What are the essentialcomponents of a DNA extraction Procedure? 1. Maximize DNA recovery 2. Remove inhibitors 3. Remove or inhibit nucleases 4. Maximize the quality of DNA
How Much DNA Can We Recover?• A Diploid Cell contains approximately 6 pg of DNA• The average WBC of an adult is 5 - 10 X 106 cells per ml of blood. Therefore, the theoretical recovery of DNA per ul of blood is 30 - 60 ng.
How Much DNA Do We Need?• The PCR reactions call for on average 1 ng of DNA (single or double stranded).• Many of the commercially available kits are sensitive below 1 ng of DNA (100-250 pg).
Basic steps for DNA extraction1. Breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by grinding, sonicating or treating the sample with lysis buffer .2. Removing membrane lipids by adding a detergent.
Purposes of the Extraction Buffer Detergents Chaotropic salts1. Dissolve cellular membranes CTAB Detergents Metal chelators Reducing agents2. Inactivation of DNase and Rnase Salts3. Assist in the removal of contaminants CTAB PVP
Extraction/Precipitation MethodUse of Detergents to Lyse Cells: Mixed micelle Plasma membrane (phospholipid bilayer) Detergent molecules + SDS
3. Removing proteins by adding a protease (optional but almost always done).4. Precipitating the DNA with an alcohol — usually ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt.
• Depending on the material and requirement the extraction process can be modified with alteration of chemicals used
Most Commonly used DNA Extraction Procedures• Organic (Phenol-Chloroform) Extraction• Non-Organic (Proteinase K and Salting out)• NCM / Nylon membrane(Collection, Storage, and Isolation) The method utilized may be sample dependant, technique dependant, or analyst preference
Extraction/Precipitation MethodStep 1: Disruption of cell walls by grinding Step 1+2: mechanical disruption and homogenization in extraction buffer Grind sample into a fine powder to shear cell walls and membranesStep 2: Lysis of cells in extraction buffer A homogenizer allows cells to be mechanically disrupted within the extraction buffer Mix thoroughly with extraction buffer to dissolve cell membranes and inhibit nuclease activity Crude lysate
Extraction/Precipitation MethodStep 3: Organic extraction Mix thoroughly with Aqueous an equal volume of organic solvent Centrifuge Collect aqueous phase e.g. phenol, chloroform, or phenol:chloroform Interphase Organic Perform additional extractions for increased purity Crude lysate containing The aqueous phase contains water- nucleic acids and other soluble molecules, including nucleic cell constituents acids. Proteins and lipids become trapped in the organic phase, and are thus separated away. Insoluble plant debris become trapped in the interphase between the two layers
Extraction/Precipitation MethodStep 4: Nucleic Acid Precipitation Before After Supernatant 70% EtOH Centrifuge Wash Centrifuge Pellet Dissolve pellet (H2O, TE, etc.)Add alcohol and salt to • Pellet down nucleic acids.precipitate nucleic acids • Wash pellet with 70% ethanol to removefrom the aqueous fraction residual salts and other contaminants. • Discard ethanol and allow pellet to dry.
RNA extraction• This method relies on phase separation by centrifugation of a mix of the aqueous sample and a solution containing water- saturated phenol,chloroform and a denaturing solution (guanidinium thiocyanate) resulting in an upper aqueous phase and a lower organic phase (mainly chloroform).
• Nearly all of the RNA is present in the aqueous phase, while DNA and protein partition in the interphase and organic phase, respectively. In a last step, RNA is recovered from the aqueous phase by precipitation with 2-propanol or ethanol. DNA will be located in the aqueous phase in the absence of guanidinium thiocyanate and thus the technique can be used for DNA purification alone.
• Guanidinium thiocyanate denatures proteins, including RNases, and separates rRNA from ribosomes, while phenol, isopropanol and water are solvents with poor solubility. In the presence of chloroform , these solvents separate entirely into two phases that are recognized by their color: a clear, upper aqueous phase (containing the nucleic acids) and a bright pink lower phase (containing the proteins dissolved in phenol and the lipids dissolved in chloroform). Other denaturing chemicals such as 2-mercaptoethanol may also be used. The major downside is that phenol and chloroform are both hazardous and inconvenient materials, and the extraction is often laborious, so in recent years many companies now offer alternative ways to isolate DNA.
EXTRACTION• Perhaps the most basic of all procedures in genetic engineering is the purification of DNA. The key step, the removal of proteins, can often be carried out simply by extracting aqueous solutions of nucleic acids with phenol and/or chloroform.
