Exercise 7
deoxyribonucleotide monomers
(1) a negatively charged phosphate group (2) a carbohydrate called deoxyribose (3) one of the four nitrogenous bases: adenine, guanine, cytosine, or thymine
Spectrophotometer
-used to determine the concentration and relative purity of DNA -consists of two instruments: a spectrometer that produces light of a selected wavelength (the wavelength is specified by the researcher), and a photometer that measures the intensity of light
4 steps of DNA extraction/isolation
1. disruption of tissues: 2. Lysis of cells 3. Removal of contaminants 4. Recovery of DNA
nitrogenous bases
Adenine, Guanine, Cytosine, Thymine
Solubility of DNA
DNA is highly soluble in water—water forms hydrogen bonds with the negatively charged phosphate groups of the deoxyribonucleotides such that a molecule of DNA is actually coated in water
negative charge of DNA molecules
DNA, like RNA, has a net negative charge because of the negatively charged phosphate groups.
Non-target molecules
RNA, protein, lipids, and carbohydrates
strand polarity/5' and 3'
The 5' end of the strand has a free 5' phosphate (PO4-) group, and the 3' end of the strand has a free 3' hydroxyl (OH) group. When the two strands come together to form a double-stranded molecule, they do so in an antiparallel fashion, meaning that the 5' end of one strand is physically adjacent to the 3' end of the other strand
Absorbance
amount of light absorbed by a sample
Disruption of tissues
breaking up cellular clumps, such that the researcher is working with individual cells
complementary base pairing
cytosine base pairs with guanine adenine base pairs with thymine.
Purpose of salt
deionizes into positively charged cations and negatively charged anions
phosphodiester bonds:
deoxyribonucleotides are linked together through covalent bonds called phosphodiester bonds
DNA
double-stranded molecule, where each strand is a polymer of deoxyribonucleotides monomers
extracellular matrix
extracellular matrix is excreted by cells to provide structural support, allowing for adherence to adjacent cells, biochemical support, and cell-to-cell communication
DNA purity
how "protein-free" is your sample
Purpose of alcohol
makes DNA fall out of solution and the DNA literally forms a solid salt that is visible and precipitates out of solution
Adjusted DNA concentration (from DNA concentration)
multiply the DNA concentration value by 20 to account for dilution factor
Removal of contaminants
organic solvents and/or enzymes that degrade proteins, carbohydrates, or lipids are utilized.
How to destruct/remove non-target molecules
place it in a rack in the 60oC water bath for 10 minutes. During this time, the heat is disrupting the cellular membranes and denaturing molecules like enzymes that may degrade the DNA. The detergent is solubilizing (breaking-up) the membranes.
Lysis
purpose of the lysis step is to physically break open the cells, i.e. disrupt the membranes, and release the DNA into solution
Recovery of DNA
purpose of the recovery step is to pull the DNA out of solution (leaving behind other contaminants that you don't want) and concentrating the DNA. Once more, there are numerous ways to do this, but one of the more common methods is to use a salt/alcohol precipitation
determining the purity of DNA
the closer the 260:280 ratio is to 2 (two parts DNA to one part protein), the more pure (protein-free) the sample is
Lysis of cells
to physically break open the cells and release the DNA into the cells, the combination of heat (physical method) and a detergent (chemical method) are commonly used together