Clo
Clo
Clo
DNA cloning is the starting point for many genetic engineering approaches to biotechnology research.
Large amounts of DNA are needed for genetic engineering. Multiple copies of a piece of DNA can be
made either by using polymerase chain reaction (PCR) or by cloning DNA in cells.
Get information sheet: Polymerase chain reaction (PCR)
DNA cloning
To get multiple copies of a gene or other piece of DNA you must isolate, or cut, the DNA from its source
and then paste it into a DNA vector that can replicate (or copy) itself.
The four main steps in DNA cloning are:
Step 1. The chosen piece of DNA is cut from the source organism using restriction enzymes.
Get information sheet: Restriction enzymes
Step 2. The piece of DNA is pasted into a vector and the ends of the DNA are joined with the vector
DNA by ligation.
Get information sheet: DNA ligation
Step 3. The vector is introduced into a host cell, often a bacterium or yeast, by a process
called transformation. The host cells copy the vector DNA along with their own DNA, creating multiple
copies of the inserted DNA.
Get information sheet: Bacterial transformation
Step 4. The vector DNA is isolated (or separated) from the host cells DNA and purified.
DNA that has been cut and pasted from an organism into a vector is called recombinant DNA. Because
of this, DNA cloning is also called recombinant DNA technology.
Therapeutic cloning is the process of making multiple copies of a cell to treat a disease.
Metadata
Key points:
Introduction
When you hear the word cloning, you may think of the cloning
of whole organisms, such as Dolly the sheep. However, all it
means to clone something is to make a genetically exact copy of
it. In a molecular biology lab, whats most often cloned is a gene
or other small piece of DNA.
If your friend the molecular biologist say that her cloning isnt
working, she's almost certainly talking about copying bits of DNA,
not making the next Dolly!
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.
3.
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.
3. Protein production
Once we have found a bacterial colony with the right plasmid, we
can grow a large culture of plasmid-bearing bacteria. Then, we
give the bacteria a chemical signal that instructs them to make
the target protein.
Works cited:
1.
2.
Additional references:
Affibody. (n.d.). Insulin capture from human serum. Retrieved
fromhttp://affibody.com/upload/Research%20Reagents/Application
%20notes/Insulin%20AFF%20application%20note%202007-03-30.pdf .
fromhttp://www.diabetesforecast.org/2013/jul/making-insulin.html?
referrer=https://www.google.com/.
Key points:
Restriction enzymes
Restriction enzymes are found in bacteria (and other
prokaryotes). They recognize and bind to specific
sequences of DNA, called restriction sites. Each
restriction enzyme recognizes just one or a few restriction
sites. When it finds its target sequence, a restriction
enzyme will make a double-stranded cut in the DNA
molecule. Typically, the cut is at or near the restriction site
and occurs in a tidy, predictable pattern.
[Why do bacteria have restriction enzymes?]
5'-...GAATTC...-3' 3'-...CTTAAG...-5'
EcoRIsite
When EcoRI recognizes and cuts this site, it always does so
in a very specific pattern that produces ends with singlestranded DNA overhangs:
5'-...CCC|GGG...-3' 3'-...GGG|CCC...5'
Blunt-ended fragments can be joined to each other by DNA
ligase. However, blunt-ended fragments are harder to
ligate together (the ligation reaction is less efficient and
more likely to fail) because there are no single-stranded
overhangs to hold the DNA molecules in position.
[Where do restriction enzymes get these weird names?]
3,3, comma000000
5GAATTC33
5GAATTC33
CTTAAG3
CTTAAG3
5GGATCC33
5GGATCC33
CCTAGG3
CCTAGG3
5AAGCTT33
5AAGCTT33
TTCGAA5
TTCGAA5
5CCCGGG33
5CCCGGG33
GGGCCC5
GGGCCC5
5GAGCTC33
5GAGCTC33
CTCGAG5
CTCGAG5
DNA ligase
Fragment 1 of DNA:
5'-...G 3'-...CTTAA
Fragment 2 of DNA:
AATTC...-3' G...-5'
The single-stranded regions of the two molecules can stick
together by hydrogen bonding, but there are still gaps in
the backbone:
5'-...G|AATTC...-3' 3'-...CTTAA|G...-5'
DNA ligase seals the gaps to make an unbroken molecule of
DNA:
5'-...GAATTC...-3' 3'-...CTTAAG...-5'
5'-GAATTC-3' 3'-CTTAAG-5'
Our goal is to use the enzyme EcoRI to insert the gene into
the plasmid. First, we separately digest (cut) the gene
fragment and the plasmid with EcoRI. This step produces
fragments with sticky ends: