DNA manipulative enzymes can be grouped into four classes: nucleases cut DNA, ligases join DNA, polymerases copy DNA, and modifying enzymes add or remove chemical groups from DNA. Restriction endonucleases are nucleases that cut DNA at specific recognition sequences and are important for gene cloning. They produce either blunt or sticky ends. DNA ligase joins DNA fragments together by forming phosphodiester bonds. Polymerases like Klenow fragment and Taq polymerase copy DNA. Modifying enzymes alter DNA through addition or removal of chemical groups from the DNA backbone. Together, these enzymes enable the precise cutting and joining of DNA required for gene cloning experiments.

1. RDT UNIT 1
Enzymes Used in Gene Manipulation
 Once pure samples of DNA have been prepared, the next step in a gene cloning experiment
is construction of the recombinant DNA molecule.
 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.
 DNA manipulative enzymes
 DNA manipulative enzymes can be grouped into four broad classes, depending on the type
of reaction that they catalyze:
1. Nucleases are enzymes that cut, shorten, or degrade nucleic acid molecules.
2. Ligases join nucleic acid molecules together.
3. Polymerases make copies of molecules.
4. Modifying enzymes remove or add chemical groups.
1. 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:
 Exonucleases: remove nucleotides one at a time from the end of a DNA molecule.
 Endonucleases: these 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.
For e.g. the enzyme called Bal31 (purified from the bacterium Alteromonas espejiana)
removes nucleotides from both strands of a double-stranded molecule. In contrast,
enzymes such as E. coli exonuclease III degrade just one strand of a double-stranded
molecule, leaving single stranded DNA.
 On the other hand, the special group of enzymes called restriction endonucleases cleaves
double stranded DNA only at a limited number of specific recognition sites.

2. RDT UNIT 1
☻ Restriction endonucleases
 Gene cloning requires that DNA molecules be cut in a very precise and reproducible fashion.
 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. 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. There are two reasons for
this:
a) 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 DNA molecules,
b) 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.
 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.
 Purified restriction endonucleases allow the molecular biologist to cut DNA molecules in
the precise, reproducible manner required for gene cloning.
 The bacterium produces restriction endonuclease that degrades the phage DNA before it
has time to replicate and direct synthesis of new phage particles. The bacterium’s own
DNA is protected from attack because it carries additional methyl groups that block the
degradative enzyme action.
 These degradative restriction endonucleases 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.
 Three different classes of restriction endonuclease are recognized, each
distinguished by a slightly different mode of action. Types I and III are rather complex

3. RDT UNIT 1
and have only a limited role in genetic engineering. Type II restriction endonucleases, on
the other hand, are the cutting enzymes that are extensively used in gene cloning.
☻ Nomenclature of restriction endonucleases
 A suitable nomenclature system was proposed by Smith and Nathans (1973) and a simplified
version of this is in use today. The key features are:
 The nuclease is named by the first letter of the genus name and the first two letters of
the species name of the host organism, to generate a three letter abbreviation.
 This abbreviation is always written in italics. Where a particular strain has been the
source then this is identified.
 When a particular host strain has several different R-M systems, these are identified by
roman numerals. Where it is necessary to distinguish between the restriction and
methylating activities, they are given the prefixes ‘R’ and ‘M’, respectively, e.g. R.SmaI
and M.SmaI.

4. RDT UNIT 1
☻ Recognition sites for Type II restriction endonucleases
Most type II restriction endonucleases recognize and cleave DNA within particular sequences of
four to eight nucleotides (sometimes longer) which have a twofold axis of rotational symmetry.
 Such sequences are often referred to as palindromes because of their similarity to words
that read the same backwards as forwards. For example, the restriction and modification
enzymes. EcoRI recognizes the sequence: 5′-GAA T TC-3′
3′-CT T AAG-5′
Isoschizomers: Restriction enzymes with the same sequence specificity and cut site are known
as isoschizomers.
Neoschizomers: Enzymes that recognize the same sequence but cleave at different points are
known as neoschizomers, for example SmaI (CCC/GGG) and XmaI C/CCGGG).
☻ Axis of symmetry
 The position at which the restricting enzyme cuts is usually shown by the symbol ‘/’ and
the nucleotides methylated by the modification enzyme are usually marked with an
asterisk. For EcoRI these would be represented thus:
5′-G/AA* T T C-3′
3′-C TT A*A/G-5′
 For convenience recognition sequences are usually simplified by showing only one strand
of DNA, the one which runs in the 5′ to 3′direction. Thus the EcoRI recognition sequence
would be shown as G/AATTC.
 EcoRI makes single-stranded breaks four bases apart in the opposite strands of its target
sequence so generating fragments with protruding 5′ termini:
5′-G 5′-AATTC-3′
3′-CTTAA-5′ G-5′
 Some 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.

5. RDT UNIT 1
☻ Blunt ends and sticky ends
The exact nature of the cut produced by a restriction endonuclease is of considerable
importance in the design of a gene cloning experiment.
Blunt ends: Many restriction endonucleases make a simple double-stranded cut in the
middle of the recognition sequence, resulting in a blunt end or flush end. PvuII and AluI
are examples of blunt end cutters.
Sticky ends: With other restriction endonucleases 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. 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. 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.

