gene

1. Unit 2
Molecular tools for gene cloning
Restriction and Modification systems: Restriction Endonucleases,
star activity of restriction enzymes, Methylases, Ligases.
Polynucleotide kinases, Phosphatases, DNA and RNA
polymerases, Reverse transcriptase, Terminal transferase,
DNAses (Extremophiles), Mung Bean Nuclease. RNases,
Topoisomerase.

2. Enzymes in rDNA technology
Once pure samples of DNA have been prepared, the next step in a gene cloning
experiment is construction of the recombinant DNA molecule (see Figure 1.1). To
produce this recombinant molecule, the vector, as well as the DNA to be cloned, must
be cut at specific points and then joined together in a controlled manner.
Cutting and joining are two examples of DNA manipulative techniques, a wide variety
of which have been developed over the past few years. As well as being cut and
joined, DNA molecules can be shortened, lengthened, copied into RNA or into new
DNA molecules, and modified by the addition or removal of specific chemical groups.
These manipulations, all of which can be carried out in the test tube, provide the
foundation not only for gene cloning, but also for studies of DNA biochemistry, gene
structure, and the control of gene expression.
Almost all DNA manipulative techniques make use of purified enzymes. Within the cell
these enzymes participate in essential processes such as DNA replication and
transcription, breakdown of unwanted or foreign DNA (e.g., invading virus DNA),
repair of mutated DNA, and recombination between different DNA molecules.

3. Enzymes in rDNA technology
After purification from cell extracts, many of these enzymes can
be persuaded to carry out their natural reactions, or something
closely related to them, under artificial conditions.
Although these enzymatic reactions are often straightforward,
most are absolutely impossible to perform by standard chemical
methods. Purified enzymes are therefore crucial to genetic
engineering and an important industry has sprung up around
their preparation, characterization, and marketing.
Commercial suppliers of high purity enzymes provide an essential
service to the molecular biologist. The cutting and joining
manipulations that underlie gene cloning are carried out by
enzymes called restriction endonucleases (for cutting) and
ligases (for joining).

4. The range of DNA manipulative
enzymes
DNA manipulative enzymes can be grouped into four broad
classes, depending on the type of reaction that they
catalyze:
Nucleases are enzymes that cut, shorten, or degrade nucleic
acid molecules.
Ligases join nucleic acid molecules together.
Polymerases make copies of molecules.
Modifying enzymes remove or add chemical groups.

5. Nucleases
Nucleases degrade DNA molecules by breaking the phosphodiester bonds that link
one nucleotide to the next in a DNA strand. There are two different kinds of nuclease
(Figure 4.1):
Exonucleases remove nucleotides one at a time from the end of a DNA molecule.
Endonucleases are able to break internal phosphodiester bonds within a DNA
molecule.
The main distinction between different exonucleases lies in the number of strands
that are degraded when a double-stranded molecule is attacked.
The enzyme called Bal31 (purified from the bacterium Alteromonas espejiana) is an
example of an exonuclease that removes nucleotides from both strands of a double-
stranded molecule (Figure 4.2a). The greater the length of time that Bal31 is allowed
to act on a group of DNA molecules, the shorter the resulting DNA fragments will be.
In contrast, enzymes such as E. coli exonuclease III has got 3’ to 5’ exonuclease activity.
(Figure 4.2b).

8. Nucleases
The same criterion can be used to classify endonucleases. S1
endonuclease (from the fungus Aspergillus oryzae) only cleaves
single strands (Figure 4.3a), whereas deoxyribonuclease I (DNase I),
which is prepared from cow pancreas, cuts both singleand
double-stranded molecules (Figure 4.3b).
DNase I is non-specific in that it attacks DNA at any internal
phosphodiester bond, so the end result of prolonged DNase I action is
a mixture of mononucleotides and very short oligonucleotides. On the
other hand, the special group of enzymes called restriction
endonucleases cleave doublestranded DNA only at a limited number of
specific recognition sites (Figure 4.3c).

10. Enzymes for cutting DNA—restriction endonucleases
Gene cloning requires that DNA molecules be cut in a very precise and reproducible fashion. This is
illustrated by the way in which the vector is cut during construction of a recombinant DNA molecule (Figure
4.7a).
Each vector molecule must be cleaved at a single position, to open up the circle so that new DNA can be
inserted: a molecule that is cut more than once will be broken into two or more separate fragments and will
be of no use as a cloning vector.
Furthermore, each vector molecule must be cut at exactly the same position on the circle, random cleavage
is not satisfactory. It should be clear that a very special type of nuclease is needed to carry out this
manipulation. Often it is also necessary to cleave the DNA that is to be cloned (Figure 4.7b). There are two
reasons for this.
• First, if the aim is to clone a single gene, which may consist of only 2 or 3 kb of DNA, then that gene will
have to be cut out of the large (often greater than 80 kb) DNA molecules
• Second, large DNA molecules may have to be broken down simply to produce fragments small enough
to be carried by the vector.
Most cloning vectors exhibit a preference for DNA fragments that fall into a particular size range: most
plasmid-based vectors, for example, are very inefficient at cloning DNA molecules more than 8 kb in length.
Purified restriction endonucleases allow the molecular biologist to cut DNA molecules in the precise,
reproducible manner required for gene cloning. The discovery of these enzymes, which led to Nobel Prizes
for W. Arber, H. Smith, and D. Nathans in 1978, was one of the key breakthroughs in the development of
genetic engineering.

12. The discovery and function of restriction
endonucleases
The initial observation that led to the eventual discovery of restriction endonucleases was
made in the early 1950s, when it was shown that some strains of bacteria are immune to
bacteriophage infection, a phenomenon referred to as host-controlled restriction.
The mechanism of restriction is not very complicated, even though it took over 20 years to be
fully understood. Restriction occurs because the bacterium produces an enzyme that degrades
the phage DNA before it has time to replicate and direct synthesis of new phage particles
(Figure 4.8a).
The bacterium’s own DNA, the destruction of which would of course be lethal, is protected
from attack because it carries additional methyl groups that block the degradative enzyme
action (Figure 4.8b).
These degradative enzymes are called restriction endonucleases and are synthesized by many,
perhaps all, species of bacteria: over 2500 different ones have been isolated and more than
300 are available for use in the laboratory.

