biotechnology

Restriction Endonucleases

-commonly used tool in molecular biology, aka restriction enzymes

-molecular scissors that can cut double-stranded DNA at a specific base-pair sequence

-each restriction enzyme recognizes characteristic sequence of nucleotides that is known as its recognition site

-recognition sites are usually 4-8 pairs long and are usually a pair of complementary palindromic sequence

-disrupts the phosphodiester bonds with hydrolysis reaction

-there are sticky ends which has unpaired nucleotides, and blunt ends which means all nucleotides are paired

-many restriction enzymes comes from bacteria, and its purpose was to disrupt virus DNA sequence, these enzymes are named after the bacteria of origin

Gel electrophoresis

-a process of isolating desired excised gene fragment from the rest

-DNA is negatively charged and each nucleotide which contain a phosphate group carries a charge of -1

-the charge to mass ratio between nucleotides is relatively consistent

-the DNA fragment travel at different speed through the gel, the shorter it is the faster it moves

-the DNA fragments are dyed and become visible so that the electrical currents can be turned off before they reach the positive terminal

-the DNA fragments are later stained and each set of fragment has a particular banding pattern

Plasmid

-plasmids are used to express a particularly desirable gene such as insulin

-plasmids are small, circular, double-stranded DNA molecules lacking a protein coat

-plasmid’s size ranges from 1000 to 200000 base pairs.

-bacteria benefit from plasmid because plasmid usually contain gene that codes for useful functions such as antibiotic, resistance to heavy metals, and industrious chemicals

-the copy numbers of the plasmid, which is the number of a particular plasmid within a bacteria, the higher the copy number the stronger the expression of that particular gene

Transformation

-the introduction of DNA from another source

-plasmids are used to carry desired genes into a host cell

-if a bacterium readily takes up foreign DNA it is described as a competent cell

-calcium chloride is used to chemically alter the cell membrane, freezing the bacteria physically alters the cell membrane making is easier for bacteria to pick up foreign DNA

-selective plating is used to isolate bacteria with the recombinant DNA, if the bacteria grows in a environment with the specific antibiotics it contains the recombinant DNA