6. RDT UNIT 1
☻ The frequency of recognition sequences in a DNA Molecule
 The number and size of the fragments generated by a restriction enzyme depend on the
frequency of occurrence of the target site in the DNA to be cut.
 The number of recognition sequences for a particular restriction endonuclease in a DNA
molecule of known length can be calculated mathematically assuming that the
nucleotides are ordered in a random fashion and that the four different nucleotides are
present in equal proportions (i.e., the GC content =50%).
 Assuming a DNA molecule with a 50% GC content and a random distribution of the four
bases, a tetranucleotide sequence (e.g., GATC) should occur once every 44
=256
nucleotides and a hexanucleotide (e.g., GGATCC) once every 46
= 4096 nucleotides.
 In practice, neither of these assumptions is entirely valid. For example, the λ DNA
molecule, at 49 kb, should contain about 12 sites for a restriction endonuclease with a
hexanucleotide recognition sequence. In fact, many of these recognition sites occur less
frequently (e.g., six for BglII, five for BamHI, and only two for SalI), a reflection of the
fact that the GC content for λ is rather less than 50 %.)
 Furthermore, restriction sites are generally not evenly spaced out along a DNA molecule.
If they were, then digestion with a particular restriction endonuclease would give
fragments of roughly equal sizes.
 Certain restriction endonucleases show preferential cleavage of some sites in the same
DNA molecule. For example, phage λ DNA has five sites for EcoRI but the different
sites are cleaved nonrandomly. The site nearest the right terminus is cleaved 10 times
faster than the sites in the middle of the molecule.
 Mathematical calculations give an idea of how many restriction sites to expect in a given
DNA molecule, only experimental analysis can provide the true picture. We must
therefore move on to consider how restriction endonucleases are used in the laboratory.

7. RDT UNIT 1
2. Ligases
Ligation- The final step in construction of a recombinant DNA molecule is the joining together
of the vector molecule and the DNA to be cloned. This process is referred to as ligation, and the
enzyme that catalyzes the reaction is called DNA ligase.
The mode of action of DNA ligase
 DNA ligase fills discontinuities in a DNA strand by formation of phosphodiester bond
between 3’-OH (acceptor) one nucleotide and 5’-P (donor) of other nucleotide. These
enzymes require ATP or NAD. Ligation involves three steps:
1. Adenylation- transfer of AMP to active site of enzyme resulting in formation of enzyme-
AM P complex.

8. RDT UNIT 1
2. Transfer of AMP to 5’-P of donor.
3. Formation of phosphodiester bond between 5’-P of donor and 3’-OH of acceptor.
 A discontinuity is a position where a phosphodiester bond between adjacent nucleotides
is missing (contrast this with a nick, where one or more nucleotides are absent).
 Discontinuities may arise by chance breakage of the cell’s DNA molecules or are a
natural result of processes such as DNA replication and recombination.
 Individual DNA molecules or the two ends of the same molecule can be joined together
in vitro by purified DNA ligases.
 The chemical reaction involved in ligating two molecules is exactly the same as
discontinuity repair, except that two phosphodiester bonds must be made, one for each
strand.
 The DNA ligase used in genetic engineering is usually purified from E. coli bacteria that
have been infected with T4 phage.
 E. coli DNA ligase requires NAD and T4 DNA ligase requires ATP.

9. RDT UNIT 1
Figure a : Ligation Figure b : The two reactions catalyzed by DNA ligase

10. RDT UNIT 1
Figure: Using DNA ligase to create a DNA recombinant by joining ends created by EcoRI
3. Polymerases
 DNA polymerases are enzymes that synthesize a new strand of DNA complementary to
an existing DNA or RNA template.
 Most polymerases can function only if the template possesses a double-stranded region
that acts as a primer for initiation of polymerization.

11. RDT UNIT 1
 Four types of DNA polymerase are used routinely in genetic engineering:
DNA polymerase I, Klenow fragment, Taq DNA polymerase and reverse transcriptase
A) DNA polymerase I:
 It 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.
 DNA polymerase I is an example of an enzyme with a dual activity—DNA
polymerization and DNA degradation.
B) The Klenow fragment:
 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. 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 has polymerase activity
but lacks nuclease activity, therefore it fills the nick but cannot continue the
synthesis once the nick is filled in.
 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.
C) Taq DNA polymerase:
 It is used in the polymerase chain reaction (PCR) and 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.
 Thermostability of Taq DNA polymerase 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.

12. RDT UNIT 1
D) Reverse transcriptase:
 This enzyme is involved in the replication of several kinds of virus. Reverse
transcriptase is unique in that it uses as a template not DNA but RNA.
 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.
Figure: Different types of DNA polymerases used in genetic engineering

13. RDT UNIT 1
4. 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:
a) Alkaline phosphatase (from E. coli, calf intestinal tissue, or arctic shrimp): it removes
the phosphate group present at the 5′ terminus of a DNA molecule.
b) Polynucleotide kinase (from E. coli infected with T4 phage): it has the reverse effect to
alkaline phosphatase, adding phosphate groups onto free 5′termini.
c) Terminal deoxynucleotidyl transferase (from calf thymus tissue): it adds one or more
deoxyribonucleotides onto the 3′terminus of a DNA molecule.