14. Restriction Endonuclease
Nomenclature
Restriction endonucleases are named according to the organism in which
they were discovered, using a system of letters and numbers. For example,
HindIII was discovered in Haemophilus influenza (strain d). The Roman
numerals are used to identify specific enzymes from bacteria that contain
multiple restriction enzymes indicating the order in which restriction enzymes
were discovered in a particular strain.

15. Classification of Restriction
Endonucleases
There are three major classes of restriction
endonucleases based on the types of sequences
recognized, the nature of the cut made in the
DNA, and the enzyme structure:
• Type I restriction enzymes
• Type II restriction enzymes
• Type III restriction enzymes

16. Type I restriction enzymes
• These enzymes have both restriction and modification activities.
Restriction depends upon the methylation status of the target DNA.
• Cleavage occurs approximately 1000 bp away from the recognition site.
• The recognition site is asymmetrical and is composed of two specific
portions in which one portion contain 3–4 nucleotides while another
portion contain 4–5 nucleotides and both the parts are separated by a
non-specific spacer of about 6–8 nucleotides.
• They require S-adenosylmethionine (SAM), ATP, and magnesium ions
(Mg2+) for activity.
• These enzymes are composed of mainly three subunits, a specificity
subunit that determines the DNA recognition site, a restriction subunit,
and a modification subunit

17. Type III restriction enzymes
•These enzymes recognize and methylate the same DNA
sequence but cleave 24–26 bp away.
•They have two different subunits, in which one subunit (M)
is responsible for recognition and modification of DNA
sequence and other subunit (R) has nuclease action.
• Mg+2 ions, ATP are needed for DNA cleavage and process of
cleavage is stimulated by SAM.
•Cleave only one strand. Two recognition sites in opposite
orientation are necessary to break the DNA duplex

18. Type II restriction endonucleases cut DNA at specific
nucleotide sequences
The central feature of type II restriction endonucleases (which will be referred to simply as
“restriction endonucleases” from now on) is that each enzyme has a specific recognition
sequence at which it cuts a DNA molecule.
A particular enzyme cleaves DNA at the recognition sequence and nowhere else. For
example, the restriction endonuclease called PvuI (isolated from Proteus vulgaris) cuts DNA
only at the hexanucleotide CGATCG.
In contrast, a second enzyme from the same bacterium, called PvuII, cuts at a different
hexanucleotide, in this case CAGCTG.
Many restriction endonucleases recognize hexanucleotide target sites, but others cut at
four, five, eight, or even longer nucleotide sequences. Sau3A (from Staphylococcus aureus
strain 3A) recognizes GATC, and AluI (Arthrobacter luteus) cuts at AGCT.
There are also examples of restriction endonucleases with degenerate recognition
sequences, meaning that they cut DNA at any one of a family of related sites. HinfI
(Haemophilus influenzae strain Rf), for instance, recognizes GANTC, so cuts at GAATC,
GATTC, GAGTC, and GACTC.

19. The steps involved in DNA binding and cleavage
by a type II restriction endonuclease
• These enzymes have nonspecific contact with DNA and initially bind
to DNA as dimers.
• The target site is then located by a combination of linear diffusion or
“sliding” of the enzyme along the DNA over short distances, and
hopping/jumping over longer distances.
• Once the target restriction site is located, the recognition process
(coupling) triggers large conformational changes of the enzyme and the
DNA, which leads to activation of the catalytic center.
• Catalysis results in hydrolysis of phosphodiester bond and
product release.

20. Structures of free, nonspecific, and specific DNA-bound forms of BamHI

21. Comparison chart of RE I, II and III

22. Blunt ends and sticky ends
Blunt ends
The exact nature of the cut produced by a restriction endonuclease is of considerable
importance in the design of a gene cloning experiment. Many restriction endonucleases make a
simple double-stranded cut in the middle of the recognition sequence (Figure 4.9a), resulting in
a blunt end or flush end. PvuII and AluI are examples of blunt end cutters.
Sticky Ends
Other restriction endonucleases cut DNA in a slightly different way. With these enzymes, the
two DNA strands are not cut at exactly the same position. Instead the cleavage is staggered,
usually by two or four nucleotides, so that the resulting DNA fragments have short single-
stranded overhangs at each end (Figure 4.9b).
These are called sticky or cohesive ends, as base pairing between them can stick the DNA
molecule back together again. One important feature of sticky end enzymes is that restriction
endonucleases with different recognition sequences may produce the same sticky ends.
BamHI (recognition sequence GGATCC) and BglII (AGATCT) are examples—both produce GATC
sticky ends (Figure 4.9c). The same sticky end is also produced by Sau3A, which recognizes only
the tetranucleotide GATC. Fragments of DNA produced by cleavage with either of these
enzymes can be joined to each other, as each fragment carries a complementary sticky end.

