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Dna protocols copy

  1. 1. Dr Gihan E-H Gawish, MSc, PhD. 1
  2. 2. Chapter one:Plasmid DNAWhat is Plasmid DNA?Bacterial plasmids are closed circular molecules of double-stranded DNA that range insize from 1 to >200 kb. They are found in a variety of bacterial species, where theybehave as additional genetic units inherited and replicated independently of thebacterial chromosome. However, they rely upon enzymes and proteins provided by thehost for their successful transcription and replication. Plasmids often contain genes thatcode for enzymes that can be advantageous to the host cell in some circumstances. Theencoded enzymes may be involved in resistance to, or production of, antibiotics,resistance to toxins found in the environment (e.g., complex organic compounds), orthe production of toxins by the bacteria itself. Once purified, plasmid DNA can be usedin a wide variety of downstream applications such as sequencing, PCR, expression ofproteins, transfection, and gene therapy. This chapter describes common plasmid DNAprocedures, including how to make and transform competent cells, how to culture andhandle plasmid containing cells, and how to purify and quantify plasmid DNA.Competent Cells and Transformation 2
  3. 3. Protocol 1. Preparation of competent E. coliCells that have the ability to take up DNA (from a variety of sources) are termed“competent”.MaterialsE. coli cells in glycerol stock vial Appropriate selective antibioticsLB medium TFB1 bufferLB-agar plates TFB2 buffer1. Remove a trace of E. coli cells from the glycerol stock vial with a sterile toothpickor inoculating loop, and streak it out on LB-agar plates containing an appropriateconcentration of the relevant selective antibiotic(s). If the host strain has already beencultured and stored at 2–8°C (cultures can be stored at 2–8°C for up to 3 monthswithout any significant loss of viability), streak out bacteria from those stocks.2. Incubate at 37°C overnight.3. Pick a single colony and inoculate 10 ml LB medium containing relevantantibiotic(s). Grow overnight at 37°C.4. Add 1 ml overnight culture to 100 ml prewarmed LB medium containing the relevantantibiotic(s) in a 500 ml flask, and shake at 37°C (approximately 90–120 min).5. Cool the culture on ice for 5 min, and transfer the culture to a sterile, round-bottomcentrifuge tube.6. Collect the cells by centrifugation at low speed (5 min, 4000 x g, 4°C).7. Discard the supernatant carefully. Always keep the cells on ice.8. Resuspend the cells gently in cold (4°C) TFB1 buffer (30 ml for a 100 ml culture)and keep the suspension on ice for an additional 90 min.9. Collect the cells by centrifugation (5 min, 4000 x g, 4°C). 3
  4. 4. 10. Discard the supernatant carefully. Always keep the cells on ice.11. Resuspend the cells carefully in 4 ml ice-cold TFB2 buffer.12. Prepare aliquots of 100–200 μl in sterile micro centrifuge tubes and freeze in liquidnitrogen or a dry-ice–ethanol mix. Store the competent cells at –70°C. competent cellsfor optimized transformation.Protocol 2. Transformation of competent E. coli cells 4
  5. 5. Transformation is the process in which plasmid DNA is introduced into a bacterial hostcell.MaterialsCompetent E. coli cells (see Protocol 1, above)SOC medium LB-agar plates1. Transfer an aliquot of the DNA to be transformed (10 μl or less) into a cold sterile1.5 ml microcentrifuge tube, and keep it on ice.2. Thaw an aliquot of frozen competent E. coli cells on ice.3. Gently resuspend the cells and transfer 100 μl of the cell suspension into themicrocentrifuge tube with the plasmid DNA, mix carefully, and keep on ice for 20 min.4. Transfer the tube to a 42°C water bath or heating block for 90 s.5. Add 500 μl SOC medium to the cells and incubate for 60–90 min at 37°C. Shaking increases transformation efficiency.6. Plate out 50, 100, and 200 μl aliquots on LB-agar plates containing the relevantantibiotic(s). Incubate the plates at 37°C overnight until colonies develop.Positive control to check transformation efficiencyTransform competent cells with 1 ng of a control plasmid containing an antibioticresistance gene. Plate onto LB-agar plates containing the relevant antibiotic(s).Compare the number of colonies obtained with the control plasmid to the numberobtained with the plasmid of interest to compare transformation efficiency.Negative control to check antibiotic activityTransform cells with 20 μl of TE. Plate at least 200 μl of the transformation mix on asingle LB-agar plate containing the relevant antibiotic(s). An absence of colonies onthe plates indicates that the antibiotic is active.Growth and Culture of Bacteria*Bacterial culture media and antibiotics 5
  6. 6. Liquid mediaLiquid cultures of E. coli can generally be grown in LB (Luria-Bertani) medium.Sterilizing mediaSterilize liquid or solid media by autoclaving, using a pressure and time period suitablefor the type of medium, bottle size, and autoclave type. It is advisable to autoclaveliquid medium in several small bottles rather than in one large vessel, to avoid possiblecontamination of an entire batch. Fill bottles only 3/4 full with medium and loosen thecaps before autoclaving to avoid hot medium boiling over. Tighten caps once the mediais cool (<40°C) to maintain sterility. Antibiotics and medium supplements such asamino acids are degraded by autoclaving. Antibiotics should be added to liquid mediumimmediately prior to use from stock solutions that have been filter-sterilized,distributed into aliquots, and stored in the dark at –20°CSolid mediaE. coli strains can generally be streaked and stored, for a short period of time, on LBplates containing 1.5% agar and the appropriate antibiotic(s).Preparation of LB-agar platesJust before autoclaving, add 15 grams agar per liter and mix. After autoclaving, swirlthe medium gently to distribute the melted agar evenly throughout the solution. Coolautoclaved agar medium to below 50°C (when you can hold it comfortably) beforeadding heat-sensitive antibiotics and nutrients. Mix thoroughly before pouring. Pourplates in a laminar-flow hood or on a cleaned bench surface next to a Bunsen burner.Use 30–35 ml medium per standard 90 mm petri dish. After pouring plates, any airbubbles may be removed by passing the flame of a Bunsen burner briefly over thesurface. Dry plates by removing the lids and standing the plates in a laminar-flow hoodfor 1 hour; with the covers slightly open in a 37°C incubator for 30 minutes; or leftupside down with lids on at room temperature overnight. Store plates inverted at 4°Cin a dark room or wrapped in aluminum foil to preserve light-sensitive antibiotics. Donot store for longer than 1 month as antibiotics may degrade. Label plates with the dateand the antibiotic used.AntibioticsBacterial strains carrying plasmids or genes with antibiotic selection markers shouldalways be cultured in liquid or on solid medium containing the appropriate selective 6
  7. 7. agent. Lack of antibiotic selection can lead to loss of the plasmid carrying the geneticmarker and potentially to selection of faster-growing mutants! Prepare stock solutionsof antibiotics separately from batches of liquid or solid media, sterilize by filtration,aliquot, and store in the dark at –20°C. Before adding antibiotics to freshly autoclavedmedium, ensure that the medium has cooled to below 50°C.Table 1. Concentrations of commonly used antibioticsAntibiotic Stock solutions Working concentration Concentration Storage (dilution)Ampicillin 50 mg/ml in water –20°C 100 μg/ml (1/500)(sodium salt)Chloramphenicol 34 mg/ml in ethanol –20°C 170 μg/ml (1/200)Kanamycin 10 mg/ml in water –20°C 50 μg/ml (1/200)Streptomycin 10 mg/ml in water –20°C 50 μg/ml (1/200)Tetracycline HCl 5 mg/ml in ethanol –20°C 50 μg/ml (1/100)Carbenicillin 50 mg/ml in water –20°C 50 μg/ml (1/1000)Storage of E. coli strainsThere are different methods for storing E. coli strains depending on the desired storagetime. Glycerol stocks and stab cultures enable long-term storage of bacteria, while agarplates can be used for short-term storage. When recovering a stored strain, it isadvisable to check that the antibiotic markers have not been lost by streaking the strainonto an LB-agar plate containing the appropriate antibiotic(s).Protocol 3. Preparation of glycerol stocksE. coli strains can be stored for many years at –70°C in medium containing 15%glycerol. 7
  8. 8. Prepare glycerol stocks of bacteria as follows:1. Add 0.15 ml glycerol (100%) to a 2 ml screw-cap vial and sterilize by autoclaving.Vials of sterilized glycerol can be prepared in batches and stored at room temperatureuntil required.2. Add 0.85 ml of a logarithmic-phase E. coli culture to the vial of pre-sterilizedglycerol.3. Vortex the vial vigorously to ensure even mixing of the bacterial culture and theglycerol.4. Freeze in an dry ice–ethanol bath or liquid nitrogen and store at –70°C. Avoidrepeated thawing and re-freezing of glycerol stocks as this can reduce the viability ofthe bacteria. For precious strains, storage of two stock vials is recommended.Protocol 4. Preparation of stab culturesE. coli strains can also be stored for up to 1 year as stabs in soft agar. Stab cultures canbe used to transport or send bacterial strains to other labs. 8
