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Restriction enzymes

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Restriction enzymes/endonucleases:
A class of enzymes that cleave (cut) DNA at
specific and unique internal location along
its length

Nill, K. (2006) Glossary of

biotechnology and
Nanobiotechnology Terms

Klug et al. (2006) Concepts of

Genetics 2
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Bacteriophage restriction
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Recombinant DNA cloning
procedure

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Types of endonucleases

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Nomenclature
Derivation of the PstI name
Abbreviation Meaning Description
P Providencia Genus
st stuartii Species
I First identified Order of
identification in
bacteria

Derivation of the EcoRI name

Abbreviation Meaning Description
E Escherichia Genus
co coli Species
R RY13 Strain
I First identified Order of
identification in
bacteria 7
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Examples of REs

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•  More than 200 restriction enzymes have been identified

• Restriction enzyme binds to DNA and recognizes specific

sequences in the DNA called a recognition sequence

•  The enzyme cuts both strands of DNA within the recognition

sequence in a specific cleavage pattern

•  Have been used in cloning because of their ability to

accurately and reproducibly cut DNA into fragments called
restriction fragments

•  Most recognition sequences contain a form of symmetry in

which the nucleotide read the same on both strands of the DNA
when reads in 5’ to 3’ direction - palindrome

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A palindromic sequence

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Sticky and blunt ends

•  Some REs cut DNA and produce fragments with

unpaired bases at the 5’ end – cohesive or sticky ends

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Sticky and blunt ends

•  Some REs cut symmetrically at the centre position

and produce blunt ends

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Sticky and blunt ends

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Sticky and blunt ends

•  Fragments generated with sticky ends are complementary with
one another and can be joined together with a covalent bond. The
joining of these fragments can be achieved using ligase

•  Although enzymes that generate cohesive ends are useful for

cloning, there is limitation ie can only join fragments with
compatible ends

•  For example two EcoRI fragments can be joined together but not
EcoRI fragment to BamHI fragment

•  There is an exception where compatible ends can be generated

by different REs. Example are: BamHI and BglII

•  For fragments with blunt ends, they are less efficiently ligated
but they can be joined to any blunt-ended fragment
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Sticky and blunt ends

http://library.thinkquest.org/04apr/00217/images/
content/restriction%20enzymes.jpg
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Compatible cohesive ends

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Isoschizomers
•  Some REs recognise the same sequences of bases. These are
known as isoschizomers.

•  There are two types:

i.  Recognize the same sequence and cut at the same
position of the sequence
ii.  Recognize the same sequence but cut at different
position of the sequence. For example, Acc65I and
KpnI which recognize the sequence GGTACC but cut
at different place → produce different sticky ends.
Another example are XmaI and SmaI. XmaI cuts
asymmetrically and produces sticky ends that can be
ligated to other XmaI fragments while SmaI will
generate blunt-ended fragments at the same

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Isoschizomers

http://biosiva.50webs.org/dnacloning.htm

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Performing a restriction digest in the
laboratory

Components Tube 1 Tube 2

(µl) (µl)
100 ng of plasmid ? ?
EcoRI Buffer (x10) 2 2
EcoRI enzyme (10U/µl) - 1
BSA 0.5 0.5
Sterile water ? ?
Total volume 20 20

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Restriction mapping
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Processing restriction fragments
•  After digestion, if a digest produces more than one fragment,
the fragments will normally need to be separated on agarose gel.
The desired fragment will be excised and purified

•  Or if the digestion produces only one fragment, or a complex

mixture is being digested for the production of a library, it is
normally sufficient to inactivate the enzyme the enzyme before
proceeding to the next stage

•  This is accomplished by heat-inactivation

•  If enzyme is heat-stable, phenol extraction can be performed to

ensure inactivation

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Ligation

http://ocw.mit.edu/courses/biological-engineering/
20-109-laboratory-fundamentals-in-biological-
engineering-fall-2007/labs/mod1_3/
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Ligation
•  Joining DNA fragment to a vector molecule for example a
plasmid or bacteriophage that can be replicated by the host cell
after transformation

•  Carried out by an enzyme called DNA ligase

•  If this enzyme is present when two DNA molecules having sticky

ends happen to come together, it will repair the breaks that had
been introduced by the restriction enzymes

•  Natural role of ligase is to repair single-strand breaks (nicks) in

the sugar-phosphate backbone of a double-stranded DNA
molecule such as DNA damage as well as joining molecules after
DNA replication

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Ligation

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Ligation

•  The action of ligase requires that the nick should

expose a 3’-OH group and a 5’-phosphate

•  Under proper conditions, blunt ends can be ligated

by some DNA ligases. One example is T4 DNA ligase
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Ligase reacts with ATP

to form a covalent
ligase-AMP complex,
which in turn reacts
with the 5’-phosphate
on one side of the nick,
transferring AMP to the
phosphate group

Final stage is the attack

by the 3’-OH group,
forming a new covalent
phosphodiester bond
and releasing AMP

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Optimizing ligation conditions

•  Several factors may compromise the success of ligation

which include the presence of inhibitory material
contaminating DNA preparations, degradation of the
enzyme or the DNA (including loss of the 5’-phosphate)

•  The conditions need to be adjusted to achieve optimum

effects. One condition is the temperature. Many protocols
used 16°C or room temperature can also be used

•  Also need to adjust the relative amounts of vector and

insert as well as the overall concentration of DNA

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Optimizing ligation conditions

•  Need to create an optimum vector-insert ratio; ratio typically
would range from 3:1 to 1:3

•  The ratios are molar ratios, taking into account the relative size
of the vector and the insert

Tube Vector Vector Insert Ligase Ligase dH2O (µl)

: (µl) (µl) buffer (µl)
insert (µl)
ratio
1 3:1 1 1 Top up to 10 µl

2 1:1 1 1 Top up to 10 µl

3 1:3 1 1 Top up to 10 µl

4 3:0 0 1 1 Top up to 10 µl

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Protocol

1. Set up the following reaction in a microcentrifuge tube on ice.

(T4 DNA Ligase should be added last. Note that the table shows a ligation using a
molar ratio of 1:3 vector to insert for the indicated DNA
sizes.) Use NEBioCalculator to calculate molar ratios.* The T4 DNA Ligase Buffer
should be thawed and resuspended at room temperature.

2. Gently mix the reaction by pipetting up and down and microfuge briefly.

3. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for
10 minutes.

4. For blunt ends or single base overhangs, incubate at 16°C overnight or room
temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be
used in a 10 minute ligation).

5. Heat inactivate at 65°C for 10 minutes.

6. Chill on ice and transform 1-5 µl of the reaction into 50 µl competent cells.

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Ligation

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Alkaline phosphatase

http://escience.ws/b572/L6/L6.htm
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Alkaline phosphatase
•  5’-phosphate at the nick site can be removed by an
enzyme called alkaline phosphatase

•  The most common alkaline phosphatase used is calf

intestinal phosphatase (CIP)

•  Treatment of vector molecule with CIP will remove

5’-phosphate or dephosphorylate the vector thus
prevent self-ligation to occur

•  Before ligation, CIP can be removed by phenol

extraction and subsequent ethanol precipitation

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Dephosphorylation of vector

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Double digest
•  To avoid self ligation of vector (and insert), both components
can be cut with two different restriction enzymes

•  Most current vectors have multiple cloning sites, so this will

allow double digestion to be performed

h t t p : / /
stanxterm.aecom.yu.edu/
w i k i / d a t a /
Recombinant_DNA_engineeri
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Cloning of operon cobA-cbiX into chloroplast expression
vector, p72-psbA
EcoRI BamHI BglII BamHI

psb cobAcbi
H X

pET14b- pET14b-
pET14b-
p72-psbA
cobAcbiX cobAcbiX
cobAcbiX

Digest with BamHI and treated

Digest with BamHI and BglII
with calf intestinal phosphatase

Run the digested DNA and gel Run the digested DNA and gel
extract the right size of band extract the right size of band

Ligation

Transform into DH5α

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Processing restriction fragments

•  Modifications of generated DNA fragments by REs:

•  converting sticky ends to blunt ends

•  filling in the missing bases complementary to the unpaired

ends or by digesting away the single-stranded regions

•  Use of linkers and adaptors – short synthetic DNA fragment

that add new restriction sites to the end of a fragment

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Modification of restriction fragment ends

•  Restriction fragments with sticky ends are useful as they can

be readily ligated but there is a limitation on their usefulness.
They can only be ligated to another fragments with compatible
ends

•  This is a potential hindrance and nuisance as the vector you

are using will only have a limited number of possible sites into
which you can insert DNA. It may also not possible to generate
a suitable insert with the same enzyme

•  There a few modifications that can be done to enable DNA

fragment to be ligated with other sites

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Modification of restriction fragment ends
i.  Trimming and filling

•  Sticky ends can be converted into blunt ends either by filling

in the complementary strand or by trimming back the
unpaired sequence

•  If the fragment has a 5’ overhang, the 3’ OH group provides

a primer site that can be extended by DNA polymerases, thus
filling in the recessed end and converting it to double-
stranded

http://static.fastbleep.com/assets/notes/image/
7387_1.jpg
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Modification of restriction fragment ends

•  However, some DNA polymerases (such as E. coli DNA

polymerase I) also have 5’-3’ exonuclease activity, which remove
the 5’ overhang and are not suitable for filling in the ends

•  An enzyme lacking this 5’-3’ exonuclease activity such as the

Klenow fragment of E. coli DNA polymerase I ( a proteolytic
product of E. coli DNA polymerase I in which the region
responsible for the 5’-3’ exonuclease activity has been removed)
can be used

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Filling in

Trimming back

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Modification of restriction fragment ends

ii.  Linkers and adaptors

•  Linkers are short pieces of DNA that contain a restriction site

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Modification of restriction fragment ends

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Modification of restriction fragment ends

A possible problem with the use of linkers

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Modification of restriction fragment ends

