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