  9. 9. Prepare stab cultures as follows:1. Prepare and autoclave 0.7% LB agar (standard LB medium containing 7 g/liter agar)as described in2. Cool the LB agar to below 50°C (when you can hold it comfortably) and add theappropriate antibiotic(s). While still liquid, add 1 ml agar to a 2 ml screw-cap vial understerile conditions, then leave to solidify. Vials of agar can be prepared in batches andstored at room temperature until required.3. Using a sterile straight wire, pick a single colony from a freshly grown plate and stabit deep down into the soft agar several times.4. Incubate the vial at 37°C for 8–12 h leaving the cap slightly loose.5. Seal the vial tightly and store in the dark, preferably at 4°C.Growth of E. coli culturesFigure 1 shows the sequence of steps necessary to go from a stored stock of bacteriato a liquid culture for plasmid isolation. Bacterial stocks should always be streakedonto selective plates prior to use, to check that they give rise to healthy coloniescarrying the appropriate antibiotic resistance. Stocks can potentially contain mutantsarising from the cultures used to prepare them, or can deteriorate during storageProtocol 5. Recovery of single colonies from stored culturesPlates of streaked bacteria can be sealed with Parafilm and stored upside-down at 4°Cfor several weeks. Bacteria should always be streaked onto plates containing theappropriate antibiotic to ensure that selective markers are not lost. 9
  10. 10. To obtain isolated colonies, streak an agar plate as follows:1. Flame a wire loop, and cool on a spare sterile agar plate.2. Using the wire loop, streak an inoculum of bacteria (from a glycerol stock, stabculture, or single colony on another plate) across one corner of a fresh agar plate, asshown in Figure 2.3. Flame and cool the wire loop again. Pass it through the first streak and then streakagain across a fresh corner of the plate.4. Repeat again to form a pattern as in Figure 2.5. Incubate the plate upside down at 37°C for 12–24 h until colonies develop.6. Inoculate liquid cultures from a healthy, well-isolated colony, picked from a freshlystreaked selective plate. This will ensure that cells growing in the culture are alldescended from a single founder cell, and have the same genetic makeup. Culturevolumes >10 ml should not be inoculated directly from a plate, but with a pre-cultureof 2–5 ml diluted 1/500 to 1/1000. E. coli growth curveThe growth curve of an E. coli culture can be divided into distinct phases (Figure 3).Lag phase occurs after dilution of the starter culture into fresh medium. Cell divisionis slow as the bacteria adapt to the fresh medium. After 4–5 hours the culture enters 11
  11. 11. logarithmic (log) phase, where bacteria grow exponentially. Cells enter stationaryphase (~16 hours) when the available nutrients are used up. The cell density remainsconstant in this phase. Eventually the culture enters the phase of decline, where cellsstart to lyse, the number of viable bacteria falls, and DNA becomes partly degraded.Measuring cell densityThe growth curve of a bacterial culture can be monitored photometrically by readingthe optical density at 600 nm (Figure 3). Note however that photometricmeasurements of cell density can vary between different spectrophotometers.Calibrate your spectrophotometer by determining the number of cells per millilitergiving a particular OD600 reading. Plate serial dilutions of a culture on LB agar platesand calculate the number of cells per milliliter in the original culture. This is then setin relation to the measured OD600 value. High OD600 readings should be calculatedby diluting the sample in culture medium to enable measurement in the linear range of0.1–0.5 OD600. Another way of estimating the amount of cell harvest is to assess thepellet wet weight. Typically a 1 liter, overnight culture of E. coli within a cell densityof 3–4 x 109 cells per milliliter corresponds to a pellet wet weight of approximately 3grams.Protocol 6. Preparation of bacteria for plasmid prepsTo prepare the bacterial culture for your plasmid prep, follow the steps below. 11
  12. 12. 1. Prepare a starter culture by inoculating a single colony from a freshly grownselective plate into 2–10 ml LB medium containing the appropriate antibiotic. Grow at37°C for ~8 h with vigorous shaking (~300 rpm). It is often convenient to grow thestarter culture during the day so that the larger culture can be grown overnight forharvesting the following morning.2. Dilute the starter culture 1/500 to 1/1000 into a larger volume of selective LBmedium. Use a flask of at least 5 times the volume of culture to ensure sufficientaeration. Do not use a larger culture volume than recommended in the protocol, as useof too many cells will result in inefficient lysis and reduce the quality of thepreparation.3. Grow the culture at 37°C with vigorous shaking (~300 rpm) for 12–16 h. Growth for12–16 h corresponds to the transition from logarithmic into stationary growth phase(see Figure 3), when cell density is high (3–4 x 109 cells per ml) and RNA content ofcells is low. Growth of cultures is dependent on factors such as host strain, plasmidinsert and copy number, and culture medium. To determine the optimal harvesting timefor a particular system, monitor the cell density and the growth of the culture bymeasuring the OD600 (see previous section).4. Harvest the bacterial culture by centrifugation at 6000 x g for 15 min at 4°C. Removeall traces of the supernatant. The cells are now ready for the lysis procedure.The procedure may be stopped at this point and continued later by freezing the cellpellets obtained by centrifugation. The frozen cell pellets can be stored at –20°C forseveral weeksLysis of Bacterial Cells for Plasmid PurificationEffective lysis of bacterial cells is a key step in plasmid isolation as DNA yield and 12
  13. 13. quality depend on the quality of cell lysate used for the purification.Alkaline lysisAlkaline lysis is one of the most commonly used methods for lysing bacterial cellsprior to plasmid purification (3, 4). Production of alkaline lysates involves four basicsteps (Figure 4):1. ResuspensionHarvested bacterial cells are resuspended in Tris·Cl–EDTA buffer containing RNaseA. Ensure that bacteria are resuspended completely leaving no cell clumps in order tomaximize the number of cells exposed to the lysis reagents. Do not use a culturevolume larger than recommended in the protocol as this will lead to inefficient lysisand reduce the quality of the plasmid preparation. For large scale purification of low-copy plasmids, for which larger cultures volumes are used, it may be beneficial toincrease the lysis buffer volumes in order to increase the efficiency of alkaline lysisand thereby the DNA yield.2. LysisCells are lysed with NaOH/SDS. Sodium dodecyl sulfate (SDS) solubilizes thephospholipid and protein components of the cell membrane, leading to lysis and releaseof the cell contents. NaOH denatures the chromosomal and plasmid DNA, as well asproteins. The presence of RNase A ensures that liberated cellular RNA is digestedduring lysis. If after addition of lysis buffer (NaOH/SDS) the solution appears veryviscous and is difficult to mix, this indicates excess biomass in the lysate step. Thisresults in insufficient cell lysis and it is recommended to double the amount of lysisand neutralization buffers used. Avoid vigorous stirring or vortexing of the lysate asthis can shear the bacterial chromosome, which will then co purify with the plasmidDNA. The solution should be mixed gently but thoroughly by inverting the lysis vessel4–6 times. Do not allow the lysis to proceed for longer than 5 minutes. This is optimalfor release of the plasmid DNA, while avoiding irreversible plasmid denaturation.3. NeutralizationThe lysate is neutralized by the addition of acidic potassium acetate. The high saltconcentration causes potassium dodecyl sulfate (KDS) to precipitate, and denaturedproteins, chromosomal DNA, and cellular debris are co precipitated in insoluble salt- 13
  14. 14. detergent complexes. Plasmid DNA, being circular and covalently closed, renaturescorrectly and remains in solution. Precipitation can be enhanced by using chilledneutralization buffer and incubating on ice.4. Clearing of lysatesPrecipitated debris is removed by either centrifugation or filtration, producing clearedlysates.Purification of plasmid DNA from cleared bacterial lysates was traditionally performedusing cesium chloride (CsCl) ultracentrifugation. Today, a variety of commercially 14
