resistence is large (conductivity low), either because the cross-section of the circuit «lead» is small or because there are few ions present, the system will «use up» most of the voltage to «force» the current through. With constant current and high field strength these circuit parts will use more of the available wattage and therefore produce more Joule heat than other parts. Typically, this is in the gel, which often has both a smaller cross-section and a lower conductivity than the buffer chamber. Therefore, the cooling plate is placed under the gel. Cooling plates made of metal (NB! must be electrically insulated!) or ceramics are much more efficient than those of glass, and will allow higher effect and hence shorter analysis time. As a rule of thumb, a 1 mm thick gel on a metall or ceramic cooling plates can tolerate an applied wattage of 0.2 W/cm2 gel without substantial temperature increase (i.e., not more than 2-3 degrees centigrade higher in the gel than in the coolant).
This Joule heat produced is directly dependent on the effect applied to the electrophoresis system. The effect obeys the following simple equiation:
Effect (watt) = Voltage (volt) x current (ampere)
Tissue samples; properties and treatment of proteins
An important criterion for the choise of electrophoretic method is the type of sample which is to be analysed. One line can be drawn between denaturing methods (e.g. SDS electrophoresis) and methods where the biological activity of the protein must be preserved. Another line is between amphoteric compounds (proteins, peptides) and non-amphoteric substances. Common to them all is that the sample should not contain particles, oil drops etc because these may block the pores of the medium.
Protein extracts are usually prepared by homogenization in aquous solutions (aqua. dest. or buffer). Since e.g. enzyme loci may be differently manifested in different tissue types, it can often be useful and efficient to homogenize several tissue types together (e.g. muscle and liver) in the same vial in order to have more loci represented. Usually, a few seconds of forceful mincing of the tissue samples (1 ccm in double amount liquid) with a glass rod is sufficient tot break the cell walls and release the proteins in animal tissues (plant tissues may need more labour). It is usually desirable to centrifuge the homogenates (e.g. 10.000 G for 10 minutes) to avoid cell debris in the extracts which may block the pores of the medium.
Some proteins are very tough and can stand rough treatment in the field as well as in the laboratotium, while others are extremely sensitive for factors like elevated temperatures, oxydation, low ionic strength, too high or too low pH (low pH is usually worse than high). The properties of different proteins must be learned by experience in each organism and each organ.
However, there are a few general rules. For example, proteins (e.g. enzymes) which usually perform their function at relatively high temperatures will better tolerate high temperature and storage in the laboratory. Thus, mammalian proteins are usually more stable at room temperature than proteins from e.g. fish.
In any case, the best results are usually obtained when using fresh (not frozen) samples.
Bacterial degradation can be a serious problem. It is adviceable to strive for as sterile treatment as possible during all stages of sample preparation, to keep the samples chilled, and to avoid drying-out as well as to much sample dilution. In some cases, the use of a bacteriostat like Na-azid can be necessary to avoid bacterial growth. In addition, the pH of the extraction buffer should not be too far from the natural milieu of the protein since physiological conditions will usually increase its life-time. If samples are to be stored for prolonged periods (e.g. more than 1-2 weeks) this should take place at ultra-low temperatures (e.g., at -70 degrees C or lower) in a «bio-freezer», on dry ice, or in liquid nitrogen, and packed in a way which avoids drying-out and exposure to air oxygen. One should be aware, however, that some proteins will not tolerate the freezing/thawing process. In such cases freeze-drying may be an alternative.