DNA cloning

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)

How is DNA cloned in cells?

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.

What is cloned DNA used for?

DNA cloning is used to create a large number of copies of a gene or other piece of DNA. The cloned DNA
can be used to:

Work out the function of the gene

Investigate a genes characteristics (size, expression, tissue distribution)

Look at how mutations may affect a genes function

Make large concentrations of the protein coded for by the gene

What other types of cloning are there?

The term cloning is also used to describe other laboratory processes:

Reproductive cloning is the process of making a genetically identical copy of an organism.

Therapeutic cloning is the process of making multiple copies of a cell to treat a disease.

Metadata

Overview: DNA cloning

Definition, purpose, and basic steps of DNA cloning.
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Key points:

DNA cloning is a molecular biology technique that makes

many identical copies of a piece of DNA, such as a gene.

In a typical cloning experiment, a target gene is inserted into

a circular piece of DNA called a plasmid.

The plasmid is introduced into bacteria via process

called transformation, and bacteria carrying the plasmid are
selected using antibiotics.

Bacteria with the correct plasmid are used to make more

plasmid DNA or, in some cases, induced to express the gene and
make protein.

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!

Overview of DNA cloning

DNA cloning is the process of making multiple, identical copies

of a particular piece of DNA. In a typical DNA cloning procedure,
the gene or other DNA fragment of interest (perhaps a gene for a
medically important human protein) is first inserted into a circular
piece of DNA called a plasmid. The insertion is done using
enzymes that cut and paste DNA, and it produces a molecule
ofrecombinant DNA, or DNA assembled out of fragments from
multiple sources.

Diagram showing the construction of a recombinant DNA

molecule. A circular piece of plasmid DNA has overhangs on its
ends that match those of a gene fragment. The plasmid and gene
fragment are joined together to produce a gene-containing
plasmid. This gene-containing plasmid is an example of
recombinant DNA, or a DNA molecule assembled from DNA from
multiple sources.
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.
What is the point of making many copies of a DNA sequence in a
plasmid? 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.
[More about insulin and diabetes]

Steps of DNA cloning

DNA cloning is used for many purposes. As an example, let's see
how DNA cloning can be used to synthesize a protein (such as
human insulin) in bacteria. The basic steps are:
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.
Let's take a closer look at each step.

1. Cutting and pasting DNA

How can pieces of DNA from different sources be joined together?
A common method uses two types of enzymes: 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.

[See a diagram of restriction enzymes and DNA ligase]

Our goal in cloning is to insert a target gene (e.g., for human

insulin) 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.

Diagram depicting restriction digestion and ligation in a simplified

schematic.
We start with a circular bacterial plasmid and a target gene. On
the two ends of the target gene are restriction sites, or DNA
sequences recognized by a particular restriction enzyme. In the
plasmid, there is also a restriction site recognized by that same
enzyme, right after a promoter that will drive expression in
bacteria.
Both the plasmid and the target gene are (separately) digested
with the restriction enzyme. The fragments are purified and
combined. They have matching "sticky ends," or single-stranded
DNA overhangs, so they can stick together.
The enzyme DNA ligase joins the fragments with matching ends
together to form a single, unbroken molecule of DNA. This
produces a recombinant plasmid that contains the target gene.

2. Bacterial transformation and selection

Plasmids and other DNA can be introduced into bacteria, such as
the harmlessE. 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.

The DNA produced by ligation (which may be a mix of desired

plasmids, side-product plasmids, and linear DNA pieces) is added
to bacteria. The bacteria are given a heat shock, which makes
them more apt to take up DNA by transformation. However, only a
tiny minority of the bacteria will successfully take up a plasmid.
[Why does a heat shock make bacteria take up DNA?]

^1start superscript, 1, end superscript

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
small, dot-like group, or colony, of identical bacteria that all carry
the same plasmid.

Left panel: Diagram of plasmid, showing that it contains an

antibiotic resistance gene.
Right panel: all the bacteria from the transformation are placed on
an antibiotic plate. Bacteria without a plasmid will die due to the
antibiotic. Each bacterium with a plasmid makes a colony, or a
group of clonal bacteria that all contain the same plasmid. A
typical colony looks like a small, whitish dot the size of a pinhead.
Not all colonies will necessarily contain the right plasmid. Thats
because, during a ligation, DNA fragments dont always get
pasted in exactly the way we intend. Instead, we must collect
DNA from several colonies and see whether each one contain the
right plasmid. Methods like restriction enzyme
digestion and PCR are commonly used to check the plasmids.

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.

The bacteria serve as miniature factories," churning out large

amounts of protein. For instance, if our plasmid contained the
human insulin gene, the bacteria would start transcribing the
gene and translating the mRNA to produce many molecules of
human insulin protein.
[More about expressing human genes in bacteria]

A selected colony is grown up in a large culture (e.g., a 1-liter

flask). The bacteria in the large culture are induced to express the
gene contained in the plasmid, causing the gene to be transcribed

into mRNA, and the mRNA to be translated into protein. The

protein encoded by the gene accumulates inside of the bacteria.
Once the protein has been produced, the bacterial cells can be
split open to release it. There are many other proteins and
macromolecules floating around in bacteria besides the target
protein (e.g., insulin). Because of this, the target protein must
be purified, or separated from the other contents of the cells by
biochemical techniques. The purified protein can be used for
experiments or, in the case of insulin, administered to patients.
[Is it really that simple to make insulin?]

