Pusat Kedokteran Tropis Fakultas Kedokteran - Universitas Mulawarman DNA ISOLATION Sunarto Ang Pusat Kedokteran Tropis RSUD. A. Wahab Sjahranie Fakultas Kedokteran - Universitas Mulawarman Samarinda

DNA Isolation DNA isolation: The aim: Basic Protocol: is an extraction process of DNA from various sources. The aim: is to separate DNA present in the nucleus of the cell from other cellular components. Basic Protocol: break the cells open to expose the DNA within. remove the nuclear membrane lipids by adding a detergent. precipitate the DNA with an alcohol. This step will also remove alcohol-soluble salts.

Application of DNA isolation Scientific: use DNA in number of Applications, such as introduction of DNA into cells & animals or plants for diagnostic purposes (gene cloning) Medicine: is the most common. To identify point sources for hospital and community-based outbreaks and to predict virulence of microorganisms Forensic science: needs to recover DNA for identification of individuals, (for example rapists, petty thieves, accident, or war victims), and paternity determination.

Overview of Procedure Lyse RBCs & WBCs Lyse WBCs nuclei & Denature/digest proteins Separate contaminants (e.g., proteins, heme) Precipitate DNA Resuspend DNA in final buffer

Samples for DNA Isolation Blood Semen Saliva Urine Hair (w/Root & Shaft) Teeth Bone Tissue Cigarette Butts Envelope & Stamps Fingernail Clippings Chewing Gum Bite Marks Feces

Blood Collection Blood collected in disodium EDTA tube Samples can be stored at -20oC or -70oC Fresh samples are kept in freezer for a few hours to facilitate RBCs hemolysis Allow samples to thaw before starting the extraction

Methods of DNA Isolation Organic extraction Salting out Selective DNA binding to a solid support 1. Organic extraction DNA is polar insoluble in organic solvents. Traditionally, phenol:chloroform is used to extract DNA. When phenol is mixed with the cell lysate, two phases form. DNA partitions to the (upper) aqueous phase Denatured proteins partition to the (lower) organic phase.

Genomic DNA isolation: phenol extraction 1:1 phenol : chloroform or 25:24:1 phenol : chloroform : isoamyl alcohol Phenol: denatures proteins, precipitates form at interface between aqueous and organic layer Chloroform: increases density of organic layer Isoamyl alcohol: prevents foaming Genomic DNA is isolated as pieces up to 1 Mbp!

Salting out Digested proteins are removed by salting out with high concentrations of LiCl. However, if salts are not adequately removed, problems could occur with the RFLP procedure due to alteration of DNA mobility (band shifting)

Procedure of Salting out Cell Lysis Buffer - lyse cell membrane, nuclei are intact, pellet nuclei. Resuspend nuclei in Protein Lysis Buffer containing a high concentration of Proteinase K. Lyse nuclear membrane and digest protein at 65°C 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.

Procedure of Salting out 4. 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.

Binding to a support material Support Materials Silica Anion-exchange resin Advantages Speed and convenience No organic solvents Disadvantage DNA fragmentation

Plants Nucleus is protected within a nuclear membrane which is surrounded by a cell membrane and a cell wall. Four steps are used to remove and purify the DNA Lysis Precipitation Wash Resuspension

LYSIS In DNA extraction from plants, this step commonly refers to the breaking of the cell wall and cellular membranes (most importantly, the plasma and nuclear membranes) The cell wall (made of cellulose) is disrupted by mechanical force (for example, grinding the leaves) Then the addition of a detergent in the which breaks down the cell membranes

LYSIS Detergents are able to disrupt membranes due to the amphipathic (having both hydrophilic and hydrophobic regions) nature of both cellular membranes and detergent molecules. The detergent molecules are able to pull apart the membranes The end result of LYSIS is that the contents of the plant cells are distributed in solution.

PRECIPITATION A series of steps where DNA is separated from the rest of the cellular components The first part of precipitation uses phenol/chloroform to remove the proteins from the DNA Phenol denatures proteins and dissolves denatured proteins. Chloroform is also a protein denaturant

PRECIPITATION The second is the addition of salts The salts interrupt the hydrogen bonds between the water and DNA molecules. The DNA is then precipitated by isopropanol or ethanol ethanol induces a structural change in DNA molecules that causes them to aggregate and precipitate out of solution. The DNA is pelleted by spinning with a centrifuge and the supernatant removed

The precipitated DNA is laden with acetate salts. Washing: The precipitated DNA is laden with acetate salts. It is “washed” with a 70% ethanol solution to remove salts and other water soluble impurities but not resuspend the DNA. Resuspension: The clean DNA is now resuspended in a buffer to ensure stability and long term storage. The most commonly used buffer for resuspension is called 1xTE

Overview of DNA Extraction Break down the cell wall and membranes Centrifuge to separate the solids from the dissolved DNA Precipitate the DNA using isopropanol Centrifuge to separate the DNA from the dissolved salts and sugars Flowchart of the various steps Dissolve DNA Wash the DNA pellet with Ethanol and dry the pellet

Checking the Quality of your DNA Poor quality DNA will not perform well in PCR Assessment of the quality of DNA extraction: Mix 10 µL of DNA with 10 µL of loading buffer Load this mixture into a 1% agarose gel

Quality from UV Spectrophotometry DNA absorb maximally at 260 nm. Proteins absorb at 280 nm. Background scatter absorbs at 320 nm. A260/A280 = measure of purity (A260 – A320)/(A280 – A320) 1.7 – 2.0 = good DNA or RNA <1.7 = too much protein or other contaminant

Storage Conditions Store DNA in TE buffer at 4 °C for weeks or at –20 °C to –80 °C for long term. 2–25 °C 2–8 °C –20 °C –70 °C Recommended for long-term storage in ethanol <4 Months 1–3 Years <7 Years >7 Years

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