Garcia, Jayson F.

Bio 101- C

November 29, 2011

Assignment 1. Compare and contrast DNA isolation procedures among plant, animal and microbial (e.g. bacteria, yeast) sources. Plant cells have an extra structure, the cellulosic cell wall, for extra protection. This cell wall provides an extra rigidity to the cell. Thus, DNA is harder to extract as compared to the animal cell that lacks a tough cell wall. The extraction of DNA in plants involves an extra step to remove the cell wall. It can be done mechanically through homogenization (e.g. by grinding, by centrifugation) or by adding enzymes like cellulase. Plants also have many metabolites and sugars which can contaminate the DNA during the isolation process. After disrupting the cell wall, the key steps will now be relatively the same as extracting DNA from animal cells. The cell membrane is disrupted followed by the breakage of the nuclear membrane. These procedures are done through lysis. The DNA components are then precipitated and purified. Microorganisms, on the other hand, follow different methods for DNA isolation. Bacteria also have a tough cell wall like plants that needs to be broken in order to be extracted. Enzymatic methods, as well as mechanical means, are also applied to disrupt the cell wall. An example of an enzyme that can be used is lysozyme which promotes hydrolysis of the peptidoglycans in the cell wall. Unlike other organisms, bacteria have two types of DNA that can be isolated. The first is the genomic DNA and the other one is the plasmid DNA. Also, the genetic material of prokaryotic cells is not enclosed in a nuclear membrane.

2. Common techniques used

Centrifugation- it is a method that separates components based on density, size, and shape.
a. Cesium chloride- widely used for isopycnic centrifugation. It separates components based on differences in

their molecular weights.

b. Density gradient- particles are placed in a medium with a density gradient. The particles will collect at the area

where the density of the medium equals the density of the migrating particles.

Electrophoresis- a technique for separation of particles based on their movement in an electrical field. Positively charged ions travel towards a negative electrode and negatively-charged ions migrate toward a positive electrode.
a. Agarose gel- for separation of DNA fragments b. Palyacrylamide gel (PAGE)- for separating proteins

Freezing in liquid nitrogen- plant samples are frozen in liquid nitrogen and then crushed to lyse the cell Alkaline denaturation- this is a technique commonly used to isolate bacterial plasmid DNA from the genomic DNA. The buffer contains sodium hydroxide and SDS to denature both genetic material and was then renatured by

neutralizing it in potassium acetate solution. Since plasmid is small, it re-anneals faster than genomic DNA separating the two components.

Chelex ion exchange resin- binds multivalent metal ions and is particularly useful in removing inhibitors from DNA. The resin bind to the contaminants and will be precipitated out when centrifuged leaving out behind the DNA in the supernatant.

Use of paramagnetic beads with DNA binding capacity- samples are lysed and then subjected to Proteinase K. Lysates are exposed to beads and they bind to the DNA.

Bead beater- used in lysing the cells to make the DNA accessible. Glass beads were added to the samples contained in an eppendorf tube and then the bead beater vibrates allowing the beads to break the cells.

Commonly used reagents

1. EDTA (Ethylene diamine tetraacetic acid)- prevents the destruction of DNA by DNase activity, chelates the metal ions

which are cofactors for them. is an anionic detergent which upsets cell membrane and weakens all hydrophobic interactions that hold macromolecules in their native form.
2. Tris (Tris-hydroxymethylaminomethane)- buffers the pH of the cells at 8.0. 3. NaOH/SDS- is an anionic detergent, which also denatures the membrane proteins and weakens the cell membrane. This

agents also denatures nuclear envelope releasing the nucleoplasm. SDS also aids in preventing the action of nucleases.

4. KOAc- allows circular DNA to be renatured. Adding sodium acetate to the SDS allows for the easier elimination of the SDS from the `plasmid DNA It precipitates membrane, C, H, O, lipids, proteins, and monomers.
5. PCI- to extract DNA samples. 6. Alcohol- DNA is insoluble in alcohol and clings together. It also removes salt. 7. NaCl- helps in maintaining the osmotic balance of the cells. Chloride component enter the cells by ionic transport along

with water by diffusion expanding the cells, preventing cell aggregation and thus, weakens membrane integrity.
8. Phenol- removes nonpolar proteins and lipid residue. 9. Chloroform- removes excess phenol from DNA. 10. Mercaptoethanol- cleaves the disulfide bridges of proteins and helps in denaturation of membrane proteins and cytosolic

proteins.
11. DNAzol Reagent- a commercially available reagent for lysing of the cell making the genetic material more available. 12. C-TAB is a detergent that helps lyse the cell membrane 13. Proteinase K- destroys protein.

References: Audain, K. Retrieved November 28, 2011, from http://www.ehow.com/about_6791106_difference-extraction-between-animalplant.html

eNotes.com, Inc. Retrieved November 28, 2011, from http://www.enotes.com/dnaisolation-methods-reference/dna-isolation-methods

N.A. Retrieved November 28, 2011 from http://www.invitrogen.com/site/us/en/home/References/protocols/nucleic-acidpurification-and-analysis/dna-extraction-protocols/extraction-of-dna-using-reagent.html

N.A. Retrieved November 28, 2011 from http://www.scribd.com/doc/31551020/DNA-Extraction-Reagents-Functions.

Quinn, C. Retrieved November 28, 2011, from http://www.ehow.com/about_5192729_dna-extraction-techniques.html Reamillo, M.C.S., et al.2009. Cell Biology Laboratory Manual. 6th ed. Los Banos: Genetics and Molecular Biology Division, IBS, CAS, UPLB

Rice, G. Retrived November 28, 2011 from http://serc.carleton.edu/microbelife/research_methods/genomics/dnaext.html

Suzanne. Retreived November 28, 2011 from http://bitesizebio.com/articles/plasmid-v-genomic-dna-extractionthe-difference/

Tissue, B.M. Retrieved November 28, 2011 from http://elchem.kaist.ac.kr/vt/chem-ed/sep/electrop/electrop.htm.

Wikipedia. Retrieved November 28, 2011 from http://en.wikipedia.org/wiki/Gel_electrophoresis.