  15. 15. available plasmid purification kits offer easy procedures for different throughputrequirements and applications.Isopropanol Precipitation of DNAAlcohol precipitation is commonly used for concentrating, desalting, and recoveringnucleic acids. Precipitation is mediated by high concentrations of salt and the additionof either isopropanol or ethanol. Since less alcohol is required for isopropanolprecipitation, this is the preferred method for precipitating DNA from large volumes.In addition, isopropanol precipitation can be performed at room temperature, whichminimizes co-precipitation of salt that may interfere with downstream applications.This section provides hints on how to perform an effective isopropanol precipitationand to help ensure maximum recovery of DNA. The range of values given reflectsprotocol variation depending on the scale and type of preparation.Protocol 7. Isopropanol precipitation procedure1. Adjust the salt concentration if necessary, e.g., with sodium acetate (0.3 M, pH 5.2,final concentration) or ammonium acetate (2.0–2.5 M, final concentration).2. Add 0.6–0.7 volumes of room-temperature isopropanol to the DNA solution and mixwell. Use all solutions at room temperature to minimize co-precipitation of salt.3. Centrifuge the sample immediately at 10,000–15,000 x g for 15–30 min at 4°C.Centrifugation should be carried out at 4°C to prevent overheating of the sample.(When precipitating from small volumes, centrifugation may be carried out at roomtemperature.)4. Carefully decant the supernatant without disturbing the pellet. Marking the outsideof the tube or uniformly orienting microcentrifuge tubes before centrifugation allowsthe pellet to be more easily located. Pellets from isopropanol precipitation have a glassyappearance and may be more difficult to see than the fluffy salt-containing pellets thatresult from ethanol precipitation. Care should be taken when removing the supernatantas pellets from isopropanol precipitation are more loosely attached to the side of thetube. Carefully tip the tube with the pellet on the upper side to avoid dislodging thepellet. For valuable samples, the supernatant should be retained until recovery of theprecipitated DNA has been verified. 15
  16. 16. 5. Wash the DNA pellet by adding room-temperature 70% ethanol. This removes co-precipitated salt and replaces the isopropanol with the more volatile ethanol, makingthe DNA easier to redissolve.6. Centrifuge at 10,000–15,000 x g for 5–15 min at 4°C. Centrifuge the tube in thesame orientation as previously to recover the DNA in a compact pellet.7. Carefully decant the supernatant without disturbing the pellet.8. Air-dry the pellet for 5–20 min (depending on the size of the pellet). Do not overdrythe pellet (e.g., by using a vacuum evaporator) as this will make DNA, especially high-molecular-weight DNA, difficult to redissolve.9. Redissolve the DNA in a suitable buffer. Choose an appropriate volume of bufferaccording to the expected DNA yield and the desired final DNA concentration. Use abuffer with a pH ≥8.0 for redissolving, as DNA does not dissolve easily in acidicbuffers. (If using water, check pH.) Redissolve by rinsing the walls to recover all theDNA, especially if glass tubes have been used. To avoid shearing DNA do not pipet orvortex. High-molecular-weight DNA should be redissolved very gently to avoidshearing, e.g., at room temperature overnight or at 55°C for 1–2 h with gentle agitation.Analytical GelsThis section is aimed at providing useful hints for effective gel analysis of nucleicacids. Firstly, the basic steps involved in pouring an agarose gel for DNA analysis are 16
  17. 17. outlined. Subsequent sections look at loading and running the gel and visualization ofthe DNA.Principle of gel analysisGels allow separation and identification of nucleic acids based on charge migration.Migration of nucleic acid molecules in an electric field is determined by size andconformation, allowing nucleic acids of different sizes to be separated. However, therelationship between the fragment size and rate of migration is non-linear, since largerfragments have greater frictional drag and are less efficient at migrating through thepolymer. Agarose gel analysis is the most commonly used method for analyzing DNAfragments between 0.1 and 25 kb. Other specialized analytical gel methods exist foranalyzing extremely large or small DNA molecules.Pouring an agarose gelAgarose concentrationThe concentration of agarose used for the gel depends primarily on the size of the DNAfragments to be analyzed. Low agarose concentrations are used to separate large DNAfragments, while high Agarose concentrations allow resolution of small DNAfragments (Table 2).Table 2. Concentration of agarose used for separating DNA of different sizesAgarose concentration (% w/v) DNA fragment range (kb) 0.3* 5–60 0.5 1–30 0.7 0.8–12 1.0 0.5–10 1.2 0.4–7 1.5 0.2–3 2.0* 0.05–2*Most gels are run using standard agarose, although some special types of agaroseare available for particular applications and for very high or low agaroseconcentrations. For example, low-melt agarose allows in situ enzymatic reactions andcan be used for preparative gels.Electrophoresis buffersThe most commonly used buffers for agarose gel electrophoresis are TBE (Tris-borate–EDTA) and TAE (Tris-acetate–EDTA). Although more frequently used, TAE has a 17
  18. 18. lower buffering capacity than TBE and is more easily exhausted during extendedelectrophoresis. TBE gives better resolution and sharper bands, and is particularlyrecommended for analyzing fragments <1 kb. The drawback of TBE is that the borateions in the buffer form complexes with the cis-diol groups of sugar monomers andpolymers, making it difficult to extract DNA fragments from TBE gels using traditionalmethods.Protocol 8. Pouring the gel1. Prepare enough 1x running buffer both to pour the gel and fill the electrophoresistank.2. Add an appropriate amount of agarose (depending on the concentration required) toan appropriate volume of running buffer (depending on the volume of the gel tray beingused) in a flask or bottle. The vessel should not be more than half full. Loosely coverthe vessel to minimize evaporation.Note: The cover should not be airtight.Always use the same batch of buffer to prepare the agarose as to run the gel, sincesmall differences in ionic strength can affect migration of DNA.3. Heat the slurry in a microwave or boiling water bath, swirling the vesseloccasionally, until the Agarose is dissolved. Ensure that the lid of the flask is loose toavoid buildup of pressure. Be careful not to let the Agarose solution boil over as itbecomes superheated. If the volume of liquid reduces considerably during heating dueto evaporation, make up to the original volume with distilled water.4. Cool the agarose to 55–60°C. Add ethidium bromide if desired.5. Pour the agarose solution onto the gel tray to a thickness of 3–5 mm. Insert the combeither before or immediately after pouring. Leave the gel to set (30–40 min).Ensure that there is enough space between the bottom of the comb and the glass plate(0.5–1.0 mm) to allow proper formation of the wells and avoid sample leakage.6. Carefully remove the comb and adhesive tape, if used, from the gel. Fill the tankcontaining the gel with electrophoresis buffer.Add enough buffer to cover the gel with a depth of approximately 1 mm liquid abovethe surface of the gel. If too much buffer is used the electric current will flow throughthe buffer instead of the gel. 18
  19. 19. Running an agarose gelPreparation of samplesAgarose gel analysis with ethidium bromide staining allows detection of DNA amountsfrom as little as 20 ng up to 500 ng in a band (5 mm wide x 2 mm deep). Loading oflarger amounts of DNA will result in smearing of the DNA bands on the gel. Samplesmust always be mixed with gel loading buffer prior to loading. Be sure that all sampleshave the same buffer composition. High salt concentrations will retard the migration ofthe DNA fragments. Ensure that no ethanol is present in the samples, as this will causesamples to float out of the wells.Gel loading buffers and markersGel loading buffer must be added to the samples before loading and serves three mainpurposes:1. To increase the density of the samples to ensure that they sink into the wells.2. To add color to the samples through use of dyes such as bromophenol blue, OrangeG, or xylene cyanol, facilitating loading.3. To allow tracking of the electrophoresis due to co-migration of the dyes with DNAfragments of a specific size. Molecular-weight markers should always be included ona gel to enable analysis of DNA fragment sizes in the samples.Protocol 9. Electrophoresis1. Apply samples in gel loading buffer to the wells of the gel. Prior to sample loading,rinse wells with electrophoresis buffer. Make sure that the entire gel is submerged inthe running buffer. Once samples are loaded, do not move the gel tray/tank as this maycause samples to float out of the wells.2. Connect the electrodes so that the DNA will migrate towards the anode (positiveelectrode). Electrophoresis apparatus should always be covered to protect againstelectric shocks.3. Turn on the power supply and run the gel at 1–10 V/cm until the dyes have migratedan appropriate distance. This will depend on the size of DNA being analyzed, theconcentration of agarose in the gel, and the separation required. Avoid use of very highvoltages which can cause trailing and smearing of DNA bands in the gel, particularly 19
