Structure of the cell • Plasma membrane and membranes of organelles, nuclear envelope included • DNA located in nucleus • A lot of proteins around • Mitochondrial DNA

Relevance of DNA isolation • Isolation of DNA is often the first step before further analysis • DNA profiling (forensics) • cloning • disease diagnosis • DNA sequencing • genetically modified organisms (GMO) - agriculture,pharmaceutical • Environmental testing, bioterrorism

Selecting a Genomic DNA Purification Method The goal of genomic DNA isolation depends on what the applications of the DNA after isolation. Purity, source, quantity and quality of DNA are all issues that need to be addressed prior to genomic DNA extraction. A whole host of different methods, technologies and kits are available now to researchers to isolate genomic DNA from cells.

Selecting a Genomic DNA Purification Method Quantity of DNA needed Molecular weight and size of DNA Purity of DNA required Downstream applications of DNA Time available Ease of DNA extraction technique or method Expense or money available

DNA Extraction DNA isolation is a routine procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic and one optional steps in a DNA extraction:

Steps to DNA Extraction Breaking the cells open, commonly referred to as cell disruption or cell lysis, to expose the DNA within. This is commonly achieved by grinding or sonicating the sample. Removing membrane lipids by adding a detergent. Removing proteins by adding a protease (optional but almost always done). 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.

Refinements of the technique Refinements of the technique include adding a chelating agent to chelates divalent cations such as Mg2+ and Ca2+, which prevents enzymes like DNAse from degrading the DNA. If desired, the DNA can be resolubilized in a slightly alkaline buffer or in ultra-pure water. EDTA which chelates Mg++ and is a co-factor of DNAse which chews up DNA rapidly, Incubate the solution at an elevated temperature (56oC to inhibit degradation by DNAses) for 4-24 hrs.

Cell lysis

• Lysis buffer • 50 mM Tris-HCI, pH 8.0 to maintain the pH of the solution at a level where DNA is stable • 1% SDS to break open the cell and nuclear membranes, allowing the DNA to be released into the solution (SDS also denatures and unfolds proteins, making them more susceptible to protease cleavage)

Detergent

Why add detergent? Lysis separat the cells. But each cell is surrounded by a sack (the cell membrane). DNA is found inside a second sack (the nucleus) within each cell. To see the DNA, we have to break open these two sacks.

Why add detergent? We do this with detergent. Think about

Why add detergent? why you use soap to wash dishes or your hands. To remove grease and dirt, right? Soap molecules and grease molecules are made of two parts: Heads, which like water Tails, which hate water.

Why add detergent? Both soap and grease molecules organize themselves in bubbles (spheres) with their heads outside to face the water and their tails inside to hide from the water.

Why add detergent? When soap comes close to grease, their similar structures cause them to combine, forming a greasy soapy ball.

Why add detergent? A cell's membranes have two layers of lipid (fat) molecules with proteins going through them.

Why add detergent? When detergent comes close to the cell, it captures the lipids and proteins.

Removal of Proteins

Why add protease? • Protease destroys nuclear proteins that bind DNA and cytoplasmic enzymes that breakdown and destroy DNA • Protease treatment increases the amount of intact DNA that is extracted

Cellular and histone proteins bound to the DNA can be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation.

Phenol-chloroform extraction Phenol-chloroform extraction is a liquid-liquid extraction technique in biochemistry. It is widely used in molecular biology for isolating DNA, RNA and protein. Equal volumes of a phenol:chloroform mixture and an aqueous sample are mixed, forming a biphasic mixture.

Phenol-chloroform extraction This method relies on phase separation by centrifugation of a mix of the aqueous sample and a solution containing water-saturated phenol, chloroform resulting in an upper aqueous phase and a lower organic phase (mainly chloroform). Nearly all of DNA will be located in the aqueous phase and thus the technique can be used for DNA purification alone.

Phenol-chloroform extraction 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 coloured lower phase (containing the proteins dissolved in phenol and the lipids dissolved in chloroform).

Other denaturing chemicals such as 2-mercaptoethanol and sarcosine 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.

Phenol: Phenol The phenol used for biochemistry comes as a water-saturated solution with Tris buffer, as a Tris-buffered 50% phenol, 50% chloroform solution, or as a Tris-buffered 50% phenol, 48% chloroform, 2% isoamyl alcohol solution (sometimes called "25:24:1"). Phenol is naturally somewhat water-soluble, and gives a fuzzy interface, which is sharpened by the presence of chloroform, and the isoamyl alcohol reduces foaming. Most solutions also have an antioxidant, as oxidized phenol damages the nucleic acids. For DNA purification, the pH is usually near 7, at which point all nucleic acids are found in the aqueous phase.