24. Performing a restriction digest in the laboratory
As an example, we will consider how to digest a sample of λ DNA (concentration 125 mg/ml) with BglII.
First, the required amount of DNA must be pipetted into a test tube. The amount of DNA that will be
restricted depends on the nature of the experiment.
In this case we will digest 2 μg of λ DNA, which is contained in 16 μl of the sample (Figure 4.11a). Very
accurate micropipettes will therefore be needed. The other main component in the reaction will be the
restriction endonuclease, obtained from a commercial supplier as a pure solution of known concentration.
But before adding the enzyme, the solution containing the DNA must be adjusted to provide the correct
conditions to ensure maximal activity of the enzyme. Most restriction endonucleases function adequately
at pH 7.4, but different enzymes vary in their requirements for ionic strength (usually provided by sodium
chloride (NaCl)) and magnesium (Mg2+) concentration (all type II restriction endonucleases require Mg2+ in
order to function).
It is also advisable to add a reducing agent, such as dithiothreitol (DTT), which stabilizes the enzyme and
prevents its inactivation. Providing the right conditions for the enzyme is very important—incorrect NaCl or
Mg2+ concentrations not only decrease the activity of the restriction endonuclease, they might also cause
changes in the specificity of the enzyme, so that DNA cleavage occurs at additional, non-standard
recognition sequences.
After adding suitable composition of the buffer, The restriction endonuclease can now be added.
By convention, 1 unit of enzyme is defined as the quantity needed to cut 1 μg of DNA in 1 hour, so we
need 2 units of BglII to cut 2 μg of λ DNA. BglII is frequently obtained at a concentration of 4 units/ μl, so 0.5
μl is sufficient to cleave the DNA.

25. The final ingredients in the reaction mixture are therefore 0.5 μl BglII + 2μl of BglII
buffer+ 1.5 μl water + 16 μl of template DNA giving a final volume of 20 μl (Figure
4.11c).
The last factor to consider is incubation temperature. Most restriction endonucleases,
including BglII, work best at 37°C, but a few have different requirements.
TaqI, for example, is a restriction enzyme from Thermus aquaticus and, like Taq DNA
polymerase, has a high working temperature. Restriction digests with TaqI must be
incubated at 65°C to obtain maximum enzyme activity.
After 1 hour the restriction should be complete (Figure 4.11d). If the DNA fragments
produced by restriction are to be used in cloning experiments, the enzyme must
somehow be destroyed so that it does not accidentally digest other DNA molecules
that may be added at a later stage. There are several ways of “killing” the enzyme. For
many a short incubation at 70°C is sufficient, for others phenol extraction or the
addition of ethylenediamine tetraacetate (EDTA), which binds Mg2+ ions preventing
restriction endonuclease action, is used (Figure 4.11e).

26. Applications of REs
In various applications related to genetic engineering DNA is
cleaved by using restriction enzymes.
•They are used in the process of insertion of genes into plasmid
vectors during gene cloning and protein expression experiments.
•Restriction enzymes can also be used to distinguish gene alleles by
specifically recognizing single base changes in DNA known as single
nucleotide polymorphisms (SNPs). This is only possible if a
mutation alters the restriction site present in the allele.
•Restriction enzymes are used for Restriction Fragment Length
Polymorphism (RFLP) analysis for identifying individuals or strains of
a particular species.

27. Ligases – joins DNA molecules together
DNA ligase catalyses the formation of phosphodiester bond between two deoxynucleotide residues of
two DNA strands.
DNA ligase enzyme requires a free hydroxyl group at the 3´ -end of one DNA chain and a phosphate group
at the 5´-end of the other and requires energy in the process.
E.coli and other bacterial DNA ligase utilizes NAD+ as energy donor, whereas in T4 bacteriophage, T4 DNA
ligase uses ATP as cofactor.
The role of DNA ligase is to seal nicks in the backbone of double-stranded DNA after lagging strand
formation to join the okazaki fragments. This joining process is essential for the normal synthesis of DNA
and for repairing damaged DNA. It has been exploited by genetic engineers to join DNA chains to form
recombinant DNA molecules. Usually single stranded break are repaired using the complimentary strand
as the template but sometimes double stranded breaks can also be repaired with the help of DNA
ligase IV.
The most widely used DNA ligase is isolated from T4 bacteriophage. T4 DNA ligase needs ATP as a
cofactor. The enzyme from E. coli uses cofactor NAD. Except this, the catalysis mechanism is somewhat
similar for both the ligases. The role of cofactor is splitting and forming an enzyme-AMP complex which
further aids in formation of phosphodiester bonds between hydroxyl and phosphate groups by exposing
them.

28. Ligases are the most commonly used enzymes for
carrying out ligations. They are part of the routine battery
of enzymes required by a cell for the maintenance of its
DNA. They are used in joining together adjacent Okazaki
fragments produced in replication, and in sealing the nicks
that arise from damage and repair processes.
T4 DNA ligase is encoded by bacteriophage T4, and is
produced on infection of E. coli cells. It can carry out both
blunt-ended and sticky-ended ligations, and requires ATP.
It requires a 3’-hydroxyl and a 5’-phosphate group on the
molecules to be joined.

29. E. coli DNA ligase is the endogenous bacterial enzyme.
Unlike T4 DNA ligase, it is unable to carry out
blunt-ended ligations (or does so only very inefficiently)
and, therefore, is particularly useful if such ligations need
to be avoided.
This might be the case if you were trying to seal nicks in
damaged DNA without also joining non-contiguous
sequences. Like the T4 ligase, E. coli DNA ligase requires
a 3’-hydroxyl and a 5’-phosphate group, but it requires
nicotinamide adenine dinucleotide (NAD+) as a cofactor.
Essentially the same reaction is catalysed by each of
these ligases

30. In both cases, adenosine monophosphate (AMP) is added to the
5’-phosphate, of one DNA molecule liberating either pyrophosphate
from ATP or nicotinamide mononucleotide from NAD+
. The AMP is
then displaced in a nucleophilic attack by the 3’-hydroxyl of the
other DNA molecule.
Mechanism of ligation by T4DNA ligase (using ATP)or E.coli ligase (using NAD+).

