WEEK 3: LAB Case of 4 pages for the course WEEK 3: LAB at Devry University (WEEK 3: LAB)

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

Powered by qwivy(www.qwivy.org)

 1 / 1

No comments found.
Login to post a comment
This item has not received any review yet.
Login to review this item
No Questions / Answers added yet.
Version latest
Category Exam (elaborations)
Included files pdf
Authors expert
Pages 4
Language English
Comments 0
Sales 0
Recently viewed items

We use cookies to understand how you use our website and to improve your experience. This includes personalizing content and advertising. To learn more, please click Here. By continuing to use our website, you accept our use of cookies, Privacy policy and terms & conditions.

Processing