Uses of DNA cloning

DNA molecules built through cloning techniques are used for
many purposes in molecular biology. A short list of examples
includes:

Biopharmaceuticals. DNA cloning can be used to make

human proteins with biomedical applications, such as the insulin
mentioned above. Other examples of recombinant proteins
include human growth hormone, which is given to patients who
are unable to synthesize the hormone, and tissue plasminogen
activator (tPA), which is used to treat strokes and prevent blood

clots. Recombinant proteins like these are often made in bacteria.

Gene therapy. In some genetic disorders, patients lack the

functional form of a particular gene. Gene therapy attempts to
provide a normal copy of the gene to the cells of a patients body.
For example, DNA cloning was used to build plasmids containing a
normal version of the gene that's nonfunctional in cystic fibrosis.
When the plasmids were delivered to the lungs of cystic fibrosis
patients, lung function deteriorated less quickly^22start
superscript, 2, end superscript.

Gene analysis. In basic research labs, biologists often use

DNA cloning to build artificial, recombinant versions of genes that

help them understand how normal genes in an organism

function.
[See an example]

These are just a few examples of how DNA cloning is used in

biology today. DNA cloning is a very common technique that is
used in a huge variety of molecular biology applications.

Works cited:
1.

Superbest. (2014, June 11). How does heat shock

transformation work? InBiology stack exchange. Retrieved
fromhttp://biology.stackexchange.com/questions/19038/how-does-heat-shocktransformation-work.

2.

Alton, E. W. F. W., Armstrong, D. K., Ashby, D., Bayfield, K. J.,

Bilton, Diana, Bloomfield, E. V., ... Wolstenholme-Hogg, P. (2015).
Repeated nebulisation of non-viral CFTR gene therapy in patients
with cystic fibrosis: A randomised, double-blind, placebocontrolled, phase 2b trial. Lancet Respiratory Medicine, 3(9), 684691. http://dx.doi.org/10.1016/S2213-2600(15)00245-3.

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 .

Amgen Biotech Experience. (2015). Student guide. Retrieved

fromhttps://www.amgenbiotechexperience.com/sites/abe.edc.org/files/abe_engli
sh_student_all_sequences_05.15.15.pdf.

Biotechnology. (2016, March 23). Retrieved March 29, 2016 from

Wikipedia:https://en.wikipedia.org/wiki/Biotechnology.
Gebel, E. (2013). Making insulin: A behind-the-scenes look at
producing a lifesaving medication. In Diabetes forecast. Retrieved

fromhttp://www.diabetesforecast.org/2013/jul/making-insulin.html?
referrer=https://www.google.com/.

Gene therapy breakthrough for cystic fibrosis. (2015, July 3).

In NHS choices. Retrieved
from http://www.nhs.uk/news/2015/07July/Pages/Gene-therapy-breakthroughfor-cystic-fibrosis.aspx.

R&D Systems. (2016). Recombinant human growth hormone (GH)

protein. InGrowth hormone. Retrieved
fromhttps://www.rndsystems.com/products/recombinant-human-growthhormone-gh-protein_1067-gh.

Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P.

V., and Jackson, R. B. (2011). DNA tools and biotechnology.
In Campbell biology (10th ed., pp. 408-435). San Francisco, CA:
Pearson.
Wilkin, D. (2016, March 23). Gene cloning - advanced. In CK-12
biology advanced concepts. Retrieved
from http://www.ck12.org/book/CK-12-Biology-Advanced-Concepts/section/9.2/ .

Restriction enzymes & DNA ligase

Restriction digestion. Sticky ends and blunt ends. Ligation reactions.
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Key points:

Restriction enzymes are DNA-cutting enzymes. Each

enzyme recognizes one or a few target sequences and cuts
DNA at or near those sequences.

Many restriction enzymes make staggered cuts,

producing ends with single-stranded DNA overhangs.

However, some produce blunt ends.

DNA ligase is a DNA-joining enzyme. If two pieces of

DNA have matching ends, ligase can link them to form a
single, unbroken molecule of DNA.

In DNA cloning, restriction enzymes and DNA ligase

are used to insert genes and other pieces of DNA into
plasmids.

How do you cut and paste DNA?

In DNA cloning, researchers make many copies of a piece
of DNA, such as a gene. In many cases, cloning involves
inserting the gene into a piece of circular DNA called
a plasmid, which can be copied in bacteria.
How can pieces of DNA from different sources (such as a
human gene and a bacterial plasmid) be joined together to
make a single DNA molecule? One common method is
based on restriction enzymes and DNA ligase.

A restriction enzyme is a DNA-cutting enzyme that

recognizes specific sites in DNA. Many restriction enzymes

make staggered cuts at or near their recognition sites,
producing ends with a single-stranded overhang.