31. Mechanism of Action of DNA Ligases
•ATP, or NAD+, reacts with the ligase enzyme to form a covalent enzyme–AMP complex
in which the AMP is linked to ε-amino group of a lysine residue in the active site of the
enzyme through a phospho-amide bond.
• The AMP moiety activates the phosphate group at the 5´-end of the DNA molecule to
be joined. It is called as the donor.
•The final step is a nucleophilic attack by the 3´-hydroxyl group on this activated
phosphorus atom which acts as the acceptor. A phosphodiester bond is formed and
AMP is released.
•The reaction is driven by the hydrolysis of the pyrophosphate released during the
formation of the enzyme–adenylate complex. Two high-energy phosphate bonds are
spent in forming a phosphodiester bond in the DNA backbone with ATP serving as energy
source.
•The temperature optimum for T4 DNA ligase mediated ligation in vitro is 16˚C. However
ligation is also achieved by incubation at 4˚C by incubating over night or at room
temperature condition by incubating for 30 minutes.
•Adenylate and DNA-adenylate are the important intermediates of the phosphodiester
bond forming pathway.

32. The mechanism of DNA joining
by DNA ligase

33. Applications of DNA
ligase
• DNA ligase enzyme is used by cells to join the “okazaki fragments”
during DNA replication process. In molecular cloning, ligase
enzyme has been routinely used to construct a recombinant DNA.
• Joining of adapters and linkers to blunt end DNA molecule.
• Cloning of restricted DNA to vector to construct recombinant vector.
Ligation of a gene fragment into the vector and transformation of the cell.

34. Use of DNA ligase to create a covalent DNA recombinant
joined through association of termini generated by EcoRI.

35. Application of alkaline phosphatase treatment to prevent
recircularization of vector plasmid without insertion of foreign
DNA.

36. Sticky ends increase the efficiency of ligation
The ligation reaction carried out by two blunt-ended fragments is not very
efficient. This is because the ligase is unable to “catch hold” of the
molecule to be ligated, and has to wait for chance associations to bring the
ends together.
If possible, blunt end ligation should be performed at high DNA
concentrations, to increase the chances of the ends of the molecules
coming together in the correct way.
In contrast, ligation of complementary sticky ends is much more efficient.
This is because compatible sticky ends can base pair with one another by
hydrogen bonding (Figure 4.20b), forming a relatively stable structure for
the enzyme to work on. If the phosphodiester bonds are not synthesized
fairly quickly then the sticky ends fall apart again. These transient, base-
paired structures do, however, increase the efficiency of ligation by
increasing the length of time the ends are in contact with one another.

37. Stick end and Blunt end
ligation

38. Putting sticky ends onto a blunt-ended molecule
For the reasons detailed in the preceding section, compatible
sticky ends are desirable on the DNA molecules to be ligated
together in a gene cloning experiment. Often these sticky
ends can be provided by digesting both the vector and the
DNA to be cloned with the same restriction endonuclease, or
with different enzymes that produce the same sticky end,
but it is not always possible to do this.
A common situation is where the vector molecule has
sticky ends, but the DNA fragments to be cloned are
blunt-ended. Under these circumstances one of three
following methods can be used to put the correct sticky
ends onto the DNA fragments.

39. Linkers
The first of these methods involves the use of linkers. These are short pieces of double
stranded DNA, of known nucleotide sequence, that are synthesized in the test tube.
A typical linker is shown in Figure 4.21a.
It is blunt-ended, but contains a restriction site, BamHI in the example shown. DNA ligase can
attach linkers to the ends of larger blunt ended DNA molecules. Although a blunt end
ligation, this particular reaction can be performed very efficiently because synthetic
oligonucleotides, such as linkers, can be made in very large amounts and added into the
ligation mixture at a high concentration.
More than one linker will attach to each end of the DNA molecule, producing the chain
structure shown in Figure 4.21b. However, digestion with BamHI cleaves the chains at the
recognition sequences, producing a large number of cleaved linkers and the original DNA
fragment, now carrying BamHI sticky ends. This modified fragment is ready for ligation into a
cloning vector restricted with BamHI.

41. A decameric linker molecule containing an EcoRI target site is joined by T4 DNA
ligase to both ends of flush ended foreign DNA. Cohesive ends are then
generated by EcoRI. This DNA can then be incorporated into a vector that has
been treated with the same restriction endonuclease

42. Adaptors
There is one potential drawback with the use of linkers. Consider what would happen if the
blunt-ended molecule shown in Figure 4.21b contained one or more BamHI recognition
sequences. If this was the case, the restriction step needed to cleave the linkers and produce
the sticky ends would also cleave the blunt-ended molecule (Figure 4.22). The resulting
fragments will have the correct sticky ends, but that is no consolation if the gene contained
in the blunt-ended fragment has now been broken into pieces.
The second method of attaching sticky ends to a blunt-ended molecule is designed to avoid
this problem. Adaptors, like linkers, are short synthetic oligonucleotides. But unlike linkers,
an adaptor is synthesized so that it already has one sticky end (Figure 4.23a). The idea is of
course to ligate the blunt end of the adaptor to the blunt ends of the DNA fragment, to
produce a new molecule with sticky ends. This may appear to be a simple method but in
practice a new problem arises. The sticky ends of individual adaptor molecules could base
pair with each other to form dimers (Figure 4.23b), so that the new DNA molecule is still
blunt-ended (Figure 4.23c). The sticky ends could be recreated by digestion with a restriction
endonuclease, but that would defeat the purpose of using adaptors in the first place.

43. The problem can be resolved by synthesizing Adaptor molecules such that the
blunt end is the same as “natural” DNA, but the sticky end is different. The 3ʹ-OH
terminus of the sticky end is the same as usual, but the 5ʹ-P terminus is modified:
it lacks the phosphate group, and is in fact a 5ʹ-OH terminus (Figure 4.25a). DNA
ligase is unable to form a phosphodiester bridge between 5ʹ-OH and 3ʹ-OH ends.
The result is that, although base pairing is always occurring between the sticky
ends of adaptor molecules, the association is never stabilized by ligation (Figure
4.25b). Adaptors can therefore be ligated to a blunt-ended DNA molecule but not
to themselves. After the adaptors have been attached, the abnormal 5ʹ-OH
terminus is converted to the natural 5ʹ-P form by treatment with the enzyme
polynucleotide kinase (p. 50), producing a sticky-ended fragment that can be
inserted into an appropriate vector.

