DNA Extraction
Outline
Purpose of DNA extraction
Review the main steps in the DNA extraction protocol and the chemistry involved in each step
Purpose of DNA Extraction
    To obtain DNA in a relatively purified form which can be used for further investigations, i.e. PCR, sequencing, etc
Basic Protocol
Most DNA extraction protocols consist of two parts
A technique to lyse the cells gently and solubilize the DNA
Enzymatic or chemical methods to remove contaminating proteins, RNA, or macromolecules
In plants, the 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 from the rest of the cell.
Lysis
Precipitation
Wash
Resuspension
A comparison of DNA extraction methods used in research labs as opposed to classroom labs
Research
Lysis: grind in Liquid N2 and use detergent
Precipitation Part I: phenol/chloroform extraction to get rid of proteins
Precipitation Part II: addition of salts to interrupt hydrogen bonding between water and phosphates on the DNA
Precipitation Part III: addition of ethanol to pull DNA out of solution
Wash and resuspend: DNA is washed in ethanol, dried, and resuspended in H20 or TE buffer.
Classroom
Lysis: grind in mortar/pestel and use detergent
Precipitation Part I: NONE  (chemical are too dangerous!)
Precipitation Part II: addition of salts to interrupt hydrogen bonding between water and phosphates on the DNA
Precipitation Part III: addition of ethanol to pull DNA out of solution
Wash and resuspend: DNA is washed in ethanol, dried, and resuspended in H20 or TE buffer.
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
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 (In a research lab): This a series of steps where DNA is separated from the rest of the cellular components
In a research lab, 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
  THIS STEP CANNOT BE PERFORMED IN CLASSROOM LABS!!
The second part of research lab DNA precipitation is the addition of salts
The salts interrupt the hydrogen bonds between the water and DNA molecules.
The DNA is then precipitated from the protein in a subsequent step with isopropanol or ethanol
In the presence of cations, 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
PRECIPITATION (In a classroom lab): This a series of steps where DNA is separated from the rest of the cellular components
In a classroom lab, DNA precipitation involves the addition of salts
The salts interrupt the hydrogen bonds between the water and DNA molecules.
The DNA is then precipitated from the protein in a subsequent step with isopropanol or ethanol
In the presence of cations, 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
    Note: because this protocol does not use phenol/chloroform, the DNA extracted in a classroom lab is not as “clean” as the DNA extracted in a research lab!
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
Checking the Quality of your DNA
The product of your DNA extraction will be used in subsequent experiments
Poor quality DNA will not perform well in PCR
You will want to assess the quality of your DNA extraction using the following simple protocol:
Mix 10 µL of DNA with 10 µL of loading buffer
Load this mixture into a 1% agarose gel
Analyze results (the following slides provide guidance)
Expected Results in a Classroom Lab
Using the protocol in the Cereal Genomics module, the genomic DNA extracted will look different than the optimized DNA extraction on the previous slide (this is mainly due to the missing phenol/chloroform step)
This is expected. Even though this genomic DNA preparation is not perfect, it is suitable for use as a PCR template
Lane A: BarleyLane B: Corn
Lane C: Oat
Lane D: Rice
Lane E: Wheat