Packaging DNA A T T A G C C G G C T A A T Protein scaffold 11 nm “Beads on a string” 30 nm Tight helical fiber Looped Domains 200 nm
Packaging DNA G C A T Protein scaffold Metaphase Chromosome 700 nm 11 nm 30 nm 200 nm 2 nm Looped Domains Nucleosomes B DNA Helix Tight helical fiber
DNA extractioin Samples SamplesTissueBloodSalivaSemenUrineHairTeethBone
How much DNA can we recover -A diploid cell contains approximately 6pg of DNA -Sperm contain approximately 3pg of DNA -Average WBC of an adult is 5-10 ×10 6 cells per ml of blood. Therefore, the theoretical recovery of DNA per 200µl of blood is 3- 12µg.
How much DNA do we need The PCR reaction call for on average 10ng of DNA This is equivalent to 1/20 of 1µl of blood or 350 sperm.
The most commonly used DNA extraction procedure are: Organic (phenol-chloroform) extraction. Extraction using columns. Non-organic (proteinase K and salting out).
Phenol-chloroform extraction Most basic procedure The key step, the removal of proteins by extracting aqueous solutions of nucleic acids with phenol and/or chloroform a liquid-liquid extraction technique. It is widely used for isolating DNA, RNA and protein. Equal volumes of a phenol:chloroform mixture and an aqueous sample are mixed, forming a biphasic mixtureliquid-liquid extractionDNARNAproteinbiphasic mixture
Guanidinium thiocyanateGuanidinium thiocyanate denatures proteins, including RNases, and separates rRNA from ribosomes, while phenol, isopropanol 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). phenolisopropanolchloroform Guanidinium thiocyanatephenolisopropanolchloroform
DNA extraction from blood samples Reagents required: Ethanol 0.1 M Sodium citrate in 10% ethanol 75% Ethanol 8 mM NaOH
procedure 1.DNA PRECIPITATION -Remove the remaining aqueous phase overlying the interphase, and precipitate the DNA from the interphase and organic phase with ethanol. Add 0.3 mL of 100% ethanol per 1 mL of TRIZOL Reagent used for the initial homogenization, and mix samples by inversion. - Next, store the samples at 15 to 30°C for 2-3 minutes and sediment DNA by centrifugation at no more than 2,000 x g for 5 minutes at 2 to 8°C.
2. DNA WASH -Remove the phenol-ethanol supernate, and if desired, save it for protein isolation. -Wash the DNA pellet twice in a solution containing 0.1 M sodium citrate in 10% ethanol. At each wash, store the DNA pellet in the washing solution for 30 minutes at 15 to 30°C (with periodic mixing) and centrifuge at 2,000 x g for 5 minutes at 2 to 8°C. -F-Following these two washes, suspend the DNA pellet in 75% ethanol (1.5-2 mL of 75% ethanol ), store for 10-20 minutes at 15 to 30°C (with periodic mixing) and centrifuge at 2,000 x g for 5 minutes at 2 to 8°C.
3. REDISSOLVING THE DNA -Air dry the DNA 5 to 15 minutes in an open tube -Dissolve DNA in 8 mM NaOH Typically add 300 – 600 μL of 8mM NaOH to DNA isolated from 10 7 cells or 50 – 70mg of tissue.
Extraction using columns Tissue lysis solution Proteinase K for lysis of protein DNA precipitation (ethanol) Filtering DNA onto a membrane Membrane wash (twice) DNA elution
Non organic DNA extraction Cell lysis buffer Protein lysis buffer (proteinase K) lyse nuclear membrane and digest protein at 65 0 c for 2 hours. Remove proteinaceous material by LiCL. Incubate on ice. Protein precipitate out by centrifugation. Transfer supernatant containing DNA to a new tube DNA is precipitated by isopropanol DNA washed with 70% ethanol Resuspended in TE buffer
Determination of DNA concentration 1-10 µl DNA solution (DNA in hydration solution) + 990µl D.W. 2-Read at 260 nm Concentration of DNA in µg/ml = reading at 260nm ×time of dilution (100) × extinction coefficient of DNA(50)
DNA purity Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280nm is used as a measure of DNA purity. DNA absorbs UV light at 260 and 280 nm, and aromatic proteins absorbs UV light at 280 nm; a pure sample of DNA has the 260/280 ratio at 1.8 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8.260 nm and 280nm UV
Examine the quality of the DNA extracted Run the DNA on an 1% agarose gel to check its integrity and quality Genomic DNA has a very high bp, so one would expect a band at the top of the gel (near the well) Poor quality DNA will not perform well in PCR or restrictive digests.
TROUBLESHOOTING GUIDE: Low yield -incomplete homogenization or lysis of samples. -Final DNA pellet incompletely redissolved. A260/280 ratio <1.70 -DNA sample was diluted in water instead of TE prior to spectrophotometric analysis. -Phenol was not sufficiently removed from the DNA preparation. Wash the DNA pellet an additional time with 0.1 M sodium citrate in 10% ethanol.
DNA degradation -Tissues were not immediately processed or frozen after removal from the animal. -Samples used for isolation, or the isolated RNA preparations were stored at -5 to-20°C, instead of -60 to -70°C. -Samples were homogenized with a Polytron or other high speed homogenizer. RNA contamination -Incomplete removal of aqueous phase. -DNA pellet insufficiently washed with 0.1 M sodium citrate in 10% ethanol.