44. Figure 4.25 The use of adaptors: (a) the actual structure of an adaptor, showing the modified 5ʹ-OH
terminus; (b) conversion of blunt ends to sticky ends through the attachment of adaptors.

45. Use of a BamHI adaptor molecule. A synthetic adaptor molecule is ligated to
the foreign DNA. The adaptor is used in the 5′-hydroxyl form to prevent
self-polymerization. The foreign DNA plus ligated adaptors is phosphorylated
at the 5′-termini and ligated into the vector previously cut with BamHI.

46. Producing sticky ends by homopolymer tailing
The technique of homopolymer tailing offers a radically different approach to the
production of sticky ends on a blunt-ended DNA molecule.
A homopolymer is simply a polymer in which all the subunits are the same. A DNA
strand made up entirely of, say, deoxyguanosine is an example of a homopolymer, and
is referred to as polydeoxyguanosine or poly(dG). Tailing involves using the enzyme
terminal deoxynucleotidyl transferase (p. 50) to add a series of nucleotides onto the
3ʹ-OH termini of a double-stranded DNA molecule.
If this reaction is carried out in the presence of just one deoxyribonucleotide, a
homopolymer tail is produced (Figure 4.26a). Of course, to be able to ligate together
two tailed molecules, the homopolymers must be complementary.
Frequently polydeoxycytosine (poly(dC)) tails are attached to the vector and poly(dG)
to the DNA to be cloned. Base pairing between the two occurs when the DNA
molecules are mixed (Figure 4.26b).
In practice, the poly(dG) and poly(dC) tails are not usually exactly the same length,
and the base-paired recombinant molecules that result have nicks as well as
discontinuities (Figure 4.26c).

47. Repair is therefore a two-step process,
using Klenow polymerase to fill in the
nicks followed by DNA ligase to
synthesize the final phosphodiester
bonds. This repair reaction does not
always have to be performed in the test
tube. If the complementary
homopolymer tails are longer than
about 20 nucleotides, then quite stable
base-paired associations are formed.
A recombinant DNA molecule, held
together by base pairing although not
completely ligated, is often stable
enough to be introduced into the host
cell in the next stage of the cloning
experiment. Once inside the host, the
cell’s own DNA polymerase and DNA
ligase repair the recombinant DNA
molecule, completing the construction
begun in the test tube.

48. Use of calf-thymus terminal deoxynucleotidyltransferase to add
complementary homopolymer tails to two DNA molecules. Use
of calf-thymus terminal deoxynucleotidyltransferase to add
complementary homopolymer tails to two DNA molecules.

49. Blunt end ligation with a DNA topoisomerase
A more sophisticated, but easier and generally more efficient way of carrying out blunt end ligation,
is to use a special type of enzyme called a DNA topoisomerase. Topoisomerase is an enzyme that
introduces or removes turns from the double helix by breakage and reunion of one or both
polynucleotides.
In the cell, DNA topoisomerases are involved in processes that require turns of the double helix to be
removed or added to a double-stranded DNA molecule. Turns are removed during DNA replication in
order to unwind the helix and enable each polynucleotide to be replicated, and are added to newly
synthesized circular molecules to introduce supercoiling. DNA topoisomerases are able to
separate the two strands of a DNA molecule without actually rotating the double helix. They achieve
this feat by causing transient single- or double-stranded breakages in the DNA backbone (Figure
4.27). DNA topoisomerases therefore have both nuclease and ligase activities.
To carry out blunt end ligation with a topoisomerase, a special type of cloning vector is needed. This
is a plasmid that has been linearized by the nuclease activity of the DNA topoisomerase enzyme
from vaccinia virus. The vaccinia topoisomerase cuts DNA at the sequence CCCTT, which is present
just once in the plasmid. After cutting the plasmid, topoisomerase enzymes remain covalently bound
to the resulting blunt ends. The reaction can be stopped at this point, enabling the vector to be
stored until it is needed.
Cleavage by the topoisomerase results in 5ʹ-OH and 3ʹ-P termini (Figure 4.28a). If the blunt-ended
molecules to be cloned have been produced from a larger molecule by cutting with a restriction
enzyme, then they will have 5ʹ-P and 3ʹ-OH ends. Before mixing these molecules with the vector,
their terminal phosphates must be removed to give 5ʹ-OH ends that can ligate to the 3ʹ-P termini of
the vector. The molecules are therefore treated with alkaline phosphatase (Figure 4.28b).

50. Figure 4.27
The mode of action of a Type 1 DNA
topoisomerase, which removes or adds turns
to a double helix by making a transient break
in one of the strands.
Adding the phosphatased molecules to the
vector reactivates the bound
topoisomerases, which proceed to the
ligation phase of their reaction. Ligation
occurs between the 3ʹ-P ends of the
vectors and the 5ʹ-OH ends of the
phosphatased molecules.
The blunt-ended molecules therefore
become inserted into the vectors. Only one
strand is ligated at each junction point
(Figure 4.28c), but this is not a problem
because the discontinuities will be repaired
by cellular enzymes after the recombinant
molecules have been introduced into the
host bacteria.

51. Figure 4.28 Blunt end ligation with a DNA topoisomerase. (a) Cleavage of the vector with the
topoisomerase leaves blunt ends with 5ʹ-OH and 3ʹ-P termini. (b) The molecule to be cloned
must therefore be treated with alkaline phosphatase to convert its 5ʹ-P ends into 5ʹ-OH
termini. (c) The topoisomerase ligates the 3ʹ-P and 5ʹ-OH ends, creating a double-stranded
molecule with two discontinuities, which are repaired by cellular enzymes after introduction
into the host bacteria.

