UNIT 3: Molecular Genetics
Chapter 8: Genetic Technologies
8.1: Manipulating and Cloning DNA
pg. 364 - 421
pg. 366 - 375
Diabetes mellitus – is a disease in which the blood glucose level is too high
because of the insufficient production or activity of the hormone insulin.
Type I diabetes – is a type of diabetes caused by an inability to produce
insulin.
Type II diabetes – is a type of diabetes caused by low insulin or an inability
to use insulin.
Genetic Engineering - is the intentional production of new genes and
alteration of genomes by the substitution or introduction of new genetic
material.
Recombinant DNA – is a DNA strand that is created using DNA pieces
from two or more sources.
Once it was discovered that insulin was a hormone that controlled
diabetes, Banting and Best (1922), insulin was harvested from the
pancreases of pigs and cows to control diabetes.
Continue scientific researched determined that the gene for human
insulin production could be inserted in bacteria DNA, and bacteria
would produce human insulin. Introducing the gene into bacteria
DNA was Genetic Engineering, which decreased the need to
extract insulin from animals.
Bacteria are ideal, because they reproduce quickly and often,
inexpensive to maintain, and contained plasmids (small ringed
DNA fragments). The human gene that codes for human insulin is
inserted into E. coli bacteria as a plasmid. Once inside the bacteria
cell the gene is transcribed and translated to synthesis human
insulin.
When an organism’s DNA combines with DNA from another
organism the newly formed DNA is called a recombinant DNA.
Restriction Enzymes
Restriction enzymes were first discovered by DR. Hamilton Smith
in 1970, as he studied bacteria and their ability to resisted viral
infections. Since the start of his research there have been 2500
restriction enzymes classified with a 200 target sequences.
The primary functions of restriction enzymes are to protect cells
from infections or viruses. The restriction enzymes will cut up the
foreign DNA or RNA that may enter their cell.
They are very important to microbiologists, because the enzymes
are highly specific to their recognition sites.
Restriction enzymes are name after their cell of origin, and a
Roman numeral, if there is more then one enzyme from the same
cell. Foe example EcoRI (eco-R-one) was the first restriction
enzyme isolated from E. coli.
Restriction Enzyme – is an enzyme that cuts DNA at a specific location in a
base sequence, also called restriction endonuclease.
Recognition Site – is a sequence of bases on a DNA strand that restriction
enzymes bind to.
Restriction Fragment – is a fragment that is produced when a DNA strand is
cut by a restriction enzyme.
Blunt End – is the end that remains after restriction enzymes cut straight
across a DNA strand; a blunt end is more difficult that a sticky end to
recombine with another strand.
Sticky End – is the end that remains after restriction enzymes cut on a
zigzag across a DNA strand; a sticky end of a DNA fragment can form
hydrogen bonds with a complementary sticky end on any other DNA
molecule that has been cut by the same enzyme.
The process of creating a recombinant DNA occurs through a
number of steps.
The first step is the isolation of a DNA fragment that contains the
desired gene. The chemical scissors (not actual scissors) used by
scientists to cut out the DNA fragment is called a restriction
enzymes (restriction endonuclease). These occur naturally within
bacteria cells. The restriction enzymes recognize specific
sequences (recognition site) of DNA and it is here that they will
snip DNA creating restriction fragments.
There are different types of restriction enzymes, each recognizing
their own recognition site, going in one direction. Each recognition
site is made up of a palindromic sequence (GAATTC) specific to
the restriction enzyme. The restriction enzyme cuts the DNA along
the backbone, at a phosphodiester bond between nucleotides.
These restriction enzymes can create either blunt or stick ends,
depending on the enzyme and the palindromic sequence. Straight
cuts produce blunt ends and are harder to work with. Zigzag cuts
produce sticky ends, and make it easier to insert genetic
information.
Figure 1: How the restriction enzyme EcoRI cuts DNA sequence.
DNA Ligase
DNA ligase, as indicated earlier is responsible for joining cut DNA strands
together. Sticky ends are the easiest to work with when rejoining strands. T4
DNA ligase works well with blunt ends.
For stick ends to work the overhanging portions must be complementary to
each other. Hydrogen bonds will form between the complementary base
pairs. But the most important bond is the phosphodiester bond between the
phosphate groups of adjoining nucleotides.
Plasmids
Competent Cell – is a cell that is able to take up foreign DNA from its
surroundings.
Vector – is a DNA molecule that is used as a vehicle to transfer foreign
genetic material into a cell, for example, a plasmid.
Copy Number – is the number of plasmids of a specific type within a cell.
Host Cell – is a cell that has taken up a foreign plasmid or virus and has used
its cellular machinery to express the foreign DNA.
Cloned Gene – is an identical copy of an original target gene that can be
made by introducing the target gene into a host cell and having it copied.
Plasmids are required for the production of recombinant DNA. Plasmids are
small circular pieces if DNA that are found in bacteria. They are unique,
because they are able to replicate independently of the bacteria’s
chromosomal DNA. The plasmids can contain genes that codes for various
proteins, such as: proteins that provide antibiotic resistance or protect the
bacteria from heavy metal toxins.
Competent cell is a cell that is able to pick up foreign DNA, usually in the
form of a plasmid. The plasmid is used as a vector to deliver to transport
foreign DNA into the cell. The plasmid will have a set number of copies it is
able to make of itself, known as the copy number. The higher the copy
number the greater number of copies of a protein can be synthesized.
For the fragment (gene) to be inserted into the plasmid, it must be cut by the
same restriction enzyme to that of the plasmid, so their blunt or stick ends
will match that of the plasmid. The sticky ends allow for the complementary
nitrogen base pairs to anneal and DNA ligase is used to form the
phosphodiester bonds between the adjacent nucleotides. The recombinant
DNA has now been created, which contains the new gene. The plasmid is
then introduced to a host cell where it will be able to replicate many times,
cloning the gene as well.
Figure 3: An overview of cloning DNA fragments in a bacterial plasmid.
Restriction Maps
Restriction Map – is a diagram that shows restriction enzymes recognition
sites and the distances, measured in base pairs (bps), between the sites.
The ability to complete a plasmid map allows a molecular biologist to locate
various palindromic sequences and select the appropriate restriction
enzymes to use to cut and insert DNA fragments to create a recombinant
DNA. This technique allows scientist to select the best restriction enzyme to
use for their gene cloning experiments.
When determining the various recognition sites on a plasmid two restriction
enzymes are used and three separate mapping are done. One map for each
enzyme separately and the third one is done as a combination. The fragments
created in all three situations can be used to create the map.
Figure 4: A simple example of a restriction map.
*** Tutorials 1: problems 1 – 2, pg. 370 – 373.
Transformations
When plasmids enter bacteria cells and are able to multiply and express the
foreign gene, the event is called a transformation. The cell that received the
DNA is said to be transformed.
If the bacteria cell does not accept the plasmid, it can be made competent in
the laboratory. When bacteria cells are combined with plasmids, in a calcium
chloride solution and then is placed in an ice-water bath, the bacteria will
cool as calcium stabilizes the phospholipid layer. The solution is quickly
heated and then cooled again. With this sudden change, the membrane is
disrupted allowing the plasmid to enter the bacteria cell.