  20. 20. with high-molecular-weight DNA. Monitor the temperature of the buffer periodicallyduring the run. If the buffer becomes heated, reduce the voltage. Melting of an agarosegel during electrophoresis is a sign that the voltage is too high, that the buffer may havebeen incorrectly prepared or has become exhausted during the run.Visual analysis of the gelStainingTo allow visualization of the DNA samples, agarose gels are stained with anappropriate dye. The most commonly used dye is the intercalating fluorescent dyeethidium bromide, which can be added either before or after electrophoresis (see Table3).Alternatives include commercial dyes such as SYBR® Green. Stock solutions ofethidium bromide (generally 10 mg/ml) should be stored at 4°C in a dark bottle orbottle wrapped in aluminum foil.Addition of ethidium bromide prior to electrophoresis — add ethidium bromide toa final concentration of 0.5 μg/ml to the melted and subsequently cooled agarose, i.e.,just before pouring the gel. Mix the agarose–ethidium bromide solution well to avoidlocalized staining.Addition of ethidium bromide after electrophoresis — soak the gel in a 0.5 μg/mlsolution of ethidium bromide (in water or electrophoresis buffer) for 30–40 minutes.Rinse the gel with buffer or water before examining it to remove excess ethidiumbromide.Staining buffer can be saved and re-used.Note: Ethidium bromide is a powerful mutagen and is very toxic. Wear gloves and takeappropriate safety precautions when handling. Use of nitrile gloves is recommended aslatex gloves may not provide full protection. After use, ethidium bromide solutionsshould be decontaminated as described in commonly used manuals.Table 3. Comparison of ethidium bromide staining methodsAddition of ethidium bromide Addition of ethidium bromide prior to electrophoresis after electrophoresisFaster and more convenient procedure Slower procedure requiring additional step 21
  21. 21. Allows monitoring of migration Does not allow monitoring of migrationthroughout the procedure during electrophoresisRequires decontamination of gel tanks No decontamination of gel tanks and combMore ethidium bromide is required Usually less ethidium bromide is requiredElectrophoretic mobility of linear DNA No interference with electrophoretic mobilityfragments is reduced by ~15%VisualizationEthidium bromide–DNA complexes display increased fluorescence compared to thedye in solution. This means that illumination of a stained gel under UV light (254–366nm) allows bands of DNA to be visualized against a background of unbound dye. Thegel image can be recorded by taking a Polaroid™ photograph or using a geldocumentation system. UV light can damage the eyes and skin. Always wear suitableeye and face protection when working with a UV light source. UV light damages DNA.If DNA fragments are to be extracted from the gel, use a lower intensity UV source ifpossible and minimize exposure of the DNA to the UV lightAgarose Gel Analysis of Plasmid DNAThe main uses of agarose gels for plasmid DNA analysis are:Analysis of the size and conformation of nucleic acids in a sampleQuantification of DNASeparation and extraction of DNA fragmentsAnalysis of a purification procedureFigure 5 shows how agarose gel electrophoresis can be used to analyze the nucleicacid content of samples taken during a plasmid purification procedure. The geldemonstrates successful plasmid purification using anion-exchange columns as well assome atypical results.M: Lambda DNA digested with HindIII.1: Cleared lysate containing supercoiled (lower band) and open circular plasmid DNA(upper band) and degraded RNA (smear at the bottom of the gel).2: Flow-through fraction containing only degraded RNA (the plasmid DNA is boundto the anion-exchange resin in the column). 21
  22. 22. 3: Wash fraction to ensure that the resin in the column is cleared of RNA and othercontaminants (plasmid DNA remains bound to the column).4: Eluate containing pure plasmid DNA in supercoiled and open circular forms.Lanes A–E illustrate some atypical results that may be observed in some preparations,depending on plasmid type and host strain.A: Supercoiled (lower band) and open circular form (upper band) of the high-copyplasmid, pUC18, with an additional band of denatured supercoiled DNA migrating justbeyond the supercoiled form.B: Multimeric forms of supercoiled plasmid DNA (pTZ19) that may be observed withsome host strains and should not be mistaken for genomic DNA. Multimeric plasmidDNA is easily distinguished from genomic DNA by restriction digestion.C: Linearized form of plasmid pTZ19 after restriction digestion with EcoRI.D: Sample contaminated with bacterial chromosomal DNA (uppermost band).E: EcoRI digestion of a sample contaminated with bacterial genomic DNA, whichgives a smear above the plasmid DNA. With large-constructs such as BAC, PAC, andP1 DNA, the supercoiled form migrates at a slower rate than the linear form.Furthermore, large-construct DNA >50 kb is often difficult to distinguish fromgenomic DNA by agarose gel analysis. Analysis of the Plasmid Purification Procedure Figure 5. Agarose gel analysis of a plasmid purification procedure using anion-exchange tips. Samples were taken at different stages of the procedure. 2 μl of each sample was run on a 1% agarose gel. M: lambda- HindIII markers.Gel extractionAgarose gels can be used for separation and extraction of DNA fragments, for example,a specific DNA fragment from a PCR or restriction digestion reaction. Ensure that thepercentage of agarose used for the gel allows good separation of DNA fragments for 22
  23. 23. easy excision. Run agarose gels for DNA extraction at a low voltage. This will enableefficient separation of DNA bands without smearing, facilitating excision of the gelslice. Excise the fragment quickly under low-strength UV light to limit DNA damage.DNA fragments can be extracted quickly and efficiently from agarose gels using silica-gel–based purification.Silica-gel–based methods typically result in higher and more reproducible recoveriesthan other gel extraction methods, such as electroelution, and require no phenolextraction or ethanol precipitation. In a typical silica-gel–based purification procedure,the agarose gel slice is first solubilized. DNA is then bound to the silica-gel material inthe presence of high concentrations of chaotropic salts. A wash step removesimpurities, and DNA is then eluted in low-salt buffer.Polyacrylamide gel electrophoresis (PAGE)As an alternative to agarose gel electrophoresis, polyacrylamide gels can be used forthe analytical or preparative separation of small, double-stranded DNA fragments. Thismethod is applicable to DNA fragments from 10 to 1000 bp. The resolution andcapacity of polyacrylamide gels is higher than that of agarose gels. However, agarosegels are much easier to pour and run, and in the vast majority of cases deliveracceptable resolution.Quantification of DNAReliable measurement of DNA concentration is important for many applications inmolecular biology. Plasmid DNA quantification is generally performed byspectrophotometric measurement of the absorption at 260 nm, or by agarose gelanalysis. In this section, we examine some critical factors for quantification, such asthe effect of solvents, phenol, and RNA contamination on absorption.DNA quantification by spectophotometryPlasmid DNA concentration can be determined by measuring the absorbance at 260nm (A260) in a spectrophotometer using a quartz cuvette. For reliable DNAquantification, A260 readings should lie between 0.1 and 1.0. An absorbance of 1 unitat 260 nm corresponds to 50 μg plasmid DNA per ml 23
  24. 24. (A260 = 1 ⇒neutral pH, therefore, samples should be diluted in a low-salt buffer with neutral pH(e.g., Tris·Cl, pH 7.0). An example of the calculation involved in nucleic acidquantification when using a spectrophotometer is provided inWhen working with small amounts of DNA, such as purified PCR products or DNAfragments extracted from agarose gels, quantification via agarose gel analysis may bemore effectiveIf you will use more than one quartz cuvette to measure multiple samples, the cuvettesmust be matched.Effects of solvents on spectrophotometric readingsAbsorption of nucleic acids depends on the solvent used to dissolve the nucleic acid(6). A260 values are reproducible when using low-salt buffer, but not when using water.This is most likely due to differences in the pH of the water caused by the solvation ofCO2 from air. A260/A280 ratios measured in water also give rise to a high variabilitybetween readings (Figure 6) and the ratios obtained are typically <1.8, resulting inreduced sensitivity to protein contamination (6). In contrast, A260/A280 ratiosmeasured in a low-salt buffer with slightly alkaline pH are generally reproducible.Effect of RNA contamination on spectrophotometric readings 24