52. DNA polymerases
DNA polymerases are enzymes that synthesize a new strand of DNA complementary to an
existing DNA or RNA template (Figure 4.5a). Most polymerases can function only if the
template possesses a double-stranded region that acts as a primer for initiation of
polymerization
DNA polymerase I, which is usually prepared from E. coli. This enzyme attaches to a short
single-stranded region (or nick) in a mainly double-stranded DNA molecule, and then
synthesizes a completely new strand, degrading the existing strand as it proceeds (Figure
4.5b). DNA polymerase I is therefore an example of an enzyme with a dual activity—DNA
polymerization and DNA degradation
The polymerase and nuclease activities of DNA polymerase I are controlled by different parts
of the enzyme molecule. The nuclease activity is contained in the first 323 amino acids of the
polypeptide, so removal of this segment leaves a modified enzyme that retains the
polymerase function but is unable to degrade DNA. This modified enzyme, called the Klenow
fragment, can still synthesize a complementary DNA strand on a single-stranded template,
but as it has no nuclease activity it cannot continue the synthesis once the nick is filled in
(Figure 4.5c).
Several other enzymes—natural polymerases and modified versions—have similar properties
to the Klenow fragment. The major application of these polymerases is in DNA sequencing.

53. The Taq DNA polymerase used in the polymerase chain reaction
(PCR) is the DNA polymerase I enzyme of the bacterium
Thermus aquaticus. This organism lives in hot springs, and
many of its enzymes, including the Taq DNA polymerase, are
thermostable, meaning that they are resistant to denaturation
by heat treatment. This is the special feature of Taq DNA
polymerase that makes it suitable for PCR, because if it was not
thermostable it would be inactivated when the temperature of
the reaction is raised to 94°C to denature the DNA.
The final type of DNA polymerase that is important in genetic
engineering is reverse transcriptase, an enzyme involved in the
replication of several kinds of virus. Reverse transcriptase is
unique in that it uses as a template not DNA but RNA (Figure
4.5d). The ability of this enzyme to synthesize a DNA strand
complementary to an RNA template is central to the technique
called complementary DNA (cDNA) cloning

54. Figure 4.5 The reactions
catalyzed by DNA
polymerases. (a) The basic
reaction: a new DNA strand is
synthesized in the 5ʹ to 3ʹ
direction. (b) DNA polymerase
I, which initially fills in nicks
but then continues to
synthesize a new strand,
degrading the existing one as
it proceeds. (c) The Klenow
fragment, which only fills in
nicks. (d) Reverse
transcriptase, which uses a
template of RNA.

55. DNA modifying enzymes
There are numerous enzymes that modify DNA molecules by addition
or removal of specific chemical groups. The most important are as
follows:
Polynucleotide phosphorylase
DNAse
Alkaline phosphatase
Polynucleotide kinase
Terminal deoxy nucleotidyl
transferase Methylase
RNAse

56. Polynucleotide phosphorylase
• Polynucleotide Phosphorylase (PNPase) is a bifunctional enzyme
with a phosphorolytic 3' to 5' exoribonuclease activity and a 3'-
terminal oligonucleotide polymerase activity.
• PNPase is a bifunctional enzyme and functions in mRNA processing
and degradation inside the cell.
• Structural and physiochemical studies in enzymes showed that it is
formed of subunits. The arrangements of the subunits may vary
from species to species which would alter their properties.
• These enzyme can catalyze not only the synthesis of RNA from
the mixtures of naturally occurring ribonucleoside diphosphates,
but also that of non-naturally occurring polyribonucleotides

57. Mechanism of action
As mentioned earlier, polynucleotide phosphorylase is a bifunctional enzyme. The
mechanism of action of this enzyme can be represented by following reactions:
In E.coli, polynucleotide phosphorylase regulates mRNA processing
either by
adding ribonucleotides to the 3’ end or by cleaving bases in 3’ to 5’ direction.
The function of PNPase depends upon inorganic phosphate (Pi) concentration inside
the cell.
The transcripts are polyadenylated using enzyme polyadenylate polymerase I (PAPI).
After primary polyadenylylation of the transcript by PAP I, PNPase may bind to the 3ʹ
end of the poly(A) tail. PNPase works either degradatively or biosynthetically inside
the cell depending on the Pi concentration.
Under high Pi concentration, it degrades the poly(A) tail releasing adenine
diphosphates. If the Pi concentration is low, PAP I initiates addition of one or more
nucleotides to the existing poly (A) tail and in the process generates inorganic
phosphate. On dissociation of PNPase, the 3ʹ end again is available to PAP I for further
polymerization.

58. Schematic representation of the role of PNPase in poly(A) tail metabolism in E. coli

59. Functions of Polynucleotide phosphorylase
•It is involved in mRNA processing and degradation in bacteria, plants, and in
humans.
•It synthesizes long, highly heteropolymeric tails in vivo as well as accounts for all
of the observed residual polyadenylation in poly(A) polymerase I deficient
strains.
•PNPase function as a part of RNA degradosome in E.coli cell. RNA
degradosome is a multicomponent enzyme complex that includes RNaseE
(endoribinuclease), polynucleotide phosphorylase (3’ to 5’ exonuclease), RhlB
helicase (a DEAD box helicase) and a glycolytic enzyme enolase. This complex
catalyzes 3’ to 5’ exonuclease activity in presence of ATP. Degradsomes in bacteria
are associated with processing, control and turnover of RNA transcripts.
•In rDNA cloning technology, it has been used to synthesize radiolabelled
polyribonucleotides from nucleoside diphosphate monomers.

