Exercise No. 6 DNA Electrophoresis PDF

WORKSHEET 6- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Exercise no. 6 DNA electrophoresis Theory Student:  understands the physical basics of electrophoresis  knows the conditions necessary for electrophoresis  explains the role of a...

WORKSHEET 6- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Exercise no. 6 DNA electrophoresis Theory Student:  understands the physical basics of electrophoresis  knows the conditions necessary for electrophoresis  explains the role of an electrophoretic medium and its influence on the mobility of DNA molecules Electrophoresis is an analytical and seldom preparative technique used in chemistry and molecular biology. It is based on the movement of molecules with an electric charge, in relation to the liquid phase (solution) in an electric field created by the difference in electric potential. Therefore in a colloidal solution in which the electrodes are submerged and there is a difference in potential between them, the charged molecules move in the direction of the electrode with an opposite charge. If the colloidal molecules boast a negative charge, then they migrate to the positive electrode (anode) and the process is known as anaphoresis. On the other hand, in the case of positively charged molecules, their movement is directed towards the cathode, a negative electrode, and this process is known as cathaphoresis. However, electrophoresis performed in a free solution (liquid) is characterized by a very low effectiveness of separation. An important change was the development of electrophoresis with the use of electrophoretic media which ensure a higher:  stability,  repetition,  resolution. The medium cannot react with DNA, protein, or amino acids, since it is a separation due to physical and not chemical properties. A change in the concentrations of the medium should provide the opportunity to regulate the size of the pores and as a result a selection of the range of molecule separation. The electrophoretic medium can be tissue paper, cellulose nitrate, agarose, polyacrylamide, or others, which:  stabilize electrolyte,  contribute to better separation of macromolecules. The use of porous media strengthens the effect of separation by additional fractionation of macromolecules based on the principle of a molecular sieve. The most often used ones are currently gel electrophoreses with the use of agarose or polyacrylamide. Agarose electrophoresis. In this electrophoresis the electrophoretic medium is agarose. It is a natural polysaccharide which is a polymer of galactose derivatives. It is obtained through the purification of edible agar. Agarose is easily dissolved in water, at room temperature it turns into a gel, but the process is reversible. Page 1 of 11  WORKSHEET 6- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 The size of pores in agarose can be regulated using various concentrations (the higher the concentrations the more cross-linked and smaller the pores). Advantages of agarose electrophoresis:  possibility to separate large protein molecules,  high range of separated DNA fragments, o from several hundred bp to 40 kbp (in a stable electric field), o Mpz (in a changing electric field),  feasibility of gel preparation,  is non-toxic. Disadvantages of agarose electrophoresis  low mechanical resistance of agarose gels,  low-resolution ability of agarose gels  agarose is relatively expensive. DNA electrophoresis occurs when an electric current runs through the buffer, conducting electricity in which DNA molecules are located. This mobility is possible since DNA molecules in neutral pH have a negative charge and migrate to a positive electrode – anode. DNA electrophoresis in agarose gel is an analytical method that allows for separating DNA strand fragments of different sizes and evaluating this size (100 bp to 40 kbp). If the DNA molecules of various masses migrate in the solution, their speeds will be similar, and separation will not be possible using an electric current. However, if the migrating molecules under the influence of an electric current instead of in the buffer find themselves in the cross-linked agarose gel, then their movement will be affected. In such conditions, the size of the DNA molecules becomes the most critical factor in determining their migration speed since DNA molecules must migrate through the porous network of solidified agarose. The bigger the molecule, the slower the migration. The migration speed of linear dsDNA molecules is reversely proportional to the amount of base pairs. It is precisely reversely proportional to log10 of the molecular mass. The migration speed, therefore, depends on the size/mass of the molecule and not its charge. The relative mobility of DNA in electrophoresis depends on: 1. agarose concentration, 2. current voltage, 3. composition and ionic strength of the buffer, 4. spatial conformation of DNA Page 2 of 11  WORKSHEET 6- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Ad 1 Agarose dissolves in water after it is heated until boiling. For electrophoresis, it is dissolved in a TAE or TBE running buffer. The temperature of agarose melting, i.e., the change of gel into sol, is higher than the solidification. The slightly cooled to approx. 