  25. 25. Plasmid DNA preparations can contain RNA contamination, for example, when theRNase A treatment during alkaline lysis does not degrade all RNA species. Sincespectrophotometric measurement does not differentiate between DNA and RNA, RNAcontamination can lead to overestimation of DNA concentration. RNA contaminationcan sometimes be detected by agarose gel analysis with routine ethidium bromidestaining, although not quantified effectively.RNA bands appear faint and smeary and are only detected in amounts ≥25–30 ng (0.5:1RNA:DNA ratio). RNA contamination of plasmid DNA can be a concern dependingon the method used for plasmid preparation. Methods using alkaline lysis with phenolextraction cannot separate RNA from plasmid DNA, leading to high levels of RNAcontamination. In contrast, advanced silica-gel–membrane and anion-exchange resintechnologies that ensure plasmid DNA is virtually free of RNA (7–9).Effect of phenol on spectrophotometric readingsPhenol has an absorption maximum of 270–275 nm, which is close to that of DNA.Phenol contamination mimics both higher yields and higher purity, because of anupward shift in the A260 value leading to over-quantification of DNA (Figure 7). 25
  26. 26. DNA quantification by agarose gel analysisAgarose gel analysis enables quick and easy quantification of DNA (5), especially forsmall DNA fragments (such as PCR products). As little as 20 ng DNA can be detectedby agarose gel electrophoresis with ethidium bromide staining. The DNA sample is runon an agarose gel alongside known amounts of DNA of the same or a similar size. Theamount of sample DNA loaded can be estimated by comparison of the band intensitywith the standards either visually (Figure 8) or using a scanner or imaging system. Besure to use standards of roughly the same size as the fragment of interest to ensurereliable estimation of the DNA quantity, since large fragments interchelate more dyethan small fragments and give a greater band intensity. Agarose Gel Analysis of Plasmid DNA 26
  27. 27. Figure 8. An unknown amount of a 5.5 kb DNA fragment (U) was run alongside knownquantities (as indicated in ng) of the same DNA fragment on a 1% TAE agarose gel.The unknown sample contained ~85 ng DNA, as estimated by visual comparison withthe standards. M: 1 kb DNA ladder.More precise agarose gel quantification can be achieved by densitometric measurementof band intensity and comparison with a standard curve generated using DNA of aknown concentration. In most experiments the effective range for comparativedensitometric quantification is between 20 and 100 ng.The amount of DNA used for densitometric quantification should fall within thelinear range of the standard curve.Restriction Endonuclease Digestion of DNAPrinciple of restriction digestionDNA for downstream applications is usually digested with restriction endonucleases.This yields DNA fragments of a convenient size for downstream manipulations.Restriction endonucleases are bacterial enzymes that bind and cleave DNA at specifictarget sequences. Type II restriction enzymes are the most widely used in molecularbiology applications. They bind DNA at a specific recognition site, consisting of a shortpalindromic sequence, and cleave within this site, e.g., AGCT (for AluI), GAATTC(for EcoRI), and so on. Isoschizomers are different enzymes that share the samespecificity, and in some cases, the same cleavage pattern.Isoschizomers may have slightly different properties that can be very useful. Forexample, the enzymes MboI and Sau3A have the same sequence specificities, but MboI 27
  28. 28. does not cleave methylated DNA, while Sau3A does. Sau3A can therefore be usedinstead of MboI where necessary.Selecting suitable restriction endonucleasesThe following factors need to be considered when choosing suitable restrictionenzymes: Fragment size Blunt-ended/sticky-ended fragments Methylationsensitivity Compatibility of reaction conditionsFragment sizeRestriction enzymes with shorter recognition sequences cut more frequently than thosewith longer recognition sequences. For example, a 4 base pair (bp) cutter will cleave,on average, every 44 (256) bases, while a 6 bp cutter cleaves every 46 (4096) bases.Use 6 bp cutters for mapping genomic DNA or YACs, BACs, or P1s, as these givefragments in a suitable size range for cloning.Blunt-ended/sticky-ended fragmentsSome restriction enzymes cut in the middle of their recognition site, creating blunt-ended DNA fragments. However, the majority of enzymes make cuts staggered on eachstrand, resulting in a few base pairs of single-stranded DNA at each end of thefragment, known as “sticky” ends. Some enzymes create 5 overhangs and others create3 overhangs. The type of digestion affects the ease of downstream cloning: Sticky-ended fragments can be easily ligated to other sticky-ended fragments with compatiblesingle-stranded overhangs, resulting in efficient cloning.Blunt-ended fragments usually ligate much less efficiently, making cloning moredifficult. However, any blunt-ended fragment can be ligated to any other, so blunt-cutting enzymes are used when compatible sticky-ended fragments cannot be generated– for example, if the polylinker site of a vector does not contain an enzyme sitecompatible with the fragment being cloned.MethylationMany organisms have enzymes called methylases that methylate DNA at specificsequences. Not all restriction enzymes can cleave their recognition site when it ismethylated. Therefore the choice of restriction enzyme is affected by its sensitivity tomethylation. In addition, methylation patterns differ in different species, also affectingthe choice of restriction enzyme. 28
  29. 29. Methylation patterns differ between bacteria and eukaryotes, so restriction patterns ofcloned and uncloned DNA may differ.Methylation patterns also differ between different eukaryotes, affecting the choice ofrestriction enzyme for construction genomic DNA libraries.Compatibility of reaction conditionsIf a DNA fragment is to be cut with more than one enzyme, both enzymes can be addedto the reaction simultaneously provided that they are both active in the same buffer andat the same temperature. If the enzymes do not have compatible reaction conditions, itis necessary to carry out one digestion, purify the reaction products, and then performthe second digestion.Components of a restriction digestWater BufferDNA EnzymeDNAThe amount of DNA digested depends on the downstream application. For mapping ofcloned DNA, 0.2–1 μg DNA per reaction is adequate.DNA should be free of contaminants such as phenol, chloroform, ethanol, detergents,or salt, as these may interfere with restriction endonuclease activity.EnzymeOne unit of restriction endonuclease completely digests 1 μg of substrate DNA in 1hour. However, supercoiled plasmid DNA generally requires more than 1 unit/μg to bedigested completely. Most researchers add a ten-fold excess of enzyme to theirreactions in order to ensure complete cleavage.Ensure that the restriction enzyme does not exceed more than 10% of the total reactionvolume; otherwise the glycerol in which the enzyme is supplied may inhibit digestion.Reaction volumeMost digests are carried out in a volume between 10 and 50 μl. (Reaction volumessmaller than 10 μl are susceptible to pipetting errors, and are not recommended.)Protocol 10. Setting up a restriction digest 29
  30. 30. 1. Pipet reaction components into a tube and mix well by pipetting. Thorough mixingis extremely important. The enzyme should be kept on ice and added last. When settingup large numbers of digests, make a reaction master mix consisting of water, buffer,and enzyme, and aliquot this into tubes containing the DNA to be digested.2. Centrifuge the tube briefly to collect the liquid at the bottom.3. Incubate the digest in a water bath or heating block, usually for 1–4 h at 37°C.However, some restriction enzymes require higher (e.g., 50–65°C) while others requirelower (e.g., 25°C) incubation temperatures.4. For some downstream applications it is necessary to heat-inactivate the enzyme afterdigestion. Heating the reaction to 65°C for 20 min after digestion inactivates themajority of enzymes that have optimal incubation temperature of 37°C.Some restriction enzymes are not fully inactivated by heat treatment. Some Kitsprovide complete removal of restriction enzymes and salts following digestion.Ligation of DNAIn order to construct new DNA molecules, DNA must first be digested using restrictionendonucleases. The individual components of the desired DNA molecule are purifiedand then combined and treated with DNA ligase. The products of the ligation mixtureare introduced into competent E. coli cells and transformants are identified byappropriate genetic selection.Appropriate control ligations should also be performed (See Protocols 1 and 2, pages 2and 3).Removal of 5 phosphates from linearized vector DNA can help prevent vector self-ligation and improve ligation efficiency. To remove 5 phosphates from DNA, add calfintestinal phosphate (CIP) buffer and 1 U CIP and incubate for 30–60 minutes at 37°C.Once the reaction is complete, inactivate CIP by heating to 75°C for 15 minutes.Protocol 11. Ligation of DNA and subsequent transformation1. A typical ligation reaction is set up as follows: 31
