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
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)
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
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:
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
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: Barley Lane B: Corn
Lane C: Oat
Lane D: Rice
Lane E: Wheat
A B C D E
Ladder
Note that the DNA has sheared (particularly for wheat) – broken up into numerous fragments and is not a clean single band at the top – these are the mid-ranged sized fragments (1000-10,000bp size range)
The bright bands at the 100 - 1000 bp range are RNA, which also gets extracted using this protocol