If two DNA molecules have matching ends, they can

be joined by the enzyme DNA ligase. DNA ligase seals the
gap between the molecules, forming a single piece of DNA.
Restriction enzymes and DNA ligase are often used to
insert genes and other pieces of DNA into plasmids during
DNA cloning.

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?]

As an example of how a restriction enzyme recognizes and

cuts at a DNA sequence, let's consider EcoRI, a common
restriction enzyme used in labs.EcoRI cuts at the following
site:

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:

AnEcoRI enzyme binds to anEcoRI site in a piece of DNA

and makes a cut on both strands of the DNA. The pattern of
the cut is:
5'-...G|AATTC...-3' 3'-...CTTAA|G...-5'
Thus, it produces an overhang of 5'-AATT-3' on each end of
the cut DNA.
If another piece of DNA has matching overhangs (for
instance, because it has also been cut by EcoRI), the
overhangs can stick together by complementary base
pairing. For this reason, enzymes that leave singlestranded overhangs are said to produce sticky ends.
Sticky ends are helpful in cloning because they hold two
pieces of DNA together so they can be linked by DNA
ligase.
Not all restriction enzymes produce sticky ends. Some are
blunt cutters, which cut straight down the middle of a
target sequence and leave no overhang. The restriction
enzyme SmaI is an example of a blunt cutter:

ASmaI enzyme binds to theSmaI restriction site, which is:

5'-...CCCGGG...-3' 3'-...GGGCCC...5'
It makes a cut right in the middle of this sequence on both
strands, producing blunt ends. The cut sites are:

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

If youve learned about DNA replication, you may already

have met DNA ligase. In DNA replication, ligases job is to
join together fragments of newly synthesized DNA to form
a seamless strand. The ligases used in DNA cloning do
basically the same thing. If two pieces of DNA have
matching ends, DNA ligase can join them together to make
an unbroken molecule.

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'

How does DNA ligase do this? Using ATP as an energy

source, ligase catalyzes a reaction in which the phosphate
group sticking off the 5 end of one DNA strand is linked to
the hydroxyl group sticking off the 3 end of the other. This
reaction produces an intact sugar-phosphate backbone.

Example: Building a recombinant

plasmid
Let's see how restriction digestion and ligation can be used
to insert a gene into a plasmid. Suppose we have a target
gene, flanked with EcoRI recognition sites, and a plasmid,
containing a single EcoRI site:

We start off with a target gene and a circular plasmid. The

target gene has twoEcoRI restriction sites near its ends.
The plasmid has oneEcoRI site in it, lying just after a
promoter that drives expression in bacteria. The sequence
of theEcoRI sites is:

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:

We separately digest (cut) the gene fragment and the

plasmid withEcoRI. This step produces fragments with
sticky ends. All of the ends have an overhang of four
nucleotides, with the sequence 5'-AATT-3'. That's
becauseEcoRI's cut pattern is:
5'-G|AATTC-3' 3'-CTTAA|G-5'
Next, we take the gene fragment and the linearized
(opened-up) plasmid and combine them along with DNA
ligase. The sticky ends of the two fragments stick together
by complementary base pairing:

Next, we take the gene fragment and the linearized

(opened-up) plasmid and combine them along with DNA
ligase. The sticky ends of the two fragments stick together
by complementary base pairing. However, there are still
gaps in the sugar-phosphate backbones of the DNA double
helix at the junction sites where the gene and plasmid DNA
meet.
Once they are joined by ligase, the fragments become a
single piece of unbroken DNA. The target gene has now
been inserted into the plasmid, making a recombinant
plasmid.

Once they are joined by ligase, the fragments become a

single piece of unbroken DNA. The target gene has now
been inserted into the plasmid, making a recombinant
plasmid. In the plasmid, the gene is now flanked by
twoEcoRI sites that were generated when the cut ends were
ligated together.

Restriction digests and ligations involve

many molecules of DNA
In the example above, we saw one outcome of a ligation
between a gene and plasmid cut with EcoRI. However,
other outcomes could happen in this exact same ligation.
For instance, the cut plasmid could recircularize (close back
up) without taking in the gene. Similarly, the gene could go
into the plasmid, but flipped backwards (since its two EcoRI
sticky ends are identical).

Left: recombinant plasmid produced when gene goes in

forwards ("pointing" away from the promoter that is already
in the plasmid).
Middle: non-recombinant plasmid produced when the cut
plasmid simply closes back up (its ends ligate with each
other).
Right: recombinant plasmid produced when gene goes in
backwards ("pointing" back towards the promoter that is
already in the plasmid).
Restriction digests and ligations like this one are performed
using many copies of plasmid and gene DNA. In fact,
billions of molecules of DNA are used in a single ligation!
These molecules are all bumping into one another, and into
DNA ligase, at random in different ways. So, if multiple
products can be made, all of them will be made at some
frequency including ones we don't want.
How can we avoid the "bad" plasmids? When we transform
bacteria with DNA from a ligation, each one takes up a

different piece of DNA. We can check the bacteria after

transformation and use only the ones with the correct
plasmid. In many cases, plasmid from transformed bacteria
is analyzed using another restriction digest to see if it
contains the right insert in the right orientation.