60C solution is poured onto a flat, rectangular form with a thickness of several mm. A so-called comb should be inserted into the still-liquid solution, and the teeth of the comb should create holes/pockets in the gel into which DNA samples should be loaded. During further cooling the solution gels, creating a network of polymers stabilized by hydrogen bonds without adding the polymerizing factor. The gel is ready to use about 15-20 minutes after being poured out into the form. The ready agarose gel is transferred into the apparatus for electrophoresis. The gel is placed between electrodes in a chamber filled with the same buffer in which the agarose was dissolved. Depending on the range of masses of the separated molecules the appropriate concentration of agarose in the electrophoretic gel must be selected. There is no universal concentration of agarose, allowing for successful and simultaneous separation of molecules with high and low masses. If relatively small molecules of similar masses, e.g. 200 bp and 300 bp, are to be separated, then higher concentrations of agarose should be used. If relatively large molecules of similar masses are to be separated e.g. 5000 bp and 6000 bp then lower concentrations of agarose are to be applied. Agarose concentration (% Range of masses of optimally [w/v]) separated DNA molecules (kbp) 0.3 5 - 60 0.6 1 - 20 0.7 0.8 - 10 0.9 0.5 - 7 1.2 0.4 - 6 1.5 0.2 - 3 2.0 0.1 - 2 Ad 2 The voltage of the electric current in electrophoresis. The higher the voltage the quicker the DNA molecules migrate in the gel, nevertheless, the speed of the migration of linear dsDNA molecules is still reversely proportional to the log10 of their molecular mass. If the voltage is too high, then fragments over 12-15 kbp are smudged, and moreover, the buffer temperature is raised, which may cause melting of the gel, “smiling” of the DNA bands, and a decrease in Page 3 of 11  WORKSHEET 6- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 resolution. That is why, it is recommended not to exceed voltage of 5-8V/cm and current intensity of 75 mA in standard gels or 100 mA in mini gels. If the voltage is too low, the result is too slow migration by the molecules, during which effects of DNA molecule diffusion will be noticeable, as well as a widening/dispersion of the analyzed bands. Ad 3 Effective separation of nucleic acids in agarose electrophoresis depends on maintaining the appropriate pH in the gel; that is why buffers are an integral part of every electrophoretic technique. In addition, the electrophoretic mobility of DNA depends on the composition and ionic strength (sodium content) of the electrophoretic buffers. Without salts, buffer conductivity is minimal, so DNA molecules do not move. The most often used buffers for DNA electrophoresis are Tris/Acetate/EDTA (TAE) and Tris/Borate/EDTA (TBE) at concentrations of about 50 mM (pH 7.5-7.8). TAE is cheaper but not as stable as TBE. Nevertheless, TAE allows for a better resolution in short separation and is used when a DNA fragment must be isolated from the gel. TBE is used in longer electrophoreses when high resolution is necessary. Ad 4 DNA spatial conformation is an essential factor influencing the migration in gel and the ability to evaluate fragment mass. To properly assess a given fragment, it is necessary to know its possible conformations. DNA can be present in the following forms:  covalently closed circular (CCC) e.g... plasmid,  open circular (OC) np. Plasmid with one strand breaks (nicked circles),  linear form DNA, linear DNA fragments. Page 4 of 11  WORKSHEET 6- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 DNA with the same mass Nicked circles – – DNA migrates the slowest Linear – linear DNA fragments – speed of migration is reversely proportional to the log10 of molecular mass Supercoiled – – migrates the fastest The course of DNA electrophoresis Before placing the DNA samples onto the gel, they are mixed with the loading buffer, which increases the density of the sample and allows it to be loaded in the pocket/hole/well formed in the agarose gel. The loading buffer generally contains ficoll, glycerol, or sucrose, as well as two dyes, bromophenol blue or xylene cyanol, migrating in the electric field and allowing us to follow the course of the electrophoresis as it happens. When putting the samples into the wells, it must be remembered that the so-called marker M, meaning the molecular weight marker, should be put into one of the wells instead of a sample. The molecular weight marker is a mixture of linear DNA fragments with known masses. After applying electric voltage to the electrophoretic chamber, in which the gel with the