EXTRACTION• Cell Lysis Buffer – This buffer will lyse cell membrane, nuclei are intact, pellet nuclei.• The nuclei is Resuspended in a buffer containing Sodium Dodecly Sulfate (SDS) and Proteinase K. This will Lyse nuclear membrane and digest protein.• DNA released into solution is extracted with phenol-chloroform to remove proteinaceous material.
EXTRACTION• DNA is precipitated from the aqueous layer by the additional of ice cold 95% ethanol and salt• Precipitated DNA is washed with 70% ethanol, dried under vacuum and resuspended in TE buffer.
ORGANIC EXTRACTION REAGENTS• Cell Lysis Buffer - Non-ionic detergent , Salt, Buffer, EDTA designed to lyse outer cell membrane, but will not break down nuclear membrane.• EDTA (Ethylenediaminetetraacetic disodium salt) is a chelating agent of divalent cations such as Mg2+. Mg2+is a cofactor for Dnase nucleases. If the Mg2+is bound up by EDTA, nucleases are inactivated.
ORGANIC EXTRACTION REAGENTS• Proteinase K - it is usual to remove most of the protein by digesting with proteolytic enzymes such as proteinase K, which are active against a broad spectrum of native proteins, before extracting with organic solvents. Protienase K is approximately 10 fold more active on denatured protein. Proteins can be denatured by SDS or by heat.
ORGANIC EXTRACTION REAGENTS• Phenol/Chlorform - The standard way to remove proteins from nucleic acids solutions is to extract once with phenol, once with a 1:1 mixture of phenol and chloroform, and once with chloroform. This procedure takes advantage of the fact that deproteinization is more efficient when two different organic solvents are used instead of one.• Also, the final extraction with chloroform removes any lingering traces of phenol from the nucleic acid preparation.• Phenol is highly corrosive and can cause severe burns.
ORGANIC EXTRACTION REAGENTS• Phenol - often means phenol equilibrated with buffer (such as TE) and containing 0.1% hydroxyquinoline and 0.2% b-ercaptoethanol (added as antioxidants. The hydoxquinoline also gives the phenol a yellow color,making it easier to identify the phases (layers).• Chloroform - often means a 24:1 (v/v) mixture of chloroform and isoamyl alcohol. The isoamyl alcohol is added to help prevent foaming.• The Phenol/Chloroform/Isoamyl Alcohol ratio is 25:24:1
Concentrating DNA by Alcohol Precipitation• The most widely used method for concentrating DNA is precipitation with ethanol. The precipitate of nucleic acid, forms in the presence of moderate concentrations of monovalent cations (Salt, such as Na+), is recovered by centrifugation and redissolved in an appropriate buffer such as TE.• The technique is rapid and is quantitative even with nanogram amounts of DNA.
• phenol helps to remove non polar proteins and lipids from the solution, phenol is used to denature the proteins. ethanol change ionic potential of DNA and remove water molecules, which help in precipitation of DNA. Salt would attract the phosphate ends of DNA, therefore it pulls it way from other substances in the sample (Separation of DNA from surroundings)
Concentrating DNA Alcohol Precipitation• The four critical variables are the purity of the DNA, its molecular weight, its concentration, and the speed at which it is pelleted.• DNA a concentrations as low as 20 ng/ ml will form a precipitate that can be quantitatively recovered.• Typically 2 volumes of ice cold ethanol are added to precipitate the DNA.
Concentrating DNA Alcohol Precipitation• Very short DNA molecules (<200 bp) are precipitated inefficiently by ethanol.• The optimum pelleting conditions depend on the DNA concentration. Relatively vigorous microcentrifuge steps such as 15 minutes at or below room temperature at 12,000 rpm are designed to minimized the loss of DNA from samples with yields in the range of a few micrograms or less.
Concentrating DNA Alcohol Precipitation• Solutes that may be trapped in the precipitate may be removed by washing the DNA pellet with a solution of 70% ethanol. To make certain that no DNA is lost during washing, add 70% ethanol until the tube is 2/3 full. Vortex briefly, and recentrifuge. After the 70% ethanol wash, the pellet does not adhere tightly to the wall of thetube, so great care must be taken when removing the supernatant.
Concentrating DNA Alcohol Precipitation• Isopropanol (1 volume) may be used in place of ethanol (2 volumes) to precipitate DNA. Precipitation with isopropanol has the advantage that the volume of liquid to be centrifuged is smaller.• Isopropanol is less volatile than ethanol and it is more difficult to remove the last traces; moreover, solutes such sodium chloride are more easily coprecipitated with DNA when isopropanol is used.