60. Deoxyribonuclease (DNase):
Deoxyribonuclease (DNase):
A nuclease enzyme that can catalyze the hydrolytic cleavage of phosphodiester bonds in the
DNA backbone are known as deoxyribonuclease (DNase).
Based on the position of action, these enzymes are broadly classified as
endodeoxyribonuclease (cleave DNA sequence internally) and exodeoxyribonuclease (cleave
the terminal nucleotides).
Unlike restriction enzymes, DNase does not have any specific recognition/restriction site and
cleave DNA sequence at random locations.
There is a wide variety of deoxyribonucleases known which have different substrate
specificities, chemical mechanisms, and biological functions. They are:
▪ DNAse I
▪ DNAse II
▪ Exonuclease III
▪ Mung Bean nuclease

61. Deoxyribonuclease I (DNaseI):
An endonuclease which cleaves double-stranded DNA or single stranded DNA.
The cleavage preferentially occurs adjacent to pyrimidine (C or T) residues. The major
products are 5'-phosphorylated bi-, tri- and tetranucleotides.
It requires divalent ions (Ca2+ and Mn2+/Mg2+) for its activity and creates blunt ends or 1-2
overhang sequences.
DNaseI is the most widely used enzyme in cloning experiments to remove DNA
contamination from mRNA preparation (to be used for cDNA library preparation, northern
hybridization, RT-PCR etc). The mode of action of DNaseI varies according to the divalent
cation used.
In the presence of magnesium ions (Mg+2), DNaseI hydrolyzes each strand of duplex DNA
producing single stranded nicks in the DNA backbone, generating various random cleavages.
On the other hand, in the presence of manganese ions (Mn+2), DNaseI cleaves both strands
of a double stranded DNA at approximately the same site, producing blunt ended DNA
fragments or with 1-2 base overhangs.
The two major DNases found in metazoans are: deoxyribonuclease I and deoxyribonuclease II

62. Some of the common applications of DNase I in rDNA technology have been mentioned
below:
• Eliminating DNA contamination (e.g. plasmid) from preparations of RNA.
• Analyzing the DNA-protein interactions via DNA footprinting.
• Nicking DNA prior to radio-labeling by nick translation.
Action of DNase I in the presence of
Mg+2 and Mn+2 ions. (Arrowhead
denoting random site of cleavage in
double stranded DNA by DNase I)

63. DeoxyribonucleaseII (DNaseII)
It is a non-specific endonuclease with optimal activity at acidic pH (4.5-5.5) and conserved from
human to C.elegans.
It does not require any divalent cation for its activity.
DNaseII initially introduces multiple single stranded nicks in DNA backbone and finally generates 3’
phosphate groups by hydrolyzing phosphodiester linkages.
This enzyme releases 3’phosphate groups by hydrolyzing phosphodiester linkage and creating nicks in
the DNA backbone.
DNaseII acts by generating multiple single stranded nicks followed by production of acid soluble
nucleotides and oligonucleotides.
The catalytic site of the enzyme contains three histidine residues which are essential for
enzyme activity.
Some of the common applications of DNase II are as follows:
• DNA fragmentation
• Molecular weight marker
• Cell apoptosis assays etc.

64. Exonuclease III
Exonuclease III is a globular enzyme which has 3’→5’ exonuclease activity in
a double stranded DNA.
The template DNA should be double stranded and the enzyme does not
cleave single stranded DNA. The enzyme shows optimal activity with blunt ended
sequences or sequences with 5’ overhang.
Exonuclease III enzyme has a bound divalent cation which is essential for enzyme
activity. The mechanism of the enzyme can be affected by variation in temperature,
monovalent ion concentration in the reaction buffer, and structure and
concentration of 3’termini. The enzyme shows optimal activity at 37°C at pH 8.0.
Various application of exonuclease III in molecular cloning experiments are:
• To generate template for DNA sequencing
• To generate substrate for DNA labeling experiments
• Directed mutagenesis
•DNA-protein interaction assays (to find blockage of exonuclease III activity by
protein-DNA binding) etc.

65. Mung bean nuclease
As the name suggest, this nuclease enzyme is isolated from mung bean sprouts (Vigna radiata).
Mung bean nuclease enzymes can degrade single stranded DNA as well RNA.
Under high enzyme concentration, they can degrade double stranded DNA, RNA or
even DNA/RNA hybrids.
Mung bean nuclease can cleave single stranded DNA or RNA to produce 5’-phosphoryl mono
and oligonucleotides.
It requires Zn2+ ion for its activity and shows optimal activity at 37°C.
The enzyme works in low salt concentration (25mM ammonium acetate) and acidic pH (pH
5.0).
Treatment with EDTA or SDS results in irreversible inactivation of the
enzyme. Mung bean nuclease is less robust than S1 nuclease and easier to
handle.
It has been used to create blunt end DNA by cleaving protruding ends from 5’ ends. This

66. Phosphatase
Phosphatase catalyses the cleavage of a phosphate (PO4-2
) group from substrate
by using a water molecule (hydrolytic cleavage).
This reaction is not reversible.
On the basis of their activity there are two types of phosphatase i.e acid
phosphatase and alkaline phosphatase.
In both forms the alkaline phosphatase are most common.
Special class of phosphatase that remove a phosphate group from protein, called
“Phosphoprotein phosphatase”.
Acid phosphatase:
It shows its optimal activity at pH between 3 and 6, e.g. a lysosomal enzyme that
hydrolyze organic phosphates liberating one or more phosphate groups. They are
found in prostatic epithelial cells, erythrocyte, prostatic tissue, spleen, kidney etc

67. Alkaline
phosphatase
Homodimeric enzyme which catalyzes reactions like hydrolysis and trans
phosphophorylation of phosphate monoester.
They show their optimal activity at pH of about 10.
Alkaline phosphatase was the first zinc enzyme discovered having three closed
spaced metal ion. Two Zn+2 ions and one Mg+2 ion, in which Zn+2 ions are bridged
by Asp 51.
In human body it is present in four isoforms, in which three are tissue specific
isoform i.e. placental, germ cell, intestinal and one is non tissue specific isoform.
During post-translational modification, alkaline phosphatase is modified by N-
glycosylation. It undergoes a modification through which uptake of two Zn+2 ion
and one Mg+2 ion occurs which is important in forming active site of that enzyme.