  31. 31. Component DNAs (0.1–5 μg)Ligase buffer1 μl 10 mM ATP20–500 U T4 DNA ligase2. Incubate for 1–24 h at 15°C.Simple ligations with two fragments having 4 bp 3 or 5 overhanging ends requiremuch less ligase than more complex ligations or blunt-end ligations. The quality of theDNA will also affect the amount of ligase needed.Ligation of sticky-ends is usually carried out at 12–15°C to maintain a balance betweenannealing of the ends and the activity of the enzyme. Higher temperatures makeannealing of the ends difficult, while lower temperatures diminish ligase activity.Blunt-end ligations are usually performed at room temperature since annealing is not afactor, though the enzyme is unstable above 30°C. Blunt-end ligations require about10–100 times more enzyme than sticky-end ligations in order to achieve an equalefficiency.3. Introduce 1–10 μl of the ligated products into competent E. coli cells and select fortransformants using the genetic marker present on the vector.4. From individual E. coli transformants, purify plasmid or phage DNAs by miniprepprocedure and determine their structures by restriction mapping.It is highly recommended to include two controls in every transformation experiment:A “mock” transformation without DNA.A transformation reaction with a known amount of closed circular plasmid DNA.Controls are essential if things go wrong. For example, colonies on plates that receivemock-transformed bacteria may indicate that the medium lacks the correct antibiotic.An absence of colonies on plates receiving bacteria transformed with plasmids underconstruction can only be interpreted if a positive control using a standard DNA hasbeen included.References1. Sambrook, J. and Russell, D. (2001) Molecular Cloning: A Laboratory Manual. 3rded. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. 31
  32. 32. 2. Ausubel, F.M. et al., eds. (1999) Current Protocols in Molecular Biology, New York:John Wiley and Sons.3. Birnboim, H.C., and Doly, J. (1979) A rapid alkaline lysis procedure for screeningrecombinant plasmid DNA. Nucl. Acids. Res. 7,1513.4. Birnboim, H.C. (1983) A rapid alkaline extraction method for the isolation ofplasmid DNA. Methods Enzymol. 100, 243.6. Effect of pH and ionic strength on the spectroscopic assessment of nucleic acidpurity. (1997) BioTechniques 22, 474. 32
  33. 33. 33
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  36. 36. 36
  37. 37. Chapter two:Isolating and Purifying DNAfrom Deferent Sources of Blood forPCRThis chapter describes how to isolate and purify intact high molecularweight DNA from fresh or frozen whole blood 37
  38. 38. Isolating DNA from Fresh or Frozen Whole BloodAnticoagulants Commonly used anticoagulants such as EDTA, CPD, ACD, Citrate,or Heparin are compatible with this protocol and will not inhibit PCR. As shown inFigure 2-1, this procedure gives reproducible results with various anticoagulants.Figure 1: 1% TBE agarose gel images of gDNA isolated from humanblood in a variety of common anticoagulants. Lanes 1 and 20 show agDNA molecular weight ladder with the largest band at 40kb.Input Volume In most cases, 150 μL is the recommended maximum input volumefor fresh and frozen human whole blood. The maximum volume for blood from most 38
  39. 39. animal species is 50 μL. Volumes greater than 150 μL clog the purification tray wellsand do not increase the yield of recovered DNA.Table 1: Input volumes for fresh and frozen blood: Sample Maximum input volume Human Buffy Coat/PBMC 50 μL (1 × 106 cells per well) Human Fresh or Frozen 150 μL Chicken 20 μL Cow 50 μL Horse 50 μL Rabbit 50 μL Rat 50 μL Sheep 50 μLDNA Yields andPurity150 μL of human whole blood is expected to yield 3–8 μg of human DNA dependingon the white blood cell count of the sample. Yields of DNA from animals are listed inTable 1. For all types of fresh and frozen blood samples, the A260/280 ratios for DNApurity should be approximately 1.7–1.8 provided that volume limits and the incubationtime and temperatures are adhered to. Figure 2: Purity of DNA isolated from 3 × 96 well purifications 39
  40. 40. Figure 3: Blood gDNA yields and purity from nine different animalspeciesInhibitors in Isolated DNAIsolated DNA should free from contaminating protein such as heme and other cellularmacromolecules. Inhibitors do not carryover into the DNA even from samples that areanticoagulated with heparin.Absence of HemoglobinHeme, the non-protein iron component of hemoglobin, is a primary contaminant ofDNA from blood preparations and is detected by absorption at 410nm.Absence of ParticulatesLight-scattering particulates are easily observed as an absorption at 320nm. 41
  41. 41. Blood Volumes and Incubation Times for Animal: Sample Maximum input volume gDNA Yield Incubation (μg) Time (mins) Chicken 20 μL 2-6 10 Cow 50 μL 8-12 20 Horse 50 μL 2-4 10 Rabbit 50 μL 3-5 10 Rat 50 μL 3-6 10 Sheep 50 μL 2-3 10DNAIsolationFigure 4: 1% TBE agarose gel image of gDNA isolated from 50 μL offrozen animal blood of various species (Pig, Cow, Human, Mol Wt, Rabbit,Chicken, Dog, Pig, Sheep, Horse, Cow, Human, Mouse, Rat, Mol Wt)respectively . 20 μL of eluate (total 200 μL) was loaded into the gelshowing relative yields of gDNA from different animal speciesExtraction of DNA from Whole BloodJohn M. S. Bartlett and Anne White1. IntroductionThere are many differing protocols and a large number of commercially available kitsused for the extraction of DNA from whole blood. This procedure is one is usedroutinely in both research and clinical service provision and is cheap and robust. It can 41
  42. 42. also be applied to cell pellets from dispersed tissues or cell cultures (omitting the redblood lysis step.2. MaterialsThis method uses standard chemicals that can be obtained from any major supplier;we use Sigma.1. Waterbath set at 65°C.2. Centrifuge tubes (15 mL; Falcon).3. Microfuge (1.5 mL) tubes.4. Tube roller/rotator.5. Glass Pasteur pipets, heated to seal the end and curled to form a “loop” or “hook”for spooling DNA.6. EDTA (0.5 M), pH 8.0: Add 146.1 g of anhydrous EDTA to 800 mL of distilledwater. Adjust pH to 8.0 with NaOH pellets (this will require about 20 g). Make up to 1L with distilled water. Autoclave at 15 p.s.i. for 15 min.7. 1 M Tris-HCl, pH 7.6: Dissolve 121.1 g of Tris base in 800 mL of distilled water.Adjust pH with concentrated HCl (this requires about 60 mL).8. Reagent A: Red blood cell lysis: 0.01M Tris-HCl pH 7.4, 320 mM sucrose, 5 mMMgCl2, 1% Triton X 100.9. Add 10 mL of 1 M Tris, 109.54 g of sucrose, 0.47 g of MgCl2, and 10 mL of TritonX-100 to 800 mL of distilled water. Adjust pH to 8.0, and make up to 1 L with distilledwater. Autoclave at 10 p.s.i. for 10 min (see Note 1).10. Reagent B: Cell lysis: 0.4 M Tris-HCl, 150 mM NaCl, 0.06 M EDTA, 1% sodiumdodecyl sulphate, pH 8.0. Take 400 mL of 1 M Tris (pH 7.6), 120 mL of 0.5 M EDTA(pH 8.0),8.76 g of NaCl, and adjust pH to 8.0. Make up to 1 L with distilled water. Autoclave15 min at 15. p.s.i. After autoclaving, add 10 g of sodium dodecyl sulphate.11. 5 M sodium perchlorate: Dissolve 70 g of sodium perchlorate in 80 mL of distilledwater. Make up to 100 mL.12. TE Buffer, pH 7.6: Take 10 mL of 1 M Tris-HCl, pH 7.6, 2 mL of 0.5 M EDTA,and make up to 1 L with distilled water. Adjust pH to 7.6 and autoclave 15 min at 15.p.s.i.13. Chloroform prechilled to 4°C.14. Ethanol (100%) prechilled to 4°C. 42
  43. 43. 3. Method3.1. Blood Collection1. Collect blood in either a heparin- or EDTA-containing Vacutainer by venipuncture(see Note 2). Store at room temperature and extract within the same working day.3.2. DNA ExtractionTo extract DNA from cell cultures or disaggregated tissues, omit steps 1 through 3.1. Place 3 mL of whole blood in a 15-mL falcon tube.2. Add 12 mL of reagent A.3. Mix on a rolling or rotating blood mixer for 4 min at room temperature.4. Centrifuge at 3000g for 5 min at room temperature.5. Discard supernatant without disturbing cell pellet. Remove remaining moisture byinverting the tube and blotting onto tissue paper.6. Add 1 mL of reagent B and vortex briefly to resuspend the cell pellet.7. Add 250 μL of 5 M sodium perchlorate and mix by inverting tube several times.8. Place tube in waterbath for 15 to 20 min at 65°C.9. Allow to cool to room temperature.10. Add 2 mL of ice-cold chloroform.11. Mix on a rolling or rotating mixer for 30 to 60 min (see Note 3).12. Centrifuge at 2400g for 2 min.13. Transfer upper phase into a clean falcon tube using a sterile pipet.14. Add 2 to 3 mL of ice-cold ethanol and invert gently to allow DNA to precipitate(see Note 4).15. Using a freshly prepared flamed Pasteur pipet spool the DNA onto the hooked end(see Note 5).16. Transfer to a 1.5-mL Eppendorf tube and allow to air dry (see Note 6).17. Resuspend in 200 μL of TE buffer (see Notes 7 and 8).4. Notes1. Autoclaving sugars at high temperature can cause caramelization (browning), whichdegrades the sugars.2. As will all body fluids, blood represents a potential biohazard. Care should be takenin all steps requiring handling of blood. If the subject is from a known high riskcategory (e.g., intravenous drug abusers) additional precautions may be required. 43