•  Adaptors are pairs of short oligonucleotides that are designed to
anneal together in such a way as to create a short double-
stranded DNA fragment with different sticky ends (or with one
sticky and one blunt end)

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Modification of restriction fragment ends

iii.  Homopolymer tailing

•  Another way of adding sticky ends to DNA molecules is to use

the enzyme terminal deoxynucleotide transferase (or terminal
transferase)

•  When supplied with a single deoxynucleotide triphosphate, this

enzyme will repetitively add nucleotides to the 3’ OH end of a
DNA molecule

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Modification of restriction fragment ends

Cloning using homopolymer tailing

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Modification of restriction fragment ends

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Modification of restriction fragment ends

•  It is not possible to ensure that the tails are all exactly the same
length, so when the molecules anneal together there will be gaps
in one of the sequences.

•  This is ok. If the tails are longer than about 20 nucleotides, then
the pairing of the tails will be strong enough to be stable at room
temperature, and the product can be used for transformation
without repairing the gaps, or without sealing the nicks with
ligase

•  Both gaps and nicks will be repaired within the host cell after
transformation

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Modification of restriction fragment ends

•  The advantage of this strategy is that it is not possible for the

vector to reform without an insert as the ends of the vector are
not complementary to one another

•  The downside is that a recombinant plasmid that contains a

variable number of GC base pairs at either end of the insert is
being constructed, so it lacks the precision associated with the
formation of a recombinant using the other methods

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Other ways of joining DNA molecules

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Other ways of joining DNA molecules

i.  TA cloning of PCR products

•  Some of the polymerases used in PCR amplification

also have a terminal transferase action; they add a
single adenosine residue to the 3’ ends of the
synthesized ends

•  This provides an opportunity. There are commercially

available TA vectors which are supplied in linearized
form with a single 3’-T overhang, which is compatible
with the unpaired 3’-A on the PCR product
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Other ways of joining DNA molecules

http://www.bocascientific.com/images/
pLUG%20multi.jpg
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Other ways of joining DNA molecules

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Other ways of joining DNA molecules

http://
tools.invitrogen.com/
content/sfs/manuals/
topota_man.pdf
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ii. Designing the

PCR primers to
incorporate a
restriction site

Adding restriction sites to a PCR product

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Other ways of joining DNA molecules
•  Alternative strategies include designing the PCR primers to
incorporate a restriction site; the product can then be digested
with the appropriate enzyme before ligation

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The mode of action of type I DNA

topoisomerase
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Other ways of joining DNA molecules

iii.  DNA topoisomerase

•  Topoisomerase can be used to join two DNA molecules

•  Topoisomerases in general are responsible for controlling the

degree of supercoIling of DNA

•  Type I topoisomerases achieve this by cutting one DNA

strand, which is the free to rotate (thus reducing supercoiling)
and subsequently rejoining the cut ends of the DNA strands

•  The enzyme remains covalently attached to the phosphate

group at the end of the broken strand after cutting, thus
retaining the bond energy and being in place for the
subsequent rejoining
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Other ways of joining DNA molecules

Action of topoisomerase
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Other ways of joining DNA molecules
•  Topoisomerases will cleave DNA at specific sites, leaving a
sticky end

•  The energy released by breaking the phosphodiester bond is

stored in a covalent bond between the enzyme and one of the
cleaved strands

•  The enzyme trapped in the sticky end will then rapidly and
efficiently release its stored energy into the formation of a new
phosphodiester fragment as soon as the sticky end encounters
its complementary partner

•  Commercially available TOPO vectors offer sticky end

overhangs, from TA cloning to more complex sequences,
already bound to the TOPO enzyme

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Other ways of joining DNA molecules

•  Example: A linearised vector is supplied with Vaccinia

virus topoisomerase I covalently attached to phosphate
groups at the 3’ ends. When the prepared vector is
mixed with the DNA fragment to be cloned, the
enzyme transfers the phosphate linkages to the 5’
ends of the fragment, thus joining the insert to the
vector

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Other ways of joining DNA molecules

http://www.chem.fsu.edu/chemlab/
bch4053l/protein/cloning/TOPO.jpg
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Other ways of joining DNA molecules

•  Other enzyme systems

•  Site-specific recombinase of bacteriophage lambda and

Cre-lox system from the P1 bacteriophage

•  Cre is a highly site-specific recombinase which act only on

a specific sequence known as the lox site

•  Both enzymes will carry out recombination between two

plasmids carrying the appropriate recognition sequences,
leading to transfer of a cloned gene between two plasmids,
or fusion of the plasmids

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References:

1.  Brown, T. A. (2010). Gene cloning and DNA analysis: An

Introduction. 6th Edition. Wiley-Blackwell.

2.  Dale, J. W., Von Schantz, M. and Plant, N. (2012). From Genes
to Genomes. 3rd Edition. Wiley-Blackwell.

3.  Glick B.R & Pasternak, J.J. (2003). Molecular Biotechnology:

Principles and applications of recombinant DNA. ASM Press.

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