Resuspension and Storage of DNA• TE Buffer - Tris-EDTA Buffer: 10 mM Tris- HCl pH 8.0, 1 mM EDTA, or TE-4 which is 10 mM Tris, 0.1 mM EDTA. DNA is resuspended and stored in TE buffer. DNA must be stored in a slightly basis buffer to prevent depurination, and the EDTA chelates any Mg2+ helping to inactivate DNases.
• DNA can be stored at 4oC for extended periods, however for long term storage, - 20oC is preferable.• Avoid repetitive freeze thawing of DNA, since this can cause degradation.
Using Nucleases to Remove Unwanted DNA or RNA Add DNase + DNase (protein) Add RNase + RNase (protein)Depending on when nuclease treatment is performed, it may be necessary torepeat purification steps for protein removal (e.g. phenol/chloroform extraction).
Non-Phenol Chloroform based extraction of DNA for PCR detectionof Citrus yellow mosaic virus and Citrus greening bacterium Clear supernatent Centrifuge Supernatant Centrifuge Pellet Cell debris Add isoprpanol /alcohol and salt to precipitate nucleic acids from the aqueous fraction
Non-Organic DNA Extraction• Does not use organic reagents such as phenol or chloroform.• Digested proteins are removed by salting out with high concentrations of LiCl.• However, if salts are not adequately removed, problems could occur with the procedure due to alteration of DNA mobility (band shifting)
Non-Organic DNA Extraction Procedure• Cell Lysis Buffer Containing Proteinase - lyse cell membrane, lyse nuclear membrane and digest protein at high temperature eg. 65oC for 2 hours. Temperature helps denature proteins, and Proteinase K auto digests itself• To remove proteinaceous material, LiCl is added to a final concentration of 2.5 M, and incubated on ice. Proteins precipitate out and are pelleted by centrifugation.
Non-Organic DNA Extraction Procedure4. DNA remains in solution. Transfer supernatant to a new tube, care must be taken not to take any of protein pellet.5. DNA is precipitated by the addition of room temperature isopropanol. LiCl will not precipitate with DNA.6. Precipitated DNA is washed with 70% ethanol, dried under vacuum and resuspended in TE buffer.
Membrane based method• A spin column using a silica-based extraction method is used. This does not require the use of hazardous chemicals. Nucleic acids are attracted to the silica bead under high chaotropic salt concentrations. The sample and lysis buffer are added to a sterile tube.
The lysate is combined with alcoholand placed into the spin column,which is inserted into a tube. Theremoval of proteins and divalentcations is accomplished usingmultiple buffer washes andcentrifugation steps. Removal ofcations, such as Mg2+, preventsnucleases from further degrading theDNA. Pure DNA is eluted from themembrane into sterile water or TEbuffer.
Membrane based Nucleic acid template preparation Extract in NaOH-EDTA without use of Liquid N2 Centrifuge 15 minPCR/RT-PCR 90-100% Heat at 80 oC 10 minCG/CMBV/Viroid
Assessing the Quality and Yield of Nucleic Acids
Checking for DNAgenomicDNA Running nucleic acid sample through an agarose gel is a common method for examining the extent of DNA degradation. Good quality DNA should migrate as a high molecular weight band, with little or noRNA(degraded) evidence of smearing.
Nucleic Acid Analysis via UV Spectrophotometry DNA Absorption SpectraBy measuring the amount of light absorbed by your sample at specificwavelengths, it is possible to estimate the concentration of DNA andRNA. Nucleic acids have an absorption peak of 1 OD at ~260nm. [dsDNA] ≈ A260 x (50 µg/mL) [ssDNA] ≈ A260 x (33 µg/mL) [ssRNA] ≈ A260 x (40 µg/mL)
• 1 Optical Density (OD) unit of double- stranded DNA is 50 micrograms/ml.• 1 OD unit of single-stranded DNA is 33 micrograms/ml.• 1 OD unit of single-stranded RNA is 40 micrograms/ml.
How pure is nucleic acid sample?Nucleic acids strongly absorb at 260 nm and less strongly at 280 nmwhile proteins do the opposite.The A260/A280 ratio is ~1.8 for dsDNA, and ~2.0 for ssRNA. Ratioslower than 1.7 usually indicate significant protein contamination.The A260/A230 ratio of DNA and RNA should be roughly equal to itsA260/A280 ratio (and therefore ≥ 1.8). Lower ratios may indicatecontamination by organic compounds (e.g. phenol, alcohol, orcarbohydrates).