  44. 44. 3. Rotation for less than 30 or over 60 min can reduce the DNA yield.4. DNA should appear as a mucus-like strand in the solution phase.5. Rotating the hooked end by rolling between thumb and forefinger usually workswell. If the DNA adheres to the hook, break it off into the Eppendorf and resuspend theDNA before transferring to a fresh tube.6. Ethanol will interfere with both measurements of DNA concentration and PCRreactions. However, overdrying the pellet will prolong the resuspension time. Thesmall amount of EDTA in TE will not affect PCR. We routinely use 1 μL per PCRreaction without adverse affects.8. DNA can be quantified and diluted to a working concentration at this point or simplyuse 1 μL per PCR reaction; routinely, it is expected200 to 500 ng/μL DNA to be the yield of this procedure.Protocol 7Polymerase Chain ReactionsHaiying Grunenwald1. IntroductionThe polymerase chain reaction (PCR) is a powerful method for fast in vitro enzymaticamplifications of specific DNA sequences. PCR amplifications can be grouped intothree different categories: standard PCR, long PCR, and multiplex PCR. Standard PCRinvolves amplification of a single DNA sequence that is less than 5 kb in length and isuseful for a variety of applications, such as cycle sequencing, cloning, mutationdetection, etc. Long PCR is used for the amplification of a single sequence that islonger than 5 kb and up to 40 kb in length. Its applications include long-rangesequencing; amplification of complete genes; PCR-based detection and diagnosis ofmedically important large-gene insertions or deletions; molecular cloning; andassembly and production of larger recombinant constructions for PCR-basedmutagenesis (1,2). The third category, multiplex PCR, is used for the amplification ofmultiple sequences that are less than 5 kb in length. Its applications include forensicstudies; pathogen identification; linkage analysis; template quantitation; geneticdisease diagnosis; and population genetics (3–5). Unfortunately, there is no single setof conditions that is optimal for all PCR. Therefore, each PCR is likely to requirespecific optimization for the template/primer pairs chosen. Lack of optimization often 44
  45. 45. results in problems, such as no detectable PCR product or low efficiency amplificationof the chosen template; the presence of nonspecific bands or smeary background; theformation of “primer-dimers” that compete with the chosen template/primer set foramplification; or mutations caused by errors in nucleotide incorporation. It isparticularly important to optimize PCR that will be used for repetitive diagnostic oranalytical procedures where optimal amplification is required. The objective of thischapter is to discuss the parameters that may affect the specificity, fidelity, andefficiency of PCR, as well as approaches that can be taken to achieve optimal PCRamplifications. Optimization of a particular PCR can be time consuming andcomplicated because of the various parameters that are involved. These parametersinclude the following: (1) quality and concentration of DNA template; (2) design andconcentration of primers; (3) concentration of magnesium ions; (4) concentration ofthe four deoxynucleotides (dNTPs); (5) PCR buffer systems; (6) selection andconcentration of DNA polymerase; (7) PCR thermal cycling conditions; (8) additionand concentrations of PCR additives/cosolvents; and (9) use of the “hot start”technique. Optimization of PCR may be affected by each of these parametersindividually, as well as the combined interdependent effects of any of these parameters.2. Materials1. Template DNA (e.g., plasmid DNA, genomic DNA).2. Forward and reverse PCR primers.3. MgCl2 (25 mM).4. dNTPs (a mixture of 2.5 mM dATP, dCTP, dGTP, and dTTP).5. 10× PCR buffer: 500 mM KCl, 100 mM Tris-HCl, pH 8.3, 25°C.6. Thermal stable DNA polymerase (e.g., Taq DNA polymerase).7. PCR additives/cosolvents (optional; e.g., betaine, glycerol, DMSO, formamide,bovine serum albumin, ammonium sulfate, polyethylene glycol, gelatin, Tween-20,Triton X-100, β-mercaptoethanol, or tetramethylammonium chloride).3. Methods3.1. Setting Up PCRThe common volume of a PCR is 10, 25, 50, or 100 μL. Although larger volumes areeasier to pipet, they also use up a larger amount of reagents, which is less economical. 45
  46. 46. All of the reaction components can be mixed in together in a 0.5-mL PCR tube in anysequence except for the DNA polymerase, which should be added last. It isrecommended to mix all the components right before PCR cycling. Although it is notnecessary to set up the PCR on ice, some published protocols recommend it.For each PCR, the following components are mixed together:1. Template DNA (1–500 ng).2. Primers (0.05–1.0 μM).3. Mg2+ (0.5–5 mM).4. dNTP (20–200 μM each).5. 1× PCR buffer: 1 mM Tris-HCl and 5 mM KCl.6. DNA polymerase (0.5–2.5 U for each 50 μL of PCR).As a real-life example, the following PCR was set up to amplify the cII gene frombacteriophage lambda DNA (total volume = 50 μL):1. 1 μL of 1 ng/μL lambda DNA (final amount = 1 ng).2. 1 μL of 50 μM forward PCR primer (final concentration = 1 μM).3. 1 μL of 50 μM reverse PCR primer (final concentration = 1 μM).4. 5 μL of 25 mM MgCl2 (final concentration = 2.5 mM).5. 4 μL of 2.5 mM dNTPs (final concentration = 200 μM).6. 5 μL of 10× PCR buffer (final concentration = 1×).7. 0.25 μL of 5 U/μL Taq DNA polymerase (final amount = 1.25 U).3.2. PCR CyclingA common PCR cycling program usually starts with an initial dissociation step at 92to 95°C for 2 to 5 min to ensure the complete separation of the DNA strands. MostPCR will reach sufficient amplification after 20 to 40 cycles of strand denaturation at90 to 98°C for 10 s to 1 min, primer annealing at 55 to 70°C for 30 s to 1 min, andprimer extension at 72 to 74°C for 1 min per kilobase of expected PCR product.It is suggested that a final extension step of 5 to 10 min at 72°C will ensure that allamplicons are fully extended, although no solid evidence proves that this step isnecessary. For example, the cycling program used to amplify the previously describedlambda cII gene is as follows: initial denaturation for 4 min at 94°C, followed by 30cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C, then held at 4°C.3.3. Verifying PCR AmplificationTo measure the success of a PCR amplification, 5 to 10 μL of the final PCR product isrun on a 1 or 2% agarose gel and visualized by staining with ethidium bromide. The 46
  47. 47. critical questions are as follows: (1) Is there a band on the gel? (2) Is the band at theexpected size? (3) Are there any nonspecific bands beside the expected PCR band onthe gel? (4) Is there smear on the gel? A successful PCR amplification should displaya single band with the expected size without nonspecific bands and smear.Procedural Precautions1. Work in laboratory using DNA amplification methods should always flow in a one-way direction beginning in the specimen preparation and processing area (Area 1), thenmoving to the amplification and detection area (Area 2). Do not bring any materials orequipment from Area 2 into Area 1.2. Surface cleaning using a 1% (v/v) sodium hypochlorite solution followed by 70%(v/v) ethanol should be performed on bench tops and pipets prior to beginning the LCRAssay.3. Chlorine solutions may pit equipment and metal. Use sufficient amounts or repeatedapplications of 70% ethanol until chlorine residue is no longer visible.References1. Higuchi, R. (1989) Using PCR to engineer DNA, in PCR Technology: Principlesand Applications for DNA Amplifications (Erlich, H. A., ed.), Stockton Press, Inc., NewYork, pp. 61–70.2. Foord, O. S. and Rose, E. A. (1995) Long-distance PCR, in PCR Primer(Dieffenback, C. W. and Dveksler, G. S., ed.), Cold Spring Harbor Laboratory Press,Cold Spring, NY, pp. 63–77.3. Edwards, A., Civitello, A., Hammond, H. A., and Caskey, C. T. (1991) DNA typingand genetic mapping with trimeric and tetrameric tandem repeats. Am. J. Hum. Genet.49, 746–756.4. Edwards, A., Hammond, H. A., Jin, L., Caskey, C. T., and Chakroborty, R. (1992)Geneticvariation at five trimeric and tetrameric tandem repeat loci in four humanpopulation groups. Genomics 12, 241–253.5. Klimpton, C. P., Gill, P., Walton, A., Urquhart, A., Millican, E. S., and Adams, M.(1993) Automated DNA profiling employing multiplex amplification of short tandemrepeat loci. PCR Methods Appl. 3, 13–21. 47
  48. 48. 6. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: Alaboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NewYork.7. Lindahl, T. and Nyberg, B. (1972) Rate of depurination of native deoxyribonucleicacid. Biochemistry 11, 3610–3618.8. Rychlik, W. and Rhoads, R. E. (1989) A computer program for choosing optimaloligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA.Nucleic Acids Res. 17, 8543–8551.9. Lowe, T. M. J., Sharefkin, J., Yang, S. Q., and Dieffenback, C. W. (1990) Acomputer program for selection of oligonucleotide primers for polymerase chainreaction. Nucleic Acids Res. 18, 1757–1761.10. O’Hara, P. J. and Venezia, D. (1991) PRIMGEN, a tool for designing primersfrom multiple alignments. CABIOS 7, 533–534.11. Montpetit, M. L., Cassol, S., Salas, T., and O’Shaughnessy, M. V. (1992)OLIGOSCAN: A computer program to assist in the design of PCR primershomologous to multiple DNA sequences. J. Virol. Methods 36, 119–128.12. Suggs, S. V., Hirose, T., Myake, D. H., Kawashima, M. J., Johnson, K. I., andWallace, R. B. (1981) Using purified genes, ICN-UCLA Symp. Mol. Cell. Biol. 23,683–693.13. Breslauer, K. J., Ronald, F., Blocker, H., and Marky, L. A. (1986) PredictingDNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. USA 83, 3746–3750.14. Freier, S. M., Kierzek, R., Jaeger, J. A., Sugimoto, N., Caruthers, M. H., Neilson,T., et al. (1986) Improved free-energy parameters for predictions of RNA duplexstability. Proc. Natl. Acad. Sci. USA 83, 9373–9377.15. Innis, M. A. and Gelfand, D. H. (1990) Optimization of PCRs, in PCR Protocols:A Guide to Methods and Applications (Gelfand, D. H., Sninsky, J. J., Innis, M. A., andWhite, H., eds.), Academic Press, San Diego, CA, pp. 3–12.16. Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. A. D. (1988) DNAsequencing with Thermus aquaticus DNA polymerase and direct sequencing ofpolymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85, 9436–9440.17. Ohler, L. and Rose, E. A. (1992) Optimization of long distance PCR using atransposonbased model system. PCR Methods Appl. 2, 51–59. 48
