Isolation of DNA Biotechnology

Introductory Question Do all organisms have the same amount of DNA? Explain.

Essential Question How is DNA extracted from eukaryotic cells?

Purpose of Isolation Procedure Purify DNA – have no other macromolecules –lipids, carbohydrates, proteins, RNA present

Studying DNA Must first break open cells of the biological organism to release chromosomes (where DNA housed) DNA is isolated (precipitated) from the solution and dissolved

Fruit DNA Isolating DNA from different fruit Why fruit? Fruit are often polyploid meaning that they contain more than two copies of each chromosome A chromosome is where the DNA is housed By being polyploid, they have lots more DNA than an organism that has only two copies of each chromosome For example, strawberries are actually octoploid meaning they have 8 copies of each chromosome rather than diploid (two copies of each chromosome) like most organisms

Dissolving Cell Membrane Addition of detergent Dissolves cell membranes composed of phospholipids Denatures (unfolds) many proteins

NaCl-sodium chloride Addition of NaCl is to raise the salt concentration Under high salt, helps the cellular debris and proteins to precipitate (become a solid and fall out of solution)

Cell Bursts Dissolving cell membrane bursts open cells Releases cellular contents into test tube Precipitated proteins drop to the bottom of the vessel Addition of heat speeds up denaturation process

Protease Protease found in meat tenderizer –degrade protein contaminants in sample Protease act as scissors cutting up proteins into smaller pieces

Centrifugation Separate the precipitated proteins and cellular debris from the DNA that is still in solution During centrifugation, the solid cellular debris and protein are pulled to the bottom of the tube After centrifugation, the precipitated proteins and cellular debris will be the pellet (solid) at the bottom DNA will be in the supernatant- liquid solution on top

Centrifugation

Ethanol The DNA in the supernatant is not visible Must add ethanol to precipitate DNA (cause the DNA to come out of solution) The DNA will look like stringy white fibers Can spool on a glass rod

Dissolve Precipitated DNA DNA dissolved in 10 mM Tris, 1 mM EDTA Buffer pH 8 Also known as TE buffer pH 8 Purpose of EDTA Forms complexes with several metal ions (ex. Mg 2+) Metal ions required for majority of DNases Contaminating DNases will cut up isolated DNA Without Metal ions, DNases will not work-cannot cut up DNA Protects DNA from DNase degradation

DNA Isolation Lab Each member of lab group will choose a different fruit Perform the DNA isolation protocol Spool DNA and weigh Dissolve in TE buffer Exchange data between members of the group