DNA Extraction?

Question

What is the purpose for each of the following reagents:
a) dishwashing detergent (detergents act as surfactants)
b) meat tenderizer (it contains a protease that affects chromosome structure)
c) freezing and crushing the strawberries (these steps are required to break a structure found only in plant cells; therefore, not required to extract DNA from animal cells)

Explain the function of DNAses in cells?

Answer 1

There are three like basic steps in a DNA extraction, the details of which may vary depending on the type of sample and any substances that may interfere with the extraction and subsequent analysis.

Chelate divalent cations such as Mg2+ and Ca2+ to stop dnase enzymes functioning and degrading the DNA
Break open cells by grinding or sonication, and remove membrane lipids by adding a detergent.
Remove cellular and histone proteins bound to the DNA, by adding a protease, by precipitation with sodium or ammonium acetate, or by using a phenol-chloroform extraction step.
Precipitate DNA in cold ethanol or isopropanol, DNA is insoluble in alcohol and clings together; this step also removes salt.
Wash the resulting DNA pellet with alcohol
Solubilize the DNA in a slightly alkaline buffer

Detecting DNA

A diphenylamine (DPA) indicator will confirm the presence of DNA. This procedure involves chemical hydrolysis of DNA: when heated (e.g. ≥95oC) in acid, the reaction requires a deoxyribose sugar and therefore is specific for DNA. Under these conditions, the 2-deoxyribose is converted to w-hydroxylevulinyl aldehyde, which reacts with the compound, diphenylamine, to produce a blue-colored compound. DNA concentration can be determined measuring the intensity of absorbance of the solution at the 600 nm with a spectrophotometer and comparing to a standard curve of known DNA concentrations.

Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280nm is used as a measure of DNA purity. DNA absorbs UV light at 260 nm, and protein absorbs UV light at 280 nm; a pure sample of DNA has the 260/280 ratio at 1.8 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8.

DNA can be quantified by cutting the DNA with a restriction enzyme, running it on an agarose gel, staining with ethidium bromide or a different stain and comparing the intensity of the DNA with a DNA marker of known concentration.

Using the Southern blot technique this quantified DNA can be isolated and examined further using PCR and RFLP analysis. These procedures allow differentiation of the repeated sequences within the genome. It is these techniques which forensic scientists use for comparison and identification.

Uses of DNA
Extracted genomic DNA contains nuclear and mitochondrial DNA. If DNA is extracted from plant material it will also contain chloroplast DNA. Purified DNA may also be used for studying DNA structure and chemistry, examining DNA-protein interactions, carrying out DNA hybridizations, and for cloning and sequencing.

Answer 2

a) Since the membranes (plasma membrane and nuclear membrane, as well as any organelles) of the cell are made of lipids, you need a detergent to break it down. The detergent solubilizes the membrane, the same way that detergent takes grease off of a pan. There are other ways to break down the membranes, but this one is the least traumatic to the DNA inside the cell.

b) DNA is normally very tightly wound around histone proteins. This is what forms the very tiny X-shaped chromasomes during mitosis. Outside of mitosis, chromosomes don't usually take this form, but it is still wound up and compact. It's what allows the cell to compress ~4 meters of DNA (in a human cell) into a microscopic structure. The protease in the meat tenderizer breaks down the histone proteins and allows the DNA to freely expand.

c) Plant cells have a cell wall. It's a very tough outer layer that allows a plant to stay fairly rigid even when it becomes dehydrated (plants wilt when they don't have enough water, but they still retain some of their structure. Freezing causes ice crystals to form inside the cell, shredding the cell wall, the membranes, and possibly even the DNA. For a simple demonstration of DNA extraction, this isn't a problem, since the DNA from many, many cells will be combined into one large mass. If you were extracting DNA for analytical purposes, you would want to use a more gentle method to preserve the DNA as much as possible.

DNAses have two main purposes:
1) Apoptosis - when a cell is given a signal to commit suicide, it uses it's remaining energy to destroy it's internal processes and uses DNAse to cut up it's own DNA. The result is compact, inert remains, ready to be broken down by macrophages.

2) Virus defense - Cells contain DNAses to break down DNA outside of the nucleus, or anywhere it is not supposed to be. This provides some defense against DNA-based viruses that attempt to infect the cell.