  49. 49. 18. Gelfand, D. H. (1989) Taq DNA polymerase, in PCR Technology: Principles andApplications for DNA Amplification (Erlich, H. A., ed.), Stockton Press, New York,pp. 17–22.19. Cheng, S. (1995) Longer PCR amplifications, in PCR Strategies (Innis, M. A.,Gelfand, D. H., and Sninsky, J. J., eds.) Academic Press, San Diego, CA, pp. 313–324.20. Brock, T. D. and Freeze, H. (1969) Thermus aquaticus gene, a non-sporulatingextreme thermophile. J. Bacteriol. 98, 289–297.21. Giebel, L. B. and Spritz, R. A. (1990) Site-directed mutagenesis using the double-stranded DNA fragment as a PCR primer. Nucleic Acids Res. 18, 4947.22. Saike, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R. Horu, G. T.,Mullis, K. B., and Erlich, H. A. (1988) Primer-directed enzymatic amplification ofDNA with a thermostable DNA polymerase. Science 239, 487–491.23. Flaman, J.-M., Frebourg, T., Moreau, V., Charbonnier, R., Martin, C., Ishioka,C., et al. (1994). A rapid PCR fidelity assay. Nucleic Acids Res. 22, 3259–3260.24. Cline, J., Braman, J. C., and Hogrefe, H. H. (1996) PCR fidelity of Pfu DNApolymerase and other thermostable DNA polymerases. Nucleic Acids Res. 24, 3546–3551.25. Sardelli, A. D. (1993) Plateau effect-understanding PCR limitations, inAmplifications: A Forum for PCR Users. Perkin–Elmer Corp., Norwald, CT, pp. 1. 26.Saiki, R. K. (1989) The design and optimization of the PCR, in PCR Technology(Erlich, H. A., ed.), Stockton Press, New York, pp. 7–16.27. Kim, H. S. and Smithies, O. (1988) Recombinant fragment assay for gene targetingbased on the polymerase chain reaction. Nucleic Acids Res. 16, 8887–8903.28. McConlogue, L., Brow, M. D., and Innis, M. A. (1988) Structure-independentDNA amplification by PCR using 7-deaza-2′-deoxyguanosine. Nucleic Acids Res. 16,9869.29. Mytelka, D. S. and Chamberlin, M. J. (1996) Analysis and suppression of DNApolymerase pauses associated with a trinucleotide consensus. Nucleic Acids Res. 24,2774–2781.30. Pomp, D. and Medrano, J. F. (1991) Organic solvents as facilitators of polymerasechain reaction. BioTechniques 10, 58–59.31. Newton, C. R. and Graham, A. (1994) PCR, Bios Scientific, Publishers Ltd.,Oxford. 49
  50. 50. 32. Levinson, G., Fields, R. A., Harton, G. L., Palmer, F. T., Maddleena, A., Fugger,E. F.,et al. (1992) Reliable gender screening for human preimplantation embryos,using multiple DNA target-sequences. Hum. Reprod. 7, 1304–1313.33. Chamberlain, J. S., Gibbs, R. A., Ranier, J. E., Nguyen, P. N., and Caskey, C. T.(1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplexDNA amplification. Nucleic Acids Res. 16, 11,141–11,156.34. Uggozoli, L. and Wallace, B. (1992) Application of an allele-specific polymerasechain reaction to the direct determination of ABO blood group genotypes. Genomics12, 670–674.35. Henke, W., Herdel, K., Jung, K., Schnorr, D., and Loening, S. A. (1997) Betaineimproves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res. 25,3957–3958.36. Hengen, P. N. (1997) Optimizing multiplex and LA-PCR with betaine. TrendsBiochem. Sci. 22, 225–226.37. Bassam, B. J. and Caetano-Anolles, G. (1993) Automated “hot start” PCR usingmineral oil and paraffin wax. BioTechniques 14, 30–34.38. Wainwright, L. A. and Seifert, H. S. (1993) Paraffin beads can replace mineral oilas an evaporation barrier in PCR. BioTechniques 14, 34–36.39. Rous, K. H. (1995) Optimization and troubleshooting in PCR, in PCR Primer(Diefferbach, C. W. and Dveksler, G. S. eds.), CSH Press, New York, pp. 53–62. 51
  51. 51. DNA isolation using kit "follow the kit procedure" 51
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  54. 54. References1. Helms, C. Salting out Procedure for Human DNA extraction. In The Donis-Keller Lab - Lab Manual Homepage [online]. 24 April 1990. [cited 19 November 2002; 11:09 EST]. Available from: http://hdklab.wustl.edu/lab_manual/dna/dna2.html.2. Epplen, J.E., and T. Lubjuhn. 1999. DNA profiling and DNA fingerprinting. Birhkhauser Verlag, Berlin. p.55. 54
  55. 55. DNA Extraction from Fungi, Yeast, and BacteriaDavid Stirling1. IntroductionAlthough individual microorganisms may well require a unique DNA extractionprocedure, here it is included robust techniques for the preparation of DNA from fungi,yeast, and bacteria, which yield DNA suitable for a PCR template.2. Materials2.1. Fungal Extraction1. CTAB extraction buffer: 0.1 M Tris-HCl, pH 7.5, 1% CTAB (mixed hexadecyltrimethyl ammonium bromide), 0.7 M NaCl, 10 mM EDTA, 1% 2-mercaptoethanol.Add proteinase K to a final concentration of 0.3 mg/mL prior to use.2. Chloroform:isoamyl alcohol (24:1).2.2. Yeast Extraction1. Yeast extraction buffer A: 2% Triton X-100, 1% sodium dodecyl sulphate, 100 mMNaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0. Phenol:chloroform:isoamylalcohol: Phenol is presaturated with 10 mM Tris-HCl, pH 7.5. Prepare a mixture of25:24:1 phenol:chloroform:isoamyl alcohol (v/v/v). This solution can be stored at roomtemperature for up to 6 mo, shielded from light.2. Glass beads. Diameter range 0.04–0.07 mm (Jencons Scientific Ltd, UK), suspendedas 500 mg/mL slurry in distiller water.3. Ammonium acetate (4 M).2.3. Bacterial DNA Protocol1. Lysozyme/RNase mixture: 10 mg/mL lysozyme, 1 mg/mL RNase, 50 mM Tris-HCl(pH 8.0). Store at –20°C in small aliquots. Do not refreeze after thawing.2. STET: 8% sucrose, 5% Triton X-100, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, pH8.0. 55
  56. 56. 3. Filter sterilize and store at 4°C.3. Methods3.1. Fungal Protocol1. Grind 0.2 to 0.5 g (dry weight) of lyophilized mycellar pad in a mortar and pestle.Transfer to a 50-mL disposable centrifuge tube.2. Add 10 mL (for a 0.5 g pad) of CTAB extraction buffer.3. Gently mix to wet all the powdered pad.4. Place in 65°C water bath for 30 min.5. Cool and add an equal volume of chloroform/isoamyl alcohol (24:1).6. Mix and centrifuge at 2000g for 10 min at room temperature.7. Transfer aqueous supernatant to a new tube.8. Add an equal volume of isopropanol.9. High molecular weight DNA should precipitate upon mixing and can be spooled outwith a glass rod or hook.10. Rinse the spooled DNA with 70% ethanol.11. Air dry, add 1 to 5 mL of TE containing 20 μg/ mL RNAse A. To resuspend thesamples, place in 65°C bath or allow pellets to resuspend overnight at 4°C.3.2. Yeast Protocol1. Collect cells from fresh 5 mL culture by centrifugation at 2000g for 10 min andresuspend in 0.5 mL of water.2. Transfer cells to 1.5-mL microfuge tube and collect by centrifugation at 15,000g for10 min, pour off supernatant and resuspend in residual liquid.3. Add 0.2 mL of buffer A, 200 μL of glass beads, and 0.2 mL ofphenol:chloroform:isoamyl alcohol (25:24:1).4. Vortex for 3 min and add 0.2 mL of TE.5. Centrifuge at 15,000g for 5 min and then transfer aqueous to new tube.6. Add 1 mL of 100% EtOH (room temperature), invert tube to mix, and centrifuge at15,000g for 2 min.7. Discard supernatant and resuspend pellet in 0.4 mL of TE (no need to dry pellet).8. Add 10 μL of 4 M ammonium acetate, mix, and then add 1 mL of 100% EtOH andmix.9. Centrifuge at 15,000g for 2 min and dry pellet. Resuspend in 50 μL of TE. 56
  57. 57. 3.3. Bacterial DNA Protocol1. Collect the bacteria from a 15-mL overnight culture into a 1.5-mL microfuge tube.2. Resuspend pellet with 300 μL of STET buffer and add 30 μL of RNAse/lysozymemixture.3. Boil for 1 min 15 s.4. Centrifuge at 15,000g for at least 15 min.5. Take supernatant and phenol extract with 150 μL of STET-saturated phenol.6. Spin and take supernatant. Add 1/10 volume 4 M lithium chloride (autoclaved). Letsit on ice for 5 to 10 min.7. Spin and take supernatant. Add equal volume isopropanol at room temperature andincubate for 5 min.8. Centrifuge at 15,000g for at least 15 min. No pellet will be visible.9. IMPORTANT: wash with 80% ethanol (95% will cause the residual Triton toprecipitate).10. Resuspend pellet in 50 to 200 μL of TE.References 1. Chevillard, S. (1993) A method for sequential extraction of RNA and DNA from the same sample, specially designed for a limited supply of biological material. BioTechniques 15, 22–24. 2. Pomp, D. and Medrano, J. F. (1991) Organic solvents as facilitators of polymerase chain reaction. BioTechniques 10, 58–59. 3. Newton, C. R. and Graham, A. (1994) PCR, Bios Scientific, Publishers Ltd., Oxford. 57
  58. 58. 58