WEEK 3: LAB
DNA cloning is the process of making multiple, identical copies of a certain piece of DNA. A
gene or other DNA fragment of interest is first inserted into a circular piece of DNA called a
plasmid. The insertion is done using enzymes that are able to “cut and paste” DNA, and then a
molecule of recombinant DNA is produced. Next, the recombinant plasmid is introduced into
bacteria. Bacteria carrying the plasmid are selected and grown up. As they reproduce, they
replicate the plasmid and pass it on to their offspring, making copies of the DNA it contains.
In some cases, we need lots of DNA copies to conduct experiments or build new plasmids. In
other cases, the piece of DNA encodes a useful protein, and the bacteria are used as “factories”
to make the protein. For instance, the human insulin gene is expressed in E. coli bacteria to make
insulin used by diabetics.
The basic steps of DNA cloning:
1. Cut open the plasmid and "paste" in the gene. This process relies on restriction enzymes
(which cut DNA) and DNA ligase (which joins DNA).
2. Transform the plasmid into bacteria. Use antibiotic selection to identify the bacteria that
took up the plasmid.
3. Grow up lots of plasmid-carrying bacteria and use them as "factories" to make the
protein. Harvest the protein from the bacteria and purify it.
There are two types of enzymes that aid DNA from different sources to join, Restriction enzymes
and DNA ligase. A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target
sequence and cuts DNA into two pieces at or near that site. Many restriction enzymes produce
cut ends with short, single-stranded overhangs. If two molecules have matching overhangs, they
can base-pair and stick together. However, they won't combine to form an unbroken DNA
molecule until they are joined by DNA ligase, which seals gaps in the DNA backbone. The goal
in cloning is to insert a target gene into a plasmid. Using a carefully chosen restriction enzyme,
we digest:
The plasmid, which has a single cut site
The target gene fragment, which has a cut site near each end
Then, we combine the fragments with DNA ligase, which links them to make a recombinant
plasmid containing the gene.
Plasmids and other DNA can be introduced into bacteria, such as the harmless E. coli used in
labs, in a process called transformation. During transformation, specially prepared bacterial cells
are given a shock (such as high temperature) that encourages them to take up foreign DNA. A
plasmid typically contains an antibiotic resistance gene, which allows bacteria to survive in the
presence of a specific antibiotic. Thus, bacteria that took up the plasmid can be selected on
nutrient plates containing the antibiotic. Bacteria without a plasmid will die, while bacteria
carrying a plasmid can live and reproduce. Each surviving bacterium will give rise to a colony,
of identical bacteria that all carry the same plasmid. Not all colonies will necessarily contain the
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