Electrophoresis, cleaning up on spin-columns, labeling of PCR products and preparation extended products for sequencing

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1 Electrophoresis, cleaning up on spin-columns, labeling of PCR products and preparation extended products for sequencing PAGE electrophoresis Polyacrylamide gel electrophoresis (PAGE) is used for separating molecules by size (DNA or proteins). Pore size is determined by the ratio of acrylamide and bis-acrylamide concentra-tion. Care must be taken when working with acrylamide, since it is a strong neurotoxin, espe-cially in its powdered form. Polyacrylamide gels can separate DNA that differs by 0.2% in length, well beyond the resolving capabilities of agarose (2%). Another advantage of using polyacrylamide gels is that they can accommodate large amounts of DNA (up to 10 μg) without any loss in resolution. Depending upon the application, TBE gels can be prepared as denaturing or nondenaturing ones. Purification of amplicons Purification of nucleic acids using a specially selected silica membrane is a rapid, conven-ient and economical way to isolate DNA and RNA. The technique does not require time-consuming methods based on old technologies such as: use of extraction methods in the phe-nol-chloroform system, precipitation, or PEG-type polymers. Applied membrane technology allows to avoid problems with the silica particles used often as suspensions. The purification procedure removes primers, nucleotides, enzymes, mineral oils, salts, agarose, ethidium bro-mide, and other impurities from DNA samples. The Clean-Up kit is based on the DNA ability to adsorb to silica surface in the presence of high concentration of chaotropic salts [Fig.1]. Starting up the DNA recovery after enzymatic reaction, the binding solution G which contains chaotropic salt, is added to the reaction mix-ture. At high salt concentrations, sodium cations break hydrogen bonds between the hydro-gen atoms of water and the negatively charged oxygen ions in silica. Sodium serves as a cati-on bridge and interacts with negatively charged oxygen of the phosphate backbone of the DNA molecule.

2 During the next step the whole sample is loaded onto minicolumn with dedicated silica res-in. The DNA binds to the silica resin, while contaminants (enzymes, salts, short primers,etc.) are passing through the column freely. Leftovers of contaminants are moved away in the washing step. Pure DNA is eluted at low salt buffer or water and can be used in the following experiments without additional purification procedures (e.g. precipitation). DNA Chain Termination Sequencing The chain-terminator method (sometimes referred to as the Sanger method) is more effi-cient and uses fewer toxic chemicals and lower amounts of radioactivity than the method of Maxam and Gilbert. Generally it is method of choice for DNA sequencing. The key principle of the Sanger method was the use of dideoxynucleotides triphosphates (ddntps) as DNA chain termination [Figs 3 and 4]. The classical chain-termination method [Fig. 2.] requires a single-stranded DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides and modified nucleotides that terminate DNA strand elongation. The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (datp, dgtp, dctp and dttp) and the DNA polymerase. To each reaction added is only one of the four dideoxynucleotides (ddatp, ddgtp, ddctp, or ddttp) which are the chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, thus terminating DNA strand extension and providing various DNA fragments of varying length. Dye-terminator sequencing utilizes labelling of the chain terminator ddntps, which per-mits sequencing in a single reaction, rather than four reactions as in the labelled-primer meth-od [Fig. 3]. Dye-terminator sequencing requires each of the four dideoxynucleotide chain terminators to be labelled with different fluorescent dyes (different wavelengths of fluores-cence emission) [Fig. 4]. Owing to its greater expediency and speed, dye-terminator sequenc-ing is now the mainstay in automated sequencing. Its limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromato-gram after capillary electrophoresis. The dye-terminator sequencing method, along with au-tomated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects.

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4 Purification of chain terminated fragments The best results are obtained when unincorporated dye terminators are completely removed prior to capillary electrophoresis. Excess dye terminators, primers, proteins in sequencing reactions obscure data in the early part of the sequence and can interfere with basecalling. When unincorporated dye is present in large amount then on sequence will be visible dye blobs. Fig. 4. Example of electropherogram with visible dye blobs This are the most popular methods used for purification: Ethanol/EDTA precipitation Ethanol/EDTA/sodium acetate precipitation Plate or spin column purification Used in this training method is based on precipitation of cycle sequencing reaction products on the surface of especially designed membrane. The addition of Mix Blue reagent to the cycle sequencing reaction mixture enables an easy control of precipitation process. In the next step the binding-washing solution is added and the whole mixture is loaded onto Exterminator spin column. During the short centrifugation step the cycle sequencing products are bound by the spin column membrane while the excess of non-incorporated nucleotide terminators and sequencing primer pass through the membrane. The remaining salts and leftovers of other impurities are removed in the washing step. Subsequently the purified cycle sequencing reaction products are eluted directly by water. Literatura: Brody, J.R., Kern, S.E. (2004) History and principles of conductive media for standard DNA electrophoresis. Anal Biochem. 333(1):1-13 Smith LM, Sanders JZ, Kaiser RJ, et al (1986). "Fluorescence detection in automated DNA sequence analysis". Nature 321 (6071): Maxam AM, Gilbert W (1977). "A new method for sequencing DNA". Proc. Natl. Acad. Sci. U.S.A. 74 (2): Carter, J.M. and Milton, I.D., (1993) "An inexpensive and simple method for DNA purifications on silica particles", Nucl. Acids Res.21

5 MATERIALS REAGENTS - EQUIPMENT thermal cycler thermoblock centrifuge vortex primers NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) Ex-Terminator (A&A Biotechnology) BigDye Terminator v3.1 Cycle Sequencing Kit 1,5 ml tubes 10x TBE electrophoresis buffer (0,1 M Tris, 83 mm Boric acid, 1 mm EDTA) plates with 10% polyacrylamide gel gelloading buffer (0,25 % Bromophenol blue, 0,25 % Xylene cyanol and 40 % sucrose) vertical electrophoresis apparatus (Bio-Rad Mini Protean II) reagent A (EtOH, CH3COOH) reagent B (AgNO3-0,17 %) reagent C (NaOH - 10%, Formaldehyde CH2O - 2%) PROCEDURE PAGE Electrophoresis 1. Clamp your prepared gels and fill up buffer chambers with electrophoresis buffer 1x TBE. 2. Mix the DNA samples (3,5 µl) with the appropriate amount of gelloading buffer (1µl). 3. Load the mixture into the wells using a micropipette. (It is important not to take too long to complete loading the gel; otherwise, the samples will diffuse from the wells) 4. Connect the electrodes to a power pack, turn on the power, and begin the electrophoresis run (Adjust electric current to 30 ma/gel plate) 5. Run the gel until the marker dyes have migrated the desired distance. Turn off the electric power, disconnect the leads, and discard the electrophoresis buffer from the reservoirs. 6. Detach the glass plates. Lay the glass plates on the bench. Use a spacer or plastic wedge to lift a corner of the glass plate. 7. Put gels into the boxes filled with water. Silver staining 1. Wash the gel in H2O for at least 5 min. 2. Discard water and fill up the box with reagent A for 5 min. 3. Discard solution A and fill up the gel with reagent B for 10min. 4. Next rinse the gel with water 3x for 1 minute. 5. Fill up gel with reagent C and instantly discard it. Repeat this procedure until solution is no longer black. 6. Leave the gel in transparent reagent C until the fragments of DNA are visible. 7. Next fill up the gel with water and interpret results.

6 Clean up amplicons on spin columns

7 Use the obtained cleaned fragments to prepare reaction with BigDye. DNA Chain Termination Sequencing Reaction 1. Combine the following component for each reaction in a 0.2 ml tube (add BigDye at the end): Reagent Volume Concentration H 2O 3,75 µl - Sequencing buffer 3,5 µl 5x Primer 0,25 µl 10 µm BigDye Terminator v3.1 Ready Reaction Mix 0,5 µl - Clean PCR produkt 2 µl - 2. Use following primers to specific reaction: (EXI, Left primer), (EXII, Right primer), (Mitochondrial long HVR fragment, Left Primer) 3. Place tubes in a thermal cycler preheated to 95 C and start with proper times and temperature as stated below:

8 HVR Number of cycle 35 Initial denaturation 95ºC 5min Denaturation 95ºC 30 sec Anneling 58ºC 7 sec Extention 60ºC 4 min Hold 4ºC Purifying of extended products (ExTerminator kit) 1. Add 5 μl of Mix Blue to cycle sequencing reaction mixture. 2. Add 100 μl of bind/wash solution and mix sample by pipetting and load the whole sample onto minicolumn. 3. Spin at 1500 x g for 30 s. (the light blue colour of minicolumn membrane is a result of efficient precipitation of sequencing products.) 4. Apply on minicolumn 400 μl of bind/wash solution. 5. Spin at 6000 x g for 2 min. 6. Transfer the minicolumn to a new 1.5 ml tube 7. Apply precisely onto the membrane (center of dark blue circle) 15 μl of water. 8. Incubate at RT for 2 min. 9. Spin at 6000 x g for 2 min. (Light blue colour of eluted sample confirms proper purification of sample) 10. Dry the sample in thermoblock 55 C. 11. Dissolve dried pallet in 12µl of deionized formamide.

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