Chloroform: Chloroform is stabilized with small quantities of amylene or ethanol, because exposure of pure chloroform to oxygen and ultraviolet light produces phosgene gas. Some chloroform solutions come as pre-made a 96% chloroform, 4% isoamyl alcohol mixtures that can be mixed with an equal volume of phenol to obtain the 25:24:1 solution. Isoamyl alcohol: Isoamyl alcohol may reduce foaming and ensure deactivation of RNase.

Precipitation of DNA

Precipitation of DNA • DNA does not dissolve in alcohol. • Addition of cold alcohol makes the DNA clump together and precipitate out of solution • Precipitated DNA molecules appear as long pieces of fluffy, stringy, web-like strands. • Microscopic oxygen bubbles “aggregate” together, as the DNA precipitates • The larger, visible air bubbles “lift” the DNA out of solution, from the aqueous into the organic phase • The DNA in the glass vial can last for years

Adding salt Adding salt • The addition of NaCI allows the DNA molecules to come together instead of repelling each other, thus making it easier for DNA to precipitate out of solution when alcohol is added • Na+ ions bind to the phosphate groups of DNA molecules, neutralizing the electric charge of the DNA molecules • Many protease solution already contains salt

What is the stringy stuff? Alcohol is less dense than water, so it floats on top. Since two separate layers are formed, all of the grease and the protein that we broke up in the first two steps and the DNA have to decide which layer to go to.

What is the stringy stuff? In this case, the protein and grease parts find the bottom, watery layer the most comfortable place, while the DNA prefers the top, alcohol layer. DNA is a long, stringy molecule that likes to clump together.

What is the stringy stuff? DNA is a long, stringy molecule. The salt you added in step one helps it stick together. So what you see are clumps of tangled DNA molecules! DNA normally stays dissolved in water, but when salty DNA comes in contact with alcohol it becomes undissolved. This is called precipitation. The physical force of the DNA clumping together as it precipitates pulls more strands along with it as it rises into the alcohol. You can use a wooden stick or a straw to collect the DNA. If you want to save your DNA, you can transfer it to a small container filled with alcohol.

Wash DNA pellet to remove excess salt in 70% ethanol and air-dry. Resuspened in sterile distilled water or TE, pH7.4. Store at 4oC or frozen at -20oC long term.

Comparing the DNA Extracted from Different Cell Types Does chromosome number noticeably affect the mass of DNA you’ll see? Cells with more chromosomes contain relatively more DNA, but the difference will not likely be noticeable to the eye. The amount of DNA you will see depends more on the ratio of DNA to cell volume. For example, plant seeds yield a lot of DNA because they have very little water in the cell cytoplasm. That is, they have a small volume. So the DNA is relatively concentrated. You don’t have to use very many seeds to get a lot of DNA!

Quantifying the DNA 1 A260 O.D. unit for ssDNA = 33 or 50 µg/ml The "absorbance" (O.D.) of a chemical is the: product of its (concentration) x (optical path length) x (extinction coefficient, E). Nucleic acids have a peak absorbance in the ultraviolet range at about 260 nm       1 A260 O.D. unit for dsDNA = 50 µg/ml       1 A260 O.D. unit for ssDNA = 33 or 50 µg/ml       1 A260 O.D. unit for RNA = 40 µg/ml

Spectrophotometric analysis of DNA

DNA purity The purity of the DNA is reflected in the OD260:OD 280 ratio and must be between 1.6 and 2.00. Decreased 260:280 ratio means that contaminating protein is still present. Repurify sample.

Examining the Quality of the DNA Extracted The product of your DNA extraction will be used in subsequent experiments, poor quality DNA will not perform well in PCR or restrictive digests. You need to be able to assess the quality of your DNA extraction. Note that genomic DNA has a very high bp, so one would expect a band at the top of the gel (near the well) if it was extracted correctly. A B C D E Ladder Barley(A), Corn (B), Oat (C), and Rice (D) are all suitable Wheat (E) may need to be re-extracted

If the genomic DNA is extracted in a professional Molecular Biology laboratory, it will give clear distinct band in an electrophoresis gel Note how the bands of genomic DNA are distinct and found in the very high bp range Ladders Cereal Genomic DNA