MBGE- Introduction to genetic engineering.pdf

MBGE L 3.
INTRODUCTION TO
GENETIC
ENGINEERING

Dr. Amulya. G, The Oxford College of Engineering
Objectives & Outline
✓Basics of Genetic Engineering
✓Vectors for gene cloning:
✓Cloning and Expression vectors. Plasmids, Phages, Cosmids, Fosmids,
Phagemids, and Artificial chromosomes.
✓Viral vectors.
✓Molecular tools for gene cloning:
✓Restriction and Modification systems:
✓RestrictionEndonucleases,Methylases,Ligases.Polynucleotide
kinases,Phosphatases,DNAandRNApolymerases,Reverse
transcriptase,Terminaltransferase,DNAses(Extremophiles),
Nuclease,RNases,Topoisomerase.
✓Cloning Techniques: Restriction digestion based cloning.
✓Linkers and adapters, Strategies for cloning TA cloning.
✓Ligase free cloning.

Dr. Amulya. G, The Oxford College of Engineering
What genetic engineering is?
◼It is a technique used for artificially and deliberately modifying DNA or
Gene.
◼The study of gene manipulation through DNA, accomplished by the
processes called recombination and transformation.
◼It involves modification of genetic information of an organism by directly
changing its genome.
◼Also referred as “recombinant DNA technology” or “Gene Splicing” or
“Gene manipulation” or “Gene cloning” or “Genetic modifications”
◼In order to accomplish transfer of character of one organism to the
other.

Dr. Amulya. G, The Oxford College of Engineering
History- how it came into existance?
➢1886- Gregor Johann Mendel- “Father of Genetics” proposed that hereditary
is controlled by a paired germinal units called ‘Factors’, now called
GENES.- FIRST TO MANIPULATE ORGANISM (PISUM SATIVUM)
➢Mendel- Idea of transmission of traits.
➢1902- Sir Archibald Garrod- “Father of biochemical genetics” and “father of
human genetics”, discovered alkaptonuria- caused by a ressesive inherited
gene.- FIRST STUDY OF INBORN ERROR OF METABOLISM AND
BIOCHEMICAL GENETICS.
➢1973- Stanley Cohen (S.U/ U.S.A) and Herbert Boyer (C.U/ U.S.A) obtained
a rDNA by combining a foreign DNA containing a gene from bacterium with
plasmid of E.coli, using RE (restriction endonuclease). – FIRST GENE
TRANSFER. Fredrick Griffith- proposed transformation.

Dr. Amulya. G, The Oxford College of Engineering
EMERGENCE OF THE CONCEPT OF GE :
➢Two very significant discoveries made in bacterial research :
I. Presence of extra chromosomal DNA fragments called
‘plasmid’ in bacterial cell, replicate along with
chromosomal DNA of the bacterium.
II.Presence of ‘enzymes restriction’ nucleases that cut
DNA at specific sites also called ‘Molecular Scissors’.

Dr. Amulya. G, The Oxford College of Engineering
Purpose of Genetic Engineering:
◼For future improvement in medical activity.
◼To identify the genes that cause diseases, and provide enhanced RBCs,
WBCs and other defenses to fight disease.
◼Cure of Genetic diseases.
◼To make use of hormones for making medicines,
e.g. Creation of human insulin, HGH, Vaccines for hepatitis B.
◼To obtain disease resistant and more productive plants.
◼To get GM cell lines,GMOs and GMPs.
◼Reduce the threat to Global Warming.
◼To increase genetic diversity.
◼To save the existence of the species ( cloning).

Dr. Amulya. G, The Oxford College of Engineering
Uses of Genetic engineering:
◼To understand the molecular events in biological processes.
◼To manufacture compounds of pharmaceutical importance.
◼To produce genetically modified organisms (GMOs) or transgenic
organisms.
◼For cutting genes responsible for hereditary diseases and replace them
with normal genes (gene therapy).

Dr. Amulya. G, The Oxford College of Engineering
Process of Genetic Engineering involves:
1.Isolation of desired segments of DNA or gene of interest from the
source.
2.Inserting of the gene of interest to vector to produce rDNA.
3.Introduction of the insert into the host cell.

Dr. Amulya. G, The Oxford College of Engineering
Biological tools of Genetic Engineering:
1.Enzymes: different enzymes used to carry out different functions.
Important enzymes are :
a) Lysing enzyme- for opening the cells to get DNA, e.g. Lyzozyme.
b)Cleaving enzyme- for cleaving the DNA molecules. Three types of
cleaving enzymes are involved:
▪Exonucleases- cut off nucleotides from5’ or 3’ ends of DNA.
▪Endonucleases- Break DNA duplex at specific point except the
end.
▪Restriction endonucleases- cleave DNA duplex at specific sites
to get short single stranded free ends. E.g. ECOR-I recognizes
the base sequence GAATTC/CTTAAG in DNA and cleaves
between G and A.
c) Synthesizing enzyme- for synthesizing new strands,
complimentary to existing RNA or DNA. Two types :
▪ Reverse Transcriptase- synthesis of DNA strand on RNA
template.
▪DNA polymerases- synthesis of complimentary DNA strands on
DNA template.

Dr. Amulya. G, The Oxford College of Engineering
1.Enzymes :
d) Ligating enzyme- for joining the DNA fragment, also called ‘molecular
glues’. E.g. DNA ligase from E.coli used to ligate DNA fragment .
e) Phosphatases- Cut off phosphate group from 5’ end of linearised
circular DNA. E.g.- alkaline phosphatases.
2.Vehicle DNA or Vector DNA: carrier for transferring the insert into
suitable host. Types of vector-
a) Plasmids b) Bacteriophages c) Plant and Animal viruses
d) Transposons or Jumping genes e) Cosmids f) BACs g) YACs
3. Passenger DNA: DNA which is transferred from one organism into
another by combining with the vector DNA. E.g. cDNA, sDNA etc.

Dr. Amulya. G, The Oxford College of Engineering
Plasmid pUC19 :

Dr. Amulya. G, The Oxford College of Engineering
Technique of Genetic Engineering :
◼Cloning- producing similar population of genetically identical
individuals.
◼Gene transfer or gene therapy-use of genes to treat or prevent
disease by replacing or knocking out a mutated gene.
◼Hybrid production- producing new organisms with improved traits.

Dr. Amulya. G, The Oxford College of Engineering
Major processes :
1.Fragmentation
2.PCR
3.Restriction digestion
4.Ligation
5.Transformation
6.Plasmid isolation
7.Screening
1.FRAGMENTATION :
➢Suitable source for isolating the DNA containing gene of interest is choosed.
➢Gene of interest is extracted by lysing the cell wall using lyzozyme enzyme.
➢DNA is fragmented- breaking apart DNA strand into SS stranded free end
(sticky or blunt end) using restriction endonucleases.
➢Free ends can ligate with their complimentary DNA fragment from some
other source.
➢Selected DNA is isolated by ultra centrifugation.

Dr. Amulya. G, The Oxford College of Engineering
2. Polymerase chain reaction:
➢To amplify a single piece of DNA generating thousands to millions of
copies a particular DNA up to 10 kbps.
➢Relies on thermal cycling, consisting of cycles of repeated heating and
cooling reaction for DNA melting and enzymatic replication of DNA.
➢Primers are containing sequences complimentary to the targeted region
is selected for amplification.
➢Heat stable enzyme is used mostly Taq polymerase (thermus aquatics).
➢Used in cloning for sequencing.
➢Used in identification of genetic fingerprints.

Dr. Amulya. G, The Oxford College of Engineering
Illustration of PCR:

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
3.Restriction Digestion:
➢Recognizes DNA at specific recognition nucleotide sequences, i.e.
recognition sites.
➢Cleaves at specific site which is 4 to 8 nucleotides long.
➢Produces double stranded cut in DNA.
➢Found in bacteria and archaea.
➢Provide defense against invading viruses.
➢Used for DNA modification and DNA manipulation.
➢Used to assist insertion of genes into plasmid vectors during cloning and
protein expression.
➢Used to digest genomic DNA.
➢Used to distinguish alleles.

Dr. Amulya. G, The Oxford College of Engineering
Restriction enzymes :

Dr. Amulya. G, The Oxford College of Engineering
How RE works

Dr. Amulya. G, The Oxford College of Engineering
4.Ligation
➢Linking of two ends of DNA molecules using DNA ligase or RNA
molecules with RNA ligase.
➢Three things needed for ligation:
•DNA fragments
•Buffer which contains ATP
•T4 DNA ligase
➢For DNA ligation- T4 DNA ligase, originates from T4 bacteriophage,
ligates DNA fragments overhanging and blunts ends.

Dr. Amulya. G, The Oxford College of Engineering
Ligation :

Dr. Amulya. G, The Oxford College of Engineering
5.Transformation:
➢Transfer of genetic material to another organism (host).
➢Transfer of insert into competent cell so that it can be replicated.
➢Incorporation of DNA into bacterium genome.
➢Competent cell- cell that is capable of taking up DNA.
➢Bacterial strains- DH5-alpha, XL-1 blue, JM 109 etc.
➢Two ways-
•Electrophoration- electric charge.
•Heat shock- 42 ˚C for 65 seconds.

Dr. Amulya. G, The Oxford College of Engineering
Transformation

Dr. Amulya. G, The Oxford College of Engineering
6.Plasmid isolation
➢Separation of plasmid from the genomic DNA.
➢Based on size of DNA and lysis method- alkaline lysis.
➢Two steps
•SDS- denatures plasmid DNA, should be quick.
•Potassium acetate- to renature the plasmid.
➢Sample is then centrifuged.

Dr. Amulya. G, The Oxford College of Engineering
7.Blue and White Screening:

Dr. Amulya. G, The Oxford College of Engineering
Blue and White Screening:

Dr. Amulya. G, The Oxford College of Engineering
Summarization

Dr. Amulya. G, The Oxford College of Engineering
APPLICATIONS OF GENETIC ENGINEERING
1.Therapeutic Applications of GE
◼Subunit vaccines
◼Nonpathogenic viruses carrying genes for pathogen's antigens as
vaccines
◼Gene therapy to replace defective or missing genes
◼Human Genome Project
– Nucleotides have been sequenced
– Human Proteome Project may provide diagnostics and treatments
2.Pharmaceutical applications of GE

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
◼ Purify protein
– Insulin
– Growth factor
– Interferon
◼Generate more copies of a particular
gene: “amplify DNA”
◼ Research gene function and regulation
◼GMO- genetically modified organism.
◼GMO free food-product in which no transgenic materials were used in
its manufacture, such as soybeans used in making oils.
◼GEO-genetically enhanced organism
◼Gene therapy

Dr. Amulya. G, The Oxford College of Engineering
Vectors in rDNA technology& its salient features
Vectors can be defined in three ways depending on the function/action:
➢In epidemiology:
◼An organism or vehicle that transmits the causative agent or disease-
causing organism from the reservoir to the host.
➢In molecular biology/biotechnology:
◼A vehicle (e.g. a plasmid) used to transfer the genetic material such as
DNA sequences from the donor organism to the target cell of the recipient
organism.
➢In biological science
◼A biotic agent that disperses reproductive structures of another organism,
as a bee transmitting pollen to the stigma of a flower.

Dr. Amulya. G, The Oxford College of Engineering
Vectors
◼“A vector is a substance, usually a piece of DNA that carries a sequence of
DNA or other genetic material and introduces it into a new cell”.
◼Vectors act as vehicles to transfer genetic material from one cell to the
other for different purposes like multiplying, expressing, or isolation.
◼Vectors are used as a tool in molecular cloning procedures so as to
introduce the desired DNA insert into a host cell.
◼The DNA insert that is transmitted by a vector is termed recombinant DNA,
and the process is also known as recombinant DNA technology.
◼Usually, the vectors are DNA sequences that carry different parts involved
in different functions. Vectors usually have an insert, also known as a
transgene, that carries the recombinant DNA and a larger sequence called
the backbone of the vector responsible for the structure of the vector.

Dr. Amulya. G, The Oxford College of Engineering
◼Vectors have particular features that carry the gene sequences and
enable them to survive within the host cell.
◼The process of gene transfer also differs in different vectors where some
enter the host cell and get incorporated into the host DNA, whereas the
others just pass the genetic material into the host cell and recover
themselves.
◼Even though vectors are usually DNA sequences, viruses and other
particles can also function as vectors in processes like transduction.
◼Vectors can be reused for multiple processes as these can be recovered
at the end of the process.
◼A cloning vector is a category of vectors that are essential for cloning
procedures. These vectors have different sequences that enable them to
initiate replication in host cells as well as propagate within the host.

Dr. Amulya. G, The Oxford College of Engineering
Salient features of Vector
Origin of
replication
for replication and maintenance of vector in
host cell.
Promoter
•to drive transcription of vector's transgene
•Also to drive transcription of other genes in vector such
as the antibiotic resistance gene
Cloning
site
•allow for the insertion of foreign DNA into the
vector through ligation
Reporter
genes
that allow for identification of plasmid that
contains inserted DNA sequence.

Dr. Amulya. G, The Oxford College of Engineering
Targeting
sequence
Protein
purification tags
Antibiotic
resistance
Epitope
Genetic marker
that directs the expressed protein to a specific
organelle in the cell or specific location
Some expression vectors include proteins or peptide
sequences that allows for easier purification of
expressed protein.
allow for survival of cells that have taken up the
vector in growth media containing antibiotics
through antibiotic selection.
allows for antibody identification of cells
expressing the target protein.
allow for confirmation that the vector has
integrated with the host genomic DNA.

Dr. Amulya. G, The Oxford College of Engineering
Diagrammatic representation of vector

Dr. Amulya. G, The Oxford College of Engineering
◼Refer this diagram

Dr. Amulya. G, The Oxford College of Engineering
Vectors perform function in two ways;
1.Transcription (cloning)
2.Expression
there are two types of expression vector:
a)Prokaryotes expression vector:
◼Promoter
◼Ribosome Binding Site (RBS)
◼Translation initiation site –
b) Eukaryotes expression vector:
These require sequences that encode for:
◼Polyadenylation tail:
◼Minimal UTR length:
◼Kozak sequence:
Mode of action

Dr. Amulya. G, The Oxford College of Engineering
Characteristics or Features of vectors
◼Vectors should be capable of replicating autonomously, which, in turn, depends on the
presence of particular sequences in the vector that enables them to initiate replication and
propagation within the host cell.
◼Size of an ideal vector should be small enough for it to be incorporated into the host genome.
◼Vectors should be easy to isolate and purify as these need to be recovered and reused for
multiple processes.
◼Vector should have certain components that facilitate the process of determining whether the
host cell has received a marker genes.
◼Many vectors also require unique restriction enzyme recognition sites that enable the insertion
of the vector DNA in the presence of specific restriction enzymes
◼The introduction of vectors into the host cell should be easy.
◼The vector must be capable of integrating itself or the recombinant DNA into the genome of
the host cell.
◼The introduction of recombinant DNA into the vector doesn’t affect the replication cycle of the
vector.

Dr. Amulya. G, The Oxford College of Engineering
Types of vector
◼Types of vector- Vectors can be classified into different types depending on
different characteristics. Selection of vectors thus depends on purpose of process.
◼Binary vector - A binary vector is a standard tool in the transformation of higher
plants mediated by Agrobacterium tumefaciens.
◼Cloning vector - are vectors that are capable of replicating autonomously and
thus are used for the replication of the recombinant DNA within the host cell.
◼Shuttle vector - are that carry origins of replication from two different hosts,
which enables them to ‘shuttle’ between the two hosts. function as hybrid
vectors containing DNA sequences from bacterial plasmids and mammalian
viruses.
◼Expression vector - are vectors that enable the expression of cloned genes in
order to determine the successful cloning process
◼Viral vector - are one of the most effective means of gene transfer to modify
host cells or tissues and manipulate them to express different types of gene.
◼Secretion vectors are a type of specialized expression vector that expresses
the cloned genes in order to produce proteins at locations other than the
cytoplasm

Dr. Amulya. G, The Oxford College of Engineering
Vectors in Genetic Engineering
◼Most naturally occurring vectors do not have all the required functions for
easy propagation inside host species or accurate expression of the
recombinant DNA insert.
◼So vectors have been created by joining together segments performing
specific functions (called modules) from 2 or more natural systems.
◼There are several types of vectors of which some are natural and some
constructed. They can be grouped into different classes like plasmids,
bacteriophages, cosmids and artificial chromosomes.

Dr. Amulya. G, The Oxford College of Engineering
Cloning Vectors
◼Cloning vectors are vectors that are capable of replicating
autonomously and thus are used for the replication of the
recombinant DNA within the host cell.
◼Cloning vectors are responsible for the determination of which host
cells are appropriate for replicating a particular DNA segment.
◼Cloning vectors are of further different types that are defined by
different features unique to each type of vector.

Dr. Amulya. G, The Oxford College of Engineering
Types of Cloning Vectors
Vector Insert sizeSource Application
Plasmid ≤ 15 kbBacteria Subcloning and downstream
manipulation, cDNA cloning and
expressionassays
Phage 5-20 kbBacteriophage λ Genomic DNA cloning, cDNA cloning
andexpression library
Cosmid 35-45 kbPlasmid containing
bacteriophage λ cos site
Genomic library
construction
BAC (bacterial
artificial
chromosome)
75-300 kbPlasmid ocntaining ori
from E.coli F- plasmid
Analysis of large genomes
YAC (yeast
artificial
chromosome)
100-1000
kb
(1 Mb)
Saccharomyces
cerevisiae centromere,
telomere and
autonomously replicating
sequence
Analysis of large genome, YAC
transgenic mice
MAC
(mammalian
artificial
chromosome)
100 kb to
> 1 Mb
Mammalian centromere,
telomere and origin of
replication
Under development for use in animal
biotechnology and human gene
therapy

Dr. Amulya. G, The Oxford College of Engineering
Expression Vectors
•It enables the expression of cloned genes in order to determine the
successful cloning process.
•Usually, cloning vectors do not allow the expression of a cloned gene which
is why the use of expression vectors is required. The use of expression
vectors facilitates the processing of introns in prokaryotes as these are
designed with restriction sites next to the regulatory region.
•The restriction sites on the vectors result in splicing of the cloned gene to
permit the expression of the gene under the regulatory system.
•Expression vectors can be plasmid-based or viral-based that are
introduced into the host cells in order to code for particular mRNAs.
•The regulatory system in expression vectors consists of a promoter
sequence, a termination sequence, along a transcription termination
sequence.

Dr. Amulya. G, The Oxford College of Engineering
Plasmids
“Plasmidsare double-stranded and generally circular DNA sequences that
are capable of automatically replicating in a host cell. It is physically
separated from thechromosomal DNAand can replicate independently”.
Features
◼Plasmids are found widely in many bacteria, for example in Escherichia
coli, but may also be found in a few eukaryotes, for example in yeast such
asSaccharomyces cerevisiae.
◼In nature, plasmids often carry genes that may benefit the survival of the
organism, for exampleantibiotic resistance.
◼Plasmids usually are very small and contain only additional genes that
may be useful to the organism under certain situations or particular
conditions.

Dr. Amulya. G, The Oxford College of Engineering
Classification of plasmids
◼Plasmids can be transmitted from one bacterium to another (even of
another species) via three main mechanisms:transformation,transduction,
andconjugation.
There are five main classes of plasmids according to function:
◼FertilityF-plasmids, which containtragenes. They are capable
ofconjugation& result in the expression of sex pili. Eg. F plasmid of E. coli.
◼Resistance R- plasmids, which contain genes that provide resistance
againstantibioticsorpoisons. Eg. RP4 of Pseudomonas sp.
◼Col plasmids- which contain genes that code forbacteriocins,proteinsthat
can kill other bacteria. Eg. Col E1.
◼Degradative plasmids - which enable the digestion of unusual substances
liketolueneandsalicylicacid. Eg. TOL plasmid of Pseudomonas putida.
◼Virulence plasmids - which turn the bacterium into apathogen. Eg. Ti and
Ri plasmids.

Dr. Amulya. G, The Oxford College of Engineering
Plasmids as vectors
◼Plasmids are the most-commonly used bacterial cloning vectors.
◼ Many different E. coli plasmids are used as vectors.
◼The natural plasmids have been modified, shortened, reconstructed and
recombined both invitro and invivo to create plasmids of enhanced utility
and specific functions.
◼These plasmids serve as important tools in genetics and biotechnology
labs, where they are commonly used to clone and amplify the gene orto
express particular genes.
◼The bacteria containing the plasmids can generate millions of copies of
the vector within the bacteria in hours, and the amplified vectors can be
extracted from the bacteria for further manipulation.

Dr. Amulya. G, The Oxford College of Engineering
Cloning using plasmids
◼Plasmid cloning vectors contain a site that allows DNA fragments to be
inserted, for example amultiple cloning siteor polylinker which has several
commonly usedrestriction sites to which DNA fragments may beligated.
◼Both the plasmid and the DNA insert are digested with the same restriction
enzyme to create cohesive ends.
◼ Ligation is carried out using DNA ligase enzyme.
◼ After the gene of interest is inserted, the plasmids are introduced into
bacteria bytransformation.
◼ A plasmid cloning vector is typically used to clone DNA fragments of up to
15kbp.

Dr. Amulya. G, The Oxford College of Engineering
Selection of recombinant plasmids
It is very important to select for the low frequency of cells transformed by the
recombinant DNA from among the cells containing the unaltered vector
and the non-transformed cells.
◼These plasmids contain aselectable marker, usually 2 antibiotic
resistance genes, such as ampicillin resistance (amp
R
) and tetracycline
resistance (tet
R
) in the vector.
◼These will confer on the bacteria the ability to survive and proliferate in a
selective growth medium containing the particular antibiotics.
◼The DNA insert is integrated within one of the 2 selectable markers.

Dr. Amulya. G, The Oxford College of Engineering
◼The cells after transformation are exposed to the selective media, and
only cells containing the recombinant plasmid may survive.
◼ In this way, the antibiotics act as a filter to select only the bacteria
containing the plasmid DNA.
◼The vector may also contain other marker genesorreporter genesto
facilitate selection of plasmid with cloned insert.
◼Amplification of DNA insert – gene of interest can be amplified to a
great extent using transcription vectors, which can be used for transfer of
the gene to either bacterial, animal or plant cells.
◼Disease models - Plasmids were historically used to genetically
engineer the embryonic stem cells of rats in order to create rat genetic
disease models.

Dr. Amulya. G, The Oxford College of Engineering
◼Protein production - major use of plasmids is to make large amounts of
recombinant proteins. Just as the bacterium produces proteins to confer
its antibiotic resistance, it can also be induced to produce large amounts
of proteins from the inserted gene. This is a cheap and easy way of
mass-producing the protein the gene codes for, ex/insulin.
◼Gene therapy - Plasmid may also be used for gene transfer into human
cells as potential treatment ingene therapyso that it may express the
protein that is lacking in the cells. Some strategies ofgene
therapyrequire the insertion of therapeuticgenesat pre-
selectedchromosomaltarget sites within the humangenome. Plasmid
vectors are one of many approaches that could be used for this purpose.

Dr. Amulya. G, The Oxford College of Engineering
Plasmid vectors in molecular biology
An ideal plasmid vector must have the following functions
◼Minimum amount of DNA (<10kb to avoid problems during purification)
◼Relaxed replication control
◼Selectable marker gene for easy selection of recombinant vectors
◼Unique restriction site for at least one restriction enzyme.
◼Location of restriction site within the marker gene for easy selection of
recombinant vector

Dr. Amulya. G, The Oxford College of Engineering
pBR322
•One of the most popular and widely
used vector is pBR322.
•It was created in 1977 was named after
the Mexican postdoctoral researchers
who constructed it. The p stands for
"plasmid", and BR for "Bolivar" and
"Rodriguez".
•pBR322 is 4363 base pairs in length and
contains the ori of pMB1, a close relative
of ColE1.
•Due to this replication module each cell
accumulates upto 3000 copies of the
plasmid easily.

Dr. Amulya. G, The Oxford College of Engineering
◼It has 2 selectable markers (tetracycline, tet
R
and ampicillin, amp
R

resistance genes) which encodes two proteins which makeE.
coliresistant to ampicillin and tetracycline.
◼It also has unique restriction sites for several restriction enzymes. PstI,
SacI and PvuI restriction sites are located within the ampR gene and
BamHI, SacI within tetR gene.
Useful features of pBR322
◼Small size easy purification and manipulation
◼2 selectable markers permit easy selection of recombinant DNA
◼High copy number of 15 per cell
◼Copy number can be amplified upto 1000-3000 when protein synthesis is
blocked (by applying chloramphenicol).

Dr. Amulya. G, The Oxford College of Engineering
pUC19
•The pUC series of vectors are derivatives
of pBR322 and are much smaller.
•pUC19is one of the most widely used
plasmid vectors.
• From pBR322 it has the ampicillin
resistance gene (ampR)and the ColE1
origin derived from pMB1
•Each cell produces 500-700 copies of
plasmid without any treatment for
amplification of copy number

Dr. Amulya. G, The Oxford College of Engineering
◼The second selectable marker is due to the E. coli gene lacZ segment,
denoted as lac Zα.
◼This encodes the N-terminal fragment of β-galactosidase, the enzyme
that hydrolyses lactose.
◼A polylinker sequence or multiple cloning site (MCS) is located within the
lacZ gene providing several unique restriction sites for DNA insertion.
◼pUC18, another popular cloning vector, differs from pUC19 only in the
orientation of the MCS. The other vectors in the pUC series are pUC9,
pUC12 etc.
◼“rop” gene is removed from this vector which leads to an increase in
copy number.

Dr. Amulya. G, The Oxford College of Engineering
Blue white screening of recombinant clones
◼The lacZ gene in pUC series of vectors and several other vectors serve as
easy selectable marker for the identification of recombinant clones.
◼This gene encodes for β-galactosidase enzyme that hydrolyses lactose as
well as some synthetic substrates like X-Gal (5-bromo-4-chloro-3- indolyl- β-
D-galactoside).
◼Hydrolysis of X-gal by enzyme forms a blue dye, thus cells producing active
enzyme can be easily identified as those forming blue colonies.
◼Vectors used in cloning include the E. coli gene lacZ segment, denoted as
lac Zα which codes for the α fragment of N-terminal fragment of β-
galactosidase enzyme.

Dr. Amulya. G, The Oxford College of Engineering
◼When pUC plasmid enters host cells, gene products of the lac Z of the
plasmid and genome together produce the active enzyme.
◼Active β-galactosidase will result in formation of blue dye from X-gal, a
substrate for the enzyme. Such cells will form blue colored colonies.
◼When foreign DNA is inserted into the MCS within lacZ gene, this gene
gets disrupted and active enzyme is no longer produced.
◼Transformed cells produce white colored colonies on medium containing
amp, X-Gal and IPTG (isopropyl beta-D-1-thiogalactopyranoside) and
are easily selected.

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
BACTERIOPHAGE VECTORS
◼Bacteriophages are viruses that attack
bacteria.
◼ Most phages lyse the bacterial cells
they infect (lytic phages). But many
others follow either a lytic or lysogenic
cycle.
◼ Lysogeny is where the phage
chromosome integrates into the
bacterial chromosome and multiplies
with it ( = lysogenic phages).
◼Viruses have evolved specialized
molecular mechanisms to efficiently
transport their genome inside the cells
they infect.
◼Delivery of genetic material by a virus
into a bacterial cell is called
transduction and the infected cells are
said to be transduced.

Dr. Amulya. G, The Oxford College of Engineering
Viral vectors are tailored to specific applications but generally share a
few key properties.
◼Safety: Although viral vectors are occasionally created
frompathogenicviruses, they are modified in such a way as to minimize the
risk of handling them.
◼Low toxicity: The viral vector should have a minimal effect oncells it infects.
◼Stability: Some viruses are genetically unstable and can rapidly rearrange
their genomes. This is detrimental to predictability and reproducibility of the
work conducted using a viral vector and is avoided in their design.
◼Cell type specificity: The viral receptor can be modified to target the virus to
a specific kind of cell.
◼Identification: Viral vectors are often given certain genes that help identify
which cells took up the viral genes. A common marker is antibiotic
resistance to a certain antibiotic.

Dr. Amulya. G, The Oxford College of Engineering
Advantages of phages over plasmids
◼Phage vectors are more efficient than plasmids for cloning of large DNA fragments.
The maximum size possible with lambda λ vector is 24 kb while that for plasmid
vector is <15kb.
◼It is easier to screen a large number of phage plaques than bacterial colonies for the
identification of recombinant vectors. Screening of phage plaques by molecular
hybridization gives clearer results.
◼Plasmid vectors have to be introduced into bacterial cells, which are then cloned and
selected for the recovery of recombinant DNA. In contrast, phage vectors are directly
tested on an appropriate bacterial lawn culture (continuous bacterial growth on an
agar plate) where each phage particle forms a plaque (a clear bacteria-free zone in
the bacterial lawn).
◼Storage of viral particles is much easier than plasmid DNA.
◼Shelf life of phage particles is infinite.
◼Transformation of bacterial host cells is much easier using phages rather than
plasmids.

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
LAMBDA λ PHAGE VECTORS
The lambda λ genome is sized 48,502 bp, specifically infects E. coli cells and
resides inside cells by lysogeny. It contains
◼An origin of replication
◼Genes for head and tail proteins
◼Enzymes for DNA replication, lysis and lysogeny
◼Single stranded protruding cohesive ends (of 12 base pairs,
complementary)
◼Lambda λ genome remains linear in the phage head but within E. coli cells
the two cohesive ends anneal to form a circular molecule necessary for
replication.
◼The sealed cohesive ends are called cos sites, which are the sites of
cleavage, necessary for packaging of the mature phage DNA into phage
heads during viral assembly.

Dr. Amulya. G, The Oxford College of Engineering
◼The use of wild type lambda λ genome as a vector has 2 major problems, it
takes only 3kb insert and contains >1 recognition sites for every restriction
enzyme. The properties have therefore been modified to allow use of
phages as vectors.
◼By mutation and recombination in vivo as well as by recombinant DNA
techniques several vectors have been produced from lambda λ genome.
These modified vectors have 2 basic features
➢They can propagate as phages in E. coli cells so vector DNA can be
replicated
➢They contain restriction sites which allow removal of lysogenic segment
and also provide insertion site for DNA to be cloned.

Dr. Amulya. G, The Oxford College of Engineering
The various λ vectors are classified into 2 groups
◼Insertion vectors – here a large portion of the nonessential region is
deleted and the two arms of the λ genome are ligated. There is at least
one unique restriction site within which the DNA insert is integrated.
Eg. λgt10, λgt11, λZAP II.
◼Replacement vectors – insertion of DNA fragment is accompanied by
the deletion of all the major part of nonessential region of λ genome.
These vectors have 2 restriction sites useful for cloning. Eg. λEMBL4.

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
Cloning using λ vectors

Dr. Amulya. G, The Oxford College of Engineering
M13 VECTORS
◼M13 vectors are derived from the 6.4kb genome of E. coli filamentous
phage M13 or f1.
◼This phage has a single-stranded linear DNA genome in phage
particles. It gets converted into a ds circular molecule inside host cells.
◼ M13 infects only F+ and F’ cells, injects its genome inside through the
F-pili of these cells.
◼ M13 vectors are used to obtain ss copies of cloned DNA, especially
suited for DNA sequencing.
◼Each infected cell has ~100 copies of M13 genome and about 1000
new particles are produced during each generation of an infected cell.
◼Phage M13 does not lyse the infected cells, but it forms turbid plaques
due to growth retardation of these cells. Eg M13 mp 18.

Dr. Amulya. G, The Oxford College of Engineering
Cloning into M13 vectors
◼DNA inserts are placed into the
noncoding region which also contains
the origin of replication.
◼The vector contains the E. coli LacZ
gene which is a selection method.
◼Just like in pUC vectors, the
unchanged M13 vector produces blue
plaques on the lawn of appropriate
strain of E. coli grown on X-gal and
IPTG.
◼The recombinant DNA produces
colorless plaques which can be
readily identified

Dr. Amulya. G, The Oxford College of Engineering
Advantages of M13 vectors
•Very large DNA inserts can be cloned
•Large number of ss copies of ds DNA inserts can be obtained
•Useful in precise DNA sequencing and synthesis of specific radiolabelled
DNA probes
•Phage infected bacterial cells remain viable, so easy maintenance of
vector
•Selection of recombinants are easy (plaques are formed, also growth of
infected cells is slow) stable viral particles are formed from which
recombinant DNA can be obtained.

Dr. Amulya. G, The Oxford College of Engineering
COSMIDS
◼Cosmids are essentially engineered plasmids that combine unique
properties of plasmids and phage vectors.
◼ Acosmidis a type of hybrid plasmid constructed using recombinant DNA
technology and often used in gene cloning.
◼They contain a minimum of 250bp of λ lambda DNA, which includes the
following sequences from λ phage genome –
✓The cos site (the sequences giving cohesive ends)
✓Sequences needed for binding of and cleavage by terminase so that they
are packaged in vitro into empty λ phage particles under appropriate
conditions.

Dr. Amulya. G, The Oxford College of Engineering
◼Cosmids are usually derived from
pBR322 and can easily be maintained in
E. coli cells. Cosmids can contain 37 to
52 (normally 45) kb of DNA, limits based
on the normal bacteriophage packaging
size.
◼ A typical cosmid contains a replication
origin, unique restriction sites and
selectable markers form plasmids. Eg.
pJB8.
◼Unlike plasmids, they can also be
packaged in phage capsids, which allows
the foreign genes to be transferred into
or between cells bytransduction.

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
Cloning steps
◼Cut cosmid by appropriate RE at a unique site.
◼Mix with DNA inserts prepared using same RE (usually large DNA inserts
~40kB of eukaryotic DNA can be cloned).
◼Anneal and ligate using T4 DNA ligase.
◼Concatamers are produced (ideal precursors for packaging into viral particles).
◼Add packaging mix. DNA packaged into lambda heads in vivo.
◼Infectious particles containing recombinant DNA obtained after addition of tail
assemblies.
◼Transduction of host cells. Once inside host cells the cosmid acts like a
plasmid and replicates and propagates like a plasmid. They don’t go through
the developmental sequence of phages.
◼Transduced bacterial cells selected on medium with appropriate selection
agents.

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
◼Advantages
•Used to clone large DNA segments (upto 40kb)
•They can be packaged into lambda particles which infect host cells
(many fold more efficient than transformation with plasmid)
•Selection of recombinant DNA is simple, based on the procedures of the
concerned plasmid
•Vectors are amplified and maintained in the same easy way as the
contributing plasmid.
◼Applications
•Widely used vectors in gene cloning to construct genomic libraries of
eukaryotes
•Ideal for genome mapping.

Dr. Amulya. G, The Oxford College of Engineering
PHAGEMIDS
◼Aphagemid is avector that combines the
features of a filamentous phage and a plasmid.
◼It contains an f1origin ofreplicationfrom anf1
phage and can be used as a type
ofcloningvectorin combination with
filamentousphageM13.
Features
◼Phage f1 origin of replication
◼A portion of lacZ gene driven by lac promoter
◼A multiple cloning site (MCS) within lacZ gene
◼ColE1 origin of replication
◼ampR gene for ampicillin resistance

Dr. Amulya. G, The Oxford College of Engineering
Cloning steps
Applications
❖Phagemids were originally used to generate single stranded DNA templates for
sequencing purposes.
❖ It can also be applied into RNA transcription, restriction mapping, sequencing of
single and double stranded nucleic acids, generation of deletions etc.
❖Using phagemids, peptides and proteins can be expressed as fusions to phage
coat proteins and displayed on the viral surface and so this technique is useful to
study protein-protein interactions and other ligand/receptor combinations.

Dr. Amulya. G, The Oxford College of Engineering
pBluescript(pBS) is a commercially
availablephagemid containing
several useful sequences for use
incloningwithbacteriophage. Size
is 2958 bp and is a cloning vector
derived from pUC19 and M13
phage.
pBLUESCRIPT SK (+/-)

Dr. Amulya. G, The Oxford College of Engineering
Features
➢M13 origin of replication
➢A portion of lacZ gene driven by lac promoter
➢A multiple cloning site (MCS) located within the lacZ gene, with 21 unique
restriction enzyme recognition sites
➢Phage T7 and T3 promoter sequences flanking the MCS sequence
(promoters that can be used to synthesize RNA in vitro)
➢Col E1 origin of replication
➢ampR resistance gene
Applications
This multipurpose vector can serve as :Cloning vector,Expression vector,
Riboprobe vector &Sequencing vector.

Dr. Amulya. G, The Oxford College of Engineering
Fosmids
◼Fosmids are vectors used for cloning large fragments of DNA. They were
developed in the 1990s to improve on the capabilities of other cloning
vectors, such BACs and YACs.
◼Fosmids are particularly useful for studying large and complex genomes.
◼Fosmids are derived from F-plasmids, which are small, circular pieces of
DNA found in bacteria. F-plasmids are often used as vectors for cloning
small DNA fragments, but their usefulness is limited when it comes to
cloning larger fragments.
◼Fosmids were developed to address this limitation by incorporating
several key features that make them better suited for cloning larger
fragments of DNA.

Dr. Amulya. G, The Oxford College of Engineering
◼Fosmids can hold DNA inserts of up to 40 kb in size;
◼A fosmid library is prepared by extracting the genomic DNA from the
target organism and cloning it into the fosmid vector.

The ligation mix is
then packaged into phage particles and the DNA is transfected into the
bacterial host.
◼Bacterial clones propagate the fosmid library.
◼The low copy number offers higher stability than vectors with relatively
higher copy numbers, including cosmids.
◼Fosmids may be useful for constructing stable libraries from
complexgenomes.
◼Fosmids have high structural stability and have been found to
maintain human DNA effectively even after 100 generations of bacterial
growth.

Dr. Amulya. G, The Oxford College of Engineering
◼Discovery
◼The fertility plasmid orF-plasmidwas discovered byEsther
Lederbergand encodes information for the biosynthesis of sex pilus to
aid in bacterial conjugation.
◼Conjugation involves using the sex pilus to form a bridge between two
bacteria cells; this bridge allows the F+ cell to transfer a single-
stranded copy of the plasmid so that both cells contain a copy of the
plasmid.
◼On the way into the recipient cell, the corresponding DNA strand is
synthesized by the recipient. The donor cell maintains a functional copy
of the plasmid. It later was discovered that the F factor was the
firstepisomeand can exist as an independent plasmid making it a very
stable vector for cloning.

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
◼“Fosmids are DNA vectors that use the F-plasmid origin of replication and
partitioning mechanisms to allow cloning of large DNA fragments. A library
that provides 20–70-fold redundant coverage of the genome can easily be
prepared”.
◼Fosmids contain several functional elements:
◼OriT (Origin of Transfer): The sequence which marks the starting point of
conjugative transfer.
◼OriV (Origin of Replication): The sequence starting with which the plasmid-
DNA will be replicated in the recipient cell.
◼tra-region (transfer genes): Genes coding the F-Pilus and DNA transfer
process.
◼IS (Insertion Elements): so-called "selfish genes" (sequence fragments
which can integrate copies of themselves at different locations).

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
ARTIFICIAL CHROMOSOMES
Artificial chromosome vectors are linear or circular vectors that are stably
maintained in usually 1 or 2 copy per cell. There are several types of such
vectors.
◼BAC – bacterial artificial chromosome
◼P1 derived artificial chromosome (PAC)
◼YAC – yeast artificial chromosome
◼MAC – mammalian artificial chromosome
◼HAC – human artificial chromosome
YAC are used for cloning in yeast, while MAC and HAC are used in
mammalian and human cells.

Dr. Amulya. G, The Oxford College of Engineering
BACTERIAL ARTIFICIAL CHROMOSOMES
◼Abacterial artificial chromosome (BAC)is a vector based on a functional
fertility plasmid (or F-plasmid), used for transforming and cloning in
bacteria.
◼The bacterial artificial chromosome's usual insert size is 150-350 kb.
◼Vector pBeloBAC11 is c convenient vector of 7.4 kb, allows selection of
recombinants cloned by LacZ complementation.
◼This vector is maintained in E. coli cells at single copy per cell. It contains
❑oriS – origin of replication from E. coli F plasmid
❑repE – encodes a Rep protein required for plasmid replication and
regulation of copy number
❑parA, parB and parC loci - for partitioning F plasmid DNA to daughter
cells during division and ensures stable maintenance of the BAC

Dr. Amulya. G, The Oxford College of Engineering
•CM
R
– chloramphenicol
resisitance
•Cos N – lambda phage cos site
•Lox P – site on lambda phage P1
genome where extensive
recombination occurs
•lacZ gene – beta gal gene
•T7, bacteriophage T7 RNA
polymerase driven promoter
•SP6, bacteriophage SP6 RNA
polymerase driven promoter
Applications of BACs
1.Sequencing
2.Contribution to models of disease
- Inherited disease
3.Contribution to models of disease
- Infectious disease
4.Gene mapping

Dr. Amulya. G, The Oxford College of Engineering
YEAST ARTIFICIAL CHROMOSOME
◼Yeast artificial chromosomes (YACs)are genetically engineered
chromosomes derived from the DNA of the yeast,Saccharomyces
cerevisiae, which is then ligated into a bacterial plasmid.
◼pYAC3 is essentially a pBR322 plasmid into which the yeast sequences
have been integrated.
◼ Several yeast vectors based on pYAC3 have been constructed. The
vector is propagated in E. coli while cloning is done in yeast.
◼The primary components of a YAC are the ARS, centromere, and
telomeres fromS. cerevisiae.
◼Additionally, selectable marker genes, such as antibiotic resistance and a
visible marker, are utilized to select transformed yeast cells.

Dr. Amulya. G, The Oxford College of Engineering
Construction
Basic functional elements
•an ARS sequence
•CEN4 sequence
•Telomeric sequence
•One or two selectable markers, eg.
TRP1 and URA3
•SUP4 a selectable marker into
which the DNA insert is integrated
•Plasmid sequences for selection
and propagation in bacteria

Dr. Amulya. G, The Oxford College of Engineering
Applications
•Yeast expression vectors, such as
YACs,YIps, andYEps, have an
advantage overBACs in that they
can be used to express eukaryotic
proteins that requireposttranslational
modification.
•By being able to insert large
fragments of DNA, YACs can be
utilized to clone and assemble the
entire genomes of an organism. With
the insertion of a YAC into yeast
cells, they can be propagated as
linear artificial chromosomes, cloning
the inserted regions of DNA in the
process
•Two processes can be used to obtain
a sequenced genome, or region of
interest: physical mapping and
chromosome walking.

Dr. Amulya. G, The Oxford College of Engineering
Viral Vectors
◼Viral vectors are one of the most effective means of gene transfer to
modify host cells or tissues and manipulate them to express different
types of genes.
◼The concept of using viruses as vectors arose from the fact that viruses
are very effective in transducing their own genetic information into the
host cell.
◼During viral transduction, the non-essential viral genes are replaced with
foreign DNA sequences of therapeutic interest in order to produce
recombinant viral vectors.
◼The use of viral vectors also enables location specificity with unique
injection technology within a specific time period.

Dr. Amulya. G, The Oxford College of Engineering
◼Viral Vectors are tailored to their specific applications but generally share a few key
properties.
•Safety: Although viral vectors are occasionally created frompathogenicviruses, they
are modified in such a way as to minimize the risk of handling them. This usually
involves the deletion of a part of the viral genome critical forviral replication.
•Low toxicity: The viral vector should have a minimal effect on thephysiologyof the
cell it infects.
•Stability: Some viruses are genetically unstable and can rapidly rearrange their
genomes. This is detrimental to predictability and reproducibility of the work
conducted using a viral vector and is avoided in their design.
•Cell type specificity: Most viral vectors are engineered to infect as wide a range
ofcell typesas possible. The viral vector can be modified to target the virus to a
specific kind of cell.
•Identification: Viral vectors are often given certain genes that help identify which
cells took up the viral genes. These genes are calledmarkers. A common marker
isresistanceto a certain antibiotic.

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
Types
◼Retrovirusesare one of the mainstays of current gene therapy approaches.
The recombinant retroviruses such as the Moloneymurine leukemia
virushave the ability to integrate into the host genome in a stable fashion.
They contain areverse transcriptaseto make a DNA copy of the RNA
genome, and an integrase that allows integration into the hostgenome.
◼Lentivirusesare a subclass of retroviruses. They are sometimes used
asvectors for gene therapythanks to their ability to integrate into
thegenomeof non-dividing cells, which is a unique feature of lentiviruses,
as other retroviruses can infect only dividing cells. The viral genome in the
form ofRNAisreverse-transcribedwhen the virus enters the cell to
produceDNA, which is then inserted into the genome at a random position
by the viralintegraseenzyme.

Dr. Amulya. G, The Oxford College of Engineering
◼Adenovirus - As opposed to lentiviruses, adenoviral DNA does not
integrate into the genome and is not replicated during cell
division.
[18]:5
This limits their use in basic research, although adenoviral
vectors are still used inin vitroand alsoin vivoexperiments.
[19]
Their
primary applications are ingene therapyandvaccination.
◼Adeno-associated virus (AAV) is a small virus that infects humans and
some other primate species. AAV is not currently known to cause
disease, and causes a very mild immune response. AAV can infect both
dividing and non-dividing cells and may incorporate its genome into that
of the host cell. Adeno-associated viral vectors have been engineered to
evade virus recognition byTLR9receptors by incorporating TLR9-
inhibiting genes into the vector.

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
Uses
◼Adenoviruses have been used for the transfer of tumor suppressor genes
in cancer treatment, and retroviruses are studied for their potential use in
tissue repair and engineering.
◼A live vector vaccine is a vaccine that uses an organism (typically virus or
bacterium) that does not cause disease to transport the pathogen genes
into the body in order to stimulate an immune response.
◼Viruses expressing pathogen proteins are currently being developed as
vaccines against these pathogens, based on the same rationale as DNA
vaccines.
◼A strain of canarypox virus modified to carry feline interleukin-2 is used to
treat cats with fibrosarcoma.
◼Gene insertion, which can be done with viral vectors, is cheaper to carry
out than gene knockout

Dr. Amulya. G, The Oxford College of Engineering
Enzymes
Enzymes used in genetic engineering can be grouped into five 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 or pieces of DNA fragment together.
◼ Polymerases - make copies of molecules or make new strands of DNA.
◼ Modifying enzymes – modify the DNA by adding or removing chemical
groups
◼ Topoisomerase - remove or introduce supercoils from co-valently closed
circular DNA structures.

Dr. Amulya. G, The Oxford College of Engineering
Enzymes used in molecular cloning
•The discovery, characterization and development of a number of enzymes
have played an important role in development of techniques for modification
and analysis of DNA.
•These are required for cutting, modifying or joining DNA segments and also to
check parameters such as size of DNA molecules.
• Enzymes are major tools that catalyze specific reactions on DNA molecules.
Nucleases:
•Enzymes that break down nucleic acids are nucleases. Nucleases degrade the
DNA molecules by breaking phosphodiester bonds that hold nucleotides
together.It may be endo or exo, DNases or RNases, recombinases, ribozymes,
or RNA splicing enzymes.
•Those that break down RNA are called ribonucleases (RNases) and those that
break down DNA are called deoxyribonucleases (DNases).

Dr. Amulya. G, The Oxford College of Engineering
•In nature, they play crucial roles in genetic quality control, such as in DNA
proofreading during replication, base, nucleotide, mismatch, and double-
strand repairs, homologous recombination, and turnover.
•Nucleases have also found widespread use in molecular and cell biology
applications which require precise manipulation of nucleic acids, such as
restriction digestion, degradation of selected nucleic acids, and trimming.
•They are diverse in their functions and selectivity for types of nucleic acid
substrates, and thus can be further classified into distinct categories
•Endonucleases: Endonucleases cleave DNA and RNA from within the
middle of the chain, with varying levels of site recognition. Restriction
endonucleases, or restriction enzymes , are sequence specific, and are
widely used in cloning and gene analysis. Others, like DNase I and
Benzonase are indiscriminate and are used to fully digest DNA or RNA
samples.

Dr. Amulya. G, The Oxford College of Engineering
•Exonucleases: In contrast to endonucleases, exonucleases cleave off
nucleotides one at a time from the 3’ or 5’ ends of DNA and RNA chains.
Applications of exonucleases include selective degradation of single-
stranded DNA and removal of overhangs.
•DNases: DNases, or deoxyribonucleases, selectively digest DNA over
RNA and different types can cut either internally or from the ends. In
research, DNase I is often used in removing contaminating DNA from RNA
and protein samples, cleaning cell cultures, and in assays involving DNA
fragmentation.
•RNases: Ribonucleases, in turn, are selective to digesting RNA over DNA.
RNases (commonly RNase A and RNase H) are often used for removing
contaminating RNA from samples and certain RNA assays. RNases (with
the exception of RNase H) typically are more selective for single-stranded
than hybridized RNA.

Dr. Amulya. G, The Oxford College of Engineering
•Strand-specific Nucleases: A number of nucleases have found use for
their selectivity for either single- or double-stranded nucleic acids.
Micrococcal nuclease, S1 nuclease, and Mung Bean nuclease are
selective for single-stranded DNA and RNA (including DNA-RNA
hybridizations), and are used for selective digestion and removal of
overhangs. Duplex-specific nucleases , in turn, prefer double-stranded
DNA or RNA.
•Other nucleases: Cas9 , or “CRISPR associated protein 9,” is a
Streptococcus pyogenes-derived nuclease that is used in the
CRISPR/Cas9 genome editing technique. Cas9 nuclease, together with a
sequence-specific guide RNA, can be used to mutate, insert or delete a
specific region with the genome.

Dr. Amulya. G, The Oxford College of Engineering
◼Types of nucleases and its action.Endonucleases cleave the double-
stranded DNA at specific sequences internally while exonuclease chew
up nucleotides from either end of the double-stranded DNA.
◼
◼Exonucleases catalyse hydrolysis of terminal nucleotides from the end
of DNA or RNA molecule in either 5’to 3’ direction or 3’ to 5’ direction.
Example: exonuclease I, exonuclease II etc.
◼Endonucleases can recognize specific base sequence (restriction site)
within DNA or RNA molecule and cleave internal phosphodiester bonds
within a DNA molecule. Example: EcoRI, Hind III, BamHI etc.
Restriction enzymes are good examples of endonucleases which forms
the cutting tool of genetic engineering.

Dr. Amulya. G, The Oxford College of Engineering
What are RESTRICTION ENZYMES?
•Molecular scissors that cut double stranded DNA molecules at specific points.
•Found naturally in a wide variety of prokaryotes
•It is an important tool for manipulating DNA.
Biological Role
•Most bacteria use Restriction Enzymes as a defense against bacteriophages.
•Restriction enzymes prevent the replication of the phage by cleaving its DNA
at specific sites.
•The host DNA is protected by Methylases which add methyl groups to
adenine or cytosine bases within the recognition site thereby modifying the
site and protecting the DNA.

Dr. Amulya. G, The Oxford College of Engineering
Modification System
◼At times, it was known that methyl groups were added to bacterial DNA at limited
number of sites.
◼Most importantly the location of methyl groups varied among bacterial species.
◼Arber and collegues were able to demonstrate that modification consisted of addition
of methyl groups to protect those sites in DNA sensitive to attack by RE.
◼In E. coli adanine methylation is more common than cytosine methylation.
◼Methyl modified target sites are no longer recognized by RE and DNA is no longer
degraded.
◼Once established, methylation patterns are maintained during replication. When
resident DNA replicates, the old strand remains methylated and new strand is
unmethylated.
◼In hemimethylated state, new strand is quickly methylated by specific methylases.
◼In contrast, foreign DNA that is unmethylated or has different pattern of methylation
than the host cell DNA is degraded by RE.

Dr. Amulya. G, The Oxford College of Engineering
History Of Restriction Enzyme
•Werner Arber and Messelson were first to study type I restriction enzymes,
which cleave DNA randomly away from the recognition site.
•First restriction enzyme was isolated in 1970 by Hamilton O. Smith,Thomas
Kellyand Kent Wilcox as Hind ll, was isolated from Haemophilus influenzae.
•Later, Daniel Nathans and Kathleen Danna showed cleavage of simian virus
40 (SV40) DNA by Reyields specific fragements which could be separated
using polyacrylamide gel electrophoresis (PAGE) thus indicating RE can also
be used for mapping DNA.
•In 1978, Noble Prize for physiology or medicine was awarded to Werner
Arber, Daniel Nathans and Hamilton O. Smith.
•He also studied subsequent discovery and characterization of numerous RE.
•From then Over 3000 restriction enzymes have been studied in detail, and
more than 600 of these are available commercially and are routinely used
for DNA modification and manipulation in laboratories.

Dr. Amulya. G, The Oxford College of Engineering
Direct hydrolysis by nucleophilic attack at the
phosphorous atom
Mg
2+
is required for the catalytic activity of the enzyme. It holds the water molecule
in a position where it can attack the phosphoryl group and also helps polarize the
water molecule towards de-protonation, thus 3’OH and 5’ PO
4
3-
is produced
Mechanism of Action
Restriction Endonuclease scan the length of the DNA, binds to the DNA molecule
when it recognizes a specific sequence and makes one cut in each of the sugar
phosphate backbones of the double helix – by hydrolyzing the phoshphodiester bond.
Specifically, the bond between the 3’ O atom and the P atom is broken.

Dr. Amulya. G, The Oxford College of Engineering
Palindrome Sequences
•The mirror like palindrome in which the same forward and backwards are
on a single strand of DNA strand, as in GTAATG
• TheInverted repeat palindromes is also a sequence that reads the
same forward and backwards, but the forward and backward sequences
are found in complementary DNA strands (GTATAC being complementary
to CATATG)
• Inverted repeat palindromes are more common and have greater
biological importance than mirror- like palindromes.

Dr. Amulya. G, The Oxford College of Engineering
Ends Of Restriction Fragments
Blunt ends
Sticky ends

Dr. Amulya. G, The Oxford College of Engineering
Blunt ends
•Some restriction enzymes cut DNA at opposite base
•They leave blunt ended DNA fragments
•These blunt ended fragments can be joined to any other DNA
fragment with blunt ends.
•Enzymes useful for certain types of DNA cloning experiments.

Dr. Amulya. G, The Oxford College of Engineering
Sticky ends
•Most restriction enzymes make staggered cuts
•Staggered cuts produce single stranded “sticky-ends”
“Sticky Ends” Are Useful
DNA fragments with complimentary sticky ends can be combined to create
new molecules which allows the creation and manipulation of DNA
sequences from different sources.

Dr. Amulya. G, The Oxford College of Engineering
ISOSCHIZOMERS & NEOSCHIZOMERS
•Restriction enzymes that have the same recognition sequence as well as
the same cleavage site are Isoschizomers
•Restriction enzymes that have the same recognition sequence but cleave
the DNA at a different site within that sequence are Neoshizomers
•Isocaudomers- An enzyme that recognizes slightly different sequence but
produces the same ends. Eg- Mbo I and Bam HI

Dr. Amulya. G, The Oxford College of Engineering
NOMENCLATURE OF RESTRICTION ