This document discusses the key enzymes involved in recombinant DNA technology. It describes how restriction enzymes cut DNA at specific recognition sites, and DNA ligases join cut DNA fragments back together. The document outlines the process of recombinant DNA technology, including generating DNA fragments, inserting them into cloning vectors, introducing the vectors into host cells, and expressing the gene of interest. It provides details on various restriction enzymes and DNA-modifying enzymes used in genetic engineering applications.

1. RECOMBINANT DNA TECHNOLOGY
PART -II
ENZYMES INVOLVED
Mrs. POONAM NIKAM
ASSISTANT PROFESSOR

2. Genetic engineering primarily involves the manipulation of
genetic material (DNA) to achieve the desired goal in a pre-
determined way.
Some other terms are also in common use to describe genetic
engineering.
 Gene manipulation
 Recombinant DNA (rDNA) technology
 Gene cloning (molecular cloning)
 Genetic modifications
 New genetics.
RECOMBINANT DNA TECHNOLOGY

3. BRIEF HISTORY OF RECOMBINANT DNA TECHNOLOGY
The present day DNA technology has its roots in the experiments performed by
Herbert Boyer and Stanely Cohen in 1973.
 Successfully recombined two plasmids (pSC 101 and pSC 102) and cloned the
new plasmid in E.coli.

4. The second set of experiments of Boyer and Cohen were more
organized. This made the real beginning of modern rDNA technology
and laid foundations for the present day molecular biotechnology.

5. An outline of recombinant DNA technology
1. Generation of DNA fragments and
selection of the desired piece of DNA
(e.g. a human gene).
2. Insertion of the selected DNA into a
cloning vector (e.g. a plasmid) to
create a recombinant DNA or chimeric
DNA.
3. Introduction of the recombinant
vectors into host cells (e.g. bacteria).
4. Multiplication and selection of clones
containing the recombinant molecules.
5. Expression of the gene to produce the
desired product.

6. An engineer is a person who designs, constructs (e.g. bridges, canals, railways)
and manipulates according to a set plan.
The term genetic engineer may be appropriate for an individual who is
involved in genetic manipulations. The genetic engineer’s toolkit or molecular
tools namely the enzymes most commonly used in recombinant DNA
experiments are:
 RESTRICTION ENDONUCLEASES— DNA CUTTING ENZYMES
 DNA LIGASES — DNA JOINING ENZYMES
 ALKALINE PHOSPHATASE
 DNA MODIFYING ENZYMES
 Nucleases
Endonucleases
Exonucleases
 Polymerases
 Polynucleotide kinase

7. Restriction Enzyme (Restriction Endonuclease)
Restriction endonuclease, is a protein produced by bacteria that cleaves
DNA at specific sites along the molecule.
Restriction endonucleases cut the DNA double helix in very precise ways.
It cleaves DNA into fragments at or near specific recognition sites within
the molecule known as restriction sites.
They have the capacity to recognize specific base sequences on DNA and
then to cut each strand at a given place. Hence, they are also called as
‘molecular scissors’.

8. Source of Restriction Enzymes
• The natural source of restriction endonucleases are bacterial cells.
• These enzymes are called restriction enzymes because they
restrict infection of bacteria by certain viruses (i.e.,
bacteriophages), by degrading the viral DNA without affecting the
bacterial DNA. Thus, their function in the bacterial cell is to
destroy foreign DNA that might enter the cell.
• The restriction enzyme recognizes the foreign DNA and cuts it at
several sites along the molecule.
• Each bacterium has its own unique restriction enzymes and each
enzyme recognizes only one type of sequence.

9. Recognition Sites
The DNA sequences recognized by restriction enzymes are called
palindromes. Palindromes are the base sequences that read the same
on the two strands but in opposite directions.
• The value of restriction enzymes is that they make cuts in the DNA
molecule around this point of symmetry.
• Some enzymes cut straight across the molecule at the symmetrical
axis producing blunt ends.
• Of more value, however, are the restriction enzymes that cut
between the same two bases away from the point of symmetry on
two strands, thus, producing a staggering break.

11. Mechanism of Cleavage of Restriction Enzymes
• When a restriction endonuclease recognizes a particular
sequence, it snips through the DNA molecule by catalyzing the
hydrolysis (splitting of a chemical bond by addition of a water
molecule) of the bond between adjacent nucleotides.
• To cut DNA, all restriction enzymes make two incisions, once
through each sugar-phosphate backbone (i.e. each strand) of
the DNA double helix

12. Types of Restriction Enzymes
Traditionally, four types of restriction enzymes are recognized,
designated I, II, III, and IV, which differ primarily in structure, cleavage
site, specificity, and cofactors.
1.Type I enzymes cleave at sites remote from a recognition site;
require both ATP and S-adenosyl-L-methionine to function;
multifunctional protein with both restriction and methylase activities.
2.Type II enzymes cleave within or at short specific distances from a
recognition site; most require magnesium; single function
(restriction) enzymes independent of methylase.
3.Type III enzymes cleave at sites a short distance from a recognition
site; require ATP (but do not hydrolyze it); S-adenosyl-L-methionine
stimulates the reaction but is not required; it exists as part of a
complex with a modification methylase.
4.Type IV enzymes target modified DNA, e.g. methylated,
hydroxymethylated and glucosyl-hydroxymethylated DNA.

