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