68. Types of Alkaline phosphatase
There are several AP that are used in gene manipulation-
Bacterial alkaline phosphatase (BAP) - Bacterial alkaline phosphatase is a phospho
monoester that hydrolyzes 3’ and 5’ phosphate from nucleic acid (DNA/ RNA).
It more suitably removes phosphate group before end labeling and remove
phosphate from vector prior to insert ligation.
BAP generally shows optimum activity at temperature 65°C.
BAP is sensitive to inorganic phosphate so in presence of inorganic phosphates
activity may reduce.
Calf intestinal alkaline phosphatase (CIP) – It is isolated from calf intestine,
which catalyzes the removal of phosphate group from 5’ end of DNA as well as
RNA.
This enzyme is highly used in gene cloning experiments, as to make a
construct that could not undergo self-ligation.
Hence after the treatment with CIP, without having a phosphate group at 5’ ends a
vector cannot self ligate and recircularise. This step improves the efficiency of vector
containing desired insert

69. Shrimp alkaline phosphatase (SAP) - Shrimp alkaline phosphatase is
highly specific, heat labile phosphatase enzyme isolated from arctic
shrimp (Pandalus borealis). It removes 5’ phosphate group from
DNA, RNA, dNTPs and proteins.
SAP has similar specificity as CIP but unlike CIP, it can be
irreversibly inactivated by heat treatment at 65°C for 15mins.
SAP is used for 5’ dephosphorylation during cloning experiments
for various application as follows:
Dephosphorylate 5’-phosphate group of DNA/RNA for subsequent
labeling of the ends.
To prevent self-ligation of the linearized plasmid.
To prepare PCR product for sequencing.
To inactivate remaining dNTPs from PCR product (for
downstream sequencing appication).

70. Methylase
Methyltransferase or methylase catalyzes the transfer of methyl group (-CH3) to
its substrate. The process of transfer of methyl group to its substrate is called
methylation
Methylation is a common phenomenon in DNA and protein structure.
Methyltransferase uses a reactive methyl group that is bound to sulfur in Sadenosyl
methionine (SAM) which acts as the methyl donor.
Methylation normally occurs on cytosine (C) residue in DNA sequence.
In protein, methylation occurs on nitrogen atom either on N-terminus or on the
side chain of protein.
DNA methylation regulates gene or silence gene without changing DNA sequences,
as a part of epigenetic regulation.
In bacterial system, methylation plays a major role in preventing their genome from
degradation by restriction enzymes. It is a part of restriction – modification system
in bacteria.

71. Polynucleotide Kinase
PNK has the reverse effect to alkaline phosphatase, adding phosphate groups onto
free 5ʹ termini.
The basic residues of active site of PNK interact with the negatively charged
phosphates of the DNA.
Polynucleotide kinase (PNK) catalyzes the transfer of a phosphate group (PO4-2)
from γ position of ATP to the 5' end of either DNA or RNA and nucleoside
monophosphate.
PNK can convert 3' PO4/5' OH ends into 3' PO4/5' PO4 ends which blocks further
ligation by ligase enzyme.
PNK is used to label the ends of DNA or RNA with radioactive phosphate group.
T4 polynucleotide kinase is the most widely used PNK in molecular cloning
experiments, which was isolated from T4 bacteriophage infected E.coli.

72. PNK carries out two types of enzymatic activity:
•Forward reaction: γ-phosphate is transferred from ATP to the 5' end of a
polynucleotide (DNA or RNA). 5’ phosphate is not present either due to chemical
synthesis or dephosphorylation.
•Exchange reaction: target DNA or RNA having a 5' phosphate is incubated with an
excess of ADP - where PNK transfers the phosphate from the nucleic acid to an ADP,
forming ATP. PNK then performs a forward reaction and transfer a phosphate from ATP to
the target nucleic acid. Exchange reaction is used to label with radioactive phosphate
group. The efficiency of phosphorylation is less in exchange reaction compared to
forward reaction. Along with the phosphorylating activity, PNK also has 3' phosphatase
activity.

73. Uses of PNK
•The linkers and adopters are phosphorylated along
with the fragments of DNA before ligation, which
requires a 5' phosphate. This includes products of
polymerase chain reaction, which are generated by
using non-phosphorylated primers.
•PNK is also used for radio labelling oligonucleotides,
generally with 32P for preparing hybridization probes.

74. Ribonuclease (RNase)
Nuclease that can catalyze hydrolysis of ribonucleotides
from either single stranded or double stranded RNA
sequence are called ribonucleotides (RNase).
•RNase are classified into two types depending on position of
cleavage, i.e.endoribonuclease (cleave internal bond) and
exoribonuclease (cleave terminal bond).
• RNase is important for RNA maturation and processing.
•RNaseA and RNaseH play important role in initial defence
mechanism against RNA viral infection.

75. Types of RNAse
RibonucleaseA (RNaseA):
An endo-ribonuclease that cleaves specifically single-stranded RNA at the 3' end
of pyrimidine residues.
The RNA is degraded into 3'-phosphorylated mononucleotides C and U residues and
oligonucleotides in the form of 2', 3'-cyclic monophosphate intermediates.
Optimal temperature for RNaseA is 60˚C (activity range 15-70˚C) and optimal pH is 7.6
It is used to remove RNA contamination from DNA sample
RibonucleaseH:
Non-specific endoribonuclease that degrades RNA by hydrolytic mechanism from DNA/RNA
duplex resulting in single stranded DNA.
Enzyme bound divalent metal ion is a cofactor here. The product formed is 5’
phosphorylated ssDNA.
During cDNA library preparation from RNA sample, RNaseH enzyme is used to cleave RNA
strand of DNA-RNA duplex.