13. Nomenclature of Restriction Enzymes
Since their discovery in the 1970s, many restriction enzymes have
been identified while Type II restriction enzymes have been
characterized.
Each enzyme is named after the bacterium from which it was
isolated, using a naming system based on bacterial genus, species
and strain. For example, the name of the EcoRI restriction enzyme
was derived as:
E – Escherichia: Genus
co- coli: specific species
R- RY13: strain
I- First identified: order of identification in the bacterium

14. Applications of Restriction Enzymes
 Restriction enzymes can be isolated from bacterial cells and used
in the laboratory to manipulate fragments of DNA, such as those
that contain genes; for this reason, they are indispensable tools of
recombinant DNA technology (genetic engineering).
 The most useful aspect of restriction enzymes is that each enzyme
recognizes the same unique base sequence regardless of the
source of the DNA. It means that these enzymes establish fixed
landmarks along an otherwise very regular DNA molecule. This
allows dividing a long DNA molecule into fragments that can be
separated from each other by size with the technique of gel
electrophoresis.
 Each fragment, thus generated, are also available for further
analysis, including the sequencing.
 One value of cutting DNA molecule up into discrete fragments is
being able to locate a particular gene on the fragment where it
resides which is done by the general technique of Southern
blotting

15. • One of the most popular restriction enzymes is called EcoRI
from E. coli (bacterium).
• Hundreds of other restriction enzymes with different sequence
specificities have been isolated from several bacteria and are
commercially available.

16. DNA LIGASES — DNA JOINING ENZYMES
• The cut DNA fragments are covalently joined together by DNA
ligases. These enzymes were originally isolated from viruses. They
also occur in E.coli and eukaryotic cells. DNA ligases actively
participated in cellular DNA repair process.
• DNA ligase joins (seals) the DNA fragments by forming a
phosphodiester bond between the phosphate group of 5’-carbon of
one deoxyribose with the hydroxyl group 3’-carbon of another
deoxyribose.
• Phage T4 DNA ligase requires ATP as a cofactor while E.coli DNA
ligase is dependent on NAD*. In each case, the cofactor (ATP or
NAD*) is split to form an enzyme—AMP complex that brings about
the formation of phosphodiester bond. The action of DNA ligase is
the ultimate step in the formation of a recombinant DNA molecule.

18. • The complementary DNA strands
can be joined together by
annealing. This principle is utilized
in homopolymer tailing.
• The technique involves the
addition of oligo (dA) to 3’-ends
of some DNA molecules and the
addition of oligo (dT) to 3’-ends
of other molecules.
• The homopolymer extensions (by
adding 10-40 residues) can be
synthesized by using terminal
deoxy- nucleotidyltransferase (of
calf thymus).
Homopolymer Tailing

19. Linkers and adaptors
Linkers and adaptors are chemically
synthesized, short, double-stranded DNA
molecules.
Linkers possess restriction enzyme cleavage
sites. They can be ligated to blunt ends of
any DNA molecule and cut with specific
restriction enzymes to produce DNA
fragments with sticky ends.
Adaptors contain preformed sticky or
cohesive ends. They are useful to be ligated
to DNA fragments with blunt ends. The DNA
fragments held to linkers or adaptors are
finally ligated to vector DNA molecules

20. ALKALINE PHOSPHATASE
• Alkaline phosphatase is an enzyme
involved in the removal of phosphate
groups. This enzyme is useful to
prevent the unwanted ligation of DNA
molecules which is a frequent problem
encountered in cloning experiments.
• When the linear vector plasmid DNA is
treated with alkaline phosphatase, the
5’-terminal phosphate is removed.
• This prevents both recircularization
and plasmid DNA dimer formation. It is
now possible to insert the foreign DNA
through the participation of DNA
ligase.

21. DNA MODIFYING ENZYMES
These enzymes represent the cutting and joining functions in DNA
manipulation. They are broadly categorized as nucleases,
polymerases and enzymes modifying ends of DNA molecules.
Nucleases: The enzymes that break the phosphodiester bonds (that
hold nucleotides together) of DNA.
Endonucleases act on the internal phosphodiester bonds while
exonucleases degrade DNA from the terminal ends
• Endonucleasese :
Nuclease S1, specifically acts on single-stranded DNA or RNA molecules.
Deoxyribonuclease 1 (DNase 1) cuts either single or double-stranded
DNA molecules at random sites.
• Exonucleases:
ExonucleaseIII cuts DNA and generates molecules with protruding 5’-
ends.
Nuclease Bal 31 is a fast acting 3’-exonuclease. its action is usually
coupled with slow acting endonucleases.

29. • Genetic engineering has applications in medicine, research, industry and
agriculture and can be used on a wide range of plants, animals and
microorganisms.
• In medicine, genetic engineering has been used to mass-produce insulin,
human growth hormones, follistim (for treating infertility), human
albumin, monoclonal antibodies, antihemophilic factors, vaccines, and
many other drugs.
• In research, organisms are genetically engineered to discover the functions
of certain genes.
• Industrial applications include transforming microorganisms such as
bacteria or yeast, or insect mammalian cells with a gene coding for a useful
protein. Mass quantities of the protein can be produced by growing the
transformed organism in bioreactors using fermentation, then purifying
the protein.
• Genetic engineering is also used in agriculture to create genetically-
modified crops or genetically-modified organisms.