samples is located, the process of electrophoresis begins, meaning the migration of the charged DNA molecules (samples and standard) in the gel. During electrophoresis DNA molecules of different masses are separated due to their different rates of migration. At the same time, during electrophoresis, the dyes found in the loading buffer move similarly to the DNA in the direction of the positive anode. The most often used dyes are those that migrate faster than the DNA which allows for the determination of the front of the migrating DNA molecules and finishing of electrophoresis when the dye reaches the edge of the gel. Page 5 of 11  WORKSHEET 6- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 source: GroMar Sp. z o.o gel, wells, source of voltage, drawing representing the electrophoresis apparatus. source: GroMar Sp. z o.o. developed based on www.khanacademy.org Plate for electrophoresis M – marker I – a mixture of DNA particles from sample I II – a mixture of DNA particles from sample II source:: GroMar Sp.z o.o Page 6 of 11  WORKSHEET 6- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Dyeing Finishing the electrophoretic part or transfer does not complete the procedure of separation since most of the proteins and nucleic acids are not visible in white light. To obtain information about the distance of their migration and their amount in a band or spot (area containing the protein in 2D electrophoresis) it is necessary to dye them. To accomplish this various dyes are used, which when illuminating the gel with UV light, make the glowing DNA bands visible. Fluorescent dyes Traditionally oligonucleotides were dyed using ethidium bromide. After illuminating the gel with UV light they showed DNA bands that glowed orange. However, since ethidium bromide is a mutagenic and carcinogenic substance currently it is no longer used in most laboratories. At present, there are alternative dyes available with low toxicity such as SYBR green or Midori Green, that are often characterized by an even greater sensitivity than ethidium bromide. The sensitivity of detection is comparable to dyeing with silver or is slightly better. Sometimes dyeing with radioisotopes is used as they ensure very high sensitivity, but it is necessary to use the proper safety measures. Midori Green Ethidium bromide Documentation of electrophoretic separations The documentation of separations performed in polyacrylamide gels is the fact that they dried up. In this case, either tissue paper and/or cellophane is used. The dried gel may be stored for an infinite period of time. However, agarose gels cannot be documented in this way. The simplest way of preserving information contained in the agarose gel is by taking its photograph. Page 7 of 11  WORKSHEET 6- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Exercise no. 6 DNA electrophoresis Performing electrophoretic separation of the PCR product obtained in exercise no. 5 Practice Student:  can prepare the agarose gel, place the DNA samples, and carry out DNA electrophoresis,  can select the appropriate agarose concentration for the masses of the separated DNA fragments,  documents the result of electrophoresis evaluates the quality of the separation of DNA fragments,  estimates the masses of the separated fragments based on the mass standard. Equipment: apparatus for horizontal electrophoresis, power supply, automatic pipettes, pipettor, graduated cylinders, laboratory flasks, analytical scale, weighing containers, microwave, and GelDoc system of photographic documentation. Reagents:  agarose,  TAE buffer 50x concentrated (0.2 M Tris: octan; 0.05 M EDTA),  DNA dye: Midori Green Advance DNA Stain in a concentration of 4 - 6 µl/100 ml liquid agarose,  PCR products obtained in ex. no. 5, Preparation of two agarose gels with volumes of 40ml and different agarose content: 1% and 2.5%. 1. Prepare a working, one-time concentration of the TAE 1X buffer – final volume 700ml. 2. Prepare the form to pour out the gel (casting tray) using a support (or tape). Prepare a comb next to the form to make wells in the agarose gel. 3. Calculate the mass of agarose needed to prepare 40 ml of agarose with a concentration of 1,5 %. Calculate the mass of agarose needed to prepare 40 ml of agarose with a concentration of 2.5%. 4. Weigh the calculated agarose masses on an analytical scale. 5. Pour the weighed amount of agarose (Xg for 1,5 % gel and Yg for 2.5 % gel) into separate glass flasks and then add to each of them a one-time concentrated TAE buffer at the same time washing the weighing container. Gently stir. 6. Then boil the mixture several times in a microwave until the agarose dissolves making sure the liquid does not boil over, and then cool down to a temperature of approx. 60C. Attention! While heating in the microwave oven the bottle must not be tightly closed – a risk of explosion. Page 8 of 11  WORKSHEET 6- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 7. Add 3 l of the Midori Green dye to the dissolved agarose, and stir. 8. Pour out the liquid agarose into the casting tray prepared in the support then immerse the teeth of the comb in the liquid agarose to make wells. Leave to solidify. 9. Gently take out the comb and the support (tape), put the casting tray with the gel into the apparatus, and pour in the buffer up to the level indicated on the apparatus. Preparing the DNA samples and electrophoresis Reagent:  Molecular weight marker: A&A Biotechnology cat. 3000-500 range: 100 - 1000 bp. Concentration: 0,1 µg/µl, DNA ladder 100-1000 base pairs is a molecular weight marker, which in addition contains two dyes allowing for visual observation of the migration of DNA molecules during electrophoresis. The DNA ladder consists of fragments with precisely defined sizes from 100 to 10000 base pairs. A fragment with a size of 500 bp is marked more clearly to make identification easier. All fragments have blunt ends.  DNA samples after PCR. Since the PCR was carried out in accordance with the protocol using „PCR Mix Plus Green” in exercise no. 5, the tested samples after the reaction already contain the loading buffer and after defrosting, mixing, and centrifugation – can be directly put into the wells in the gel, in amounts of about10µl. When putting the samples onto the gel the end of the pipette should be stabilized with the finger so that it does not vibrate and damage the well in the gel. It is also necessary to pay attention to only put the end of the pipette partially into the well with the gel since otherwise there is a risk of puncturing the well and losing the sample. Page 9 of 11  WORKSHEET 6- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Placing the samples:  mass marker „DNA ladder” - 5 ,l  samples from subsequent people and controls should be placed in compliance with the order of tubes in the PCR strip. DNA electrophoresis:  close the electrophoresis apparatus and turn on the power supply setting electrophoresis parameters i.e. voltage 100 -120 V,  carry out the electrophoresis until the yellow dye of the loading buffer reaches the edge of the gel,  control the level of DNA fragment separation by putting the gel into the Gel Doc EZ System and performing DNA visualization,  optionally, if the separation of the DNA fragments is not clear, then the gel should once again be placed in the electrophoresis apparatus and the separation should be extended by a few minutes, and then once again the agarose gel should be visualized,  discuss the electrophoresis results as far as the expected results of PCR. Example result of electrophoresis of PCR products obtained with the aid of primers described in exercise no. 5 Preparation for exercises: -Theory regarding DNA electrophoresis (ex. no. 3-presentation electrophoresis, ex. no. 6 instruction) Page 10 of 11  WORKSHEET 6- MOLECULAR BIOLOGY / Faculty of Medical Sciences / Academy of Silesia / Year I; sem. 1 / 2024/25 Instruction on completing the report covering exercises 4, 5, and 6 Report elements: name, surname, group, date 1. Maximum of one page of theoretical introduction describing the essence and goal of the methods used in exercises no 4, 5, 6 (plagiarisms of the instruction will be discarded) 2. Information on the reagents used and the equipment used (assigned to a given exercise) 3. Description of the process (pipetting, volumes, incubations from exercises 4, 5, 6), it is especially important to note down and clearly mark in the report any deviations from the instructions or errors with explanations as to why they may have happened. 4. Results of DNA sample isolations for the whole group : Concentration ng/µg The purity value of the absorbency ratio is 260mm/280mm Interpretation of the results of each sample. 5. Method of adding the DNA sample to the PCR – each subgroup describes their strip of PCR tubes, and order of samples. Preparing the samples – amount of H2O, amount of ng DNA, amount of ul DNA of a given sample added to the PCR. Was the amount of DNA added to the reaction in compliance with the guidelines in the instruction? 5. Interpretation of electrophoresis results: Precise description of the picture of both gels representing the separation of the products of PCR for both subgroups, remarks regarding the quality of the separation for both gels, comparison of gels -gel description (well number/sample: initials, group) -estimating masses of DNA bands which were products of PCR -attributing significance to the observed bands, interpretation of results of each sample -commentary in case of a lack of PCR product in a given sample – possible reasons. Commentary regarding positive and negative controls, are they as expected, and do they allow for proper interpretation? If that is not the case, then what does an incorrect result of the control mean? In the case of a need for additional information or any questions regarding the report, the instructor is at your disposal – e-mail contact, meeting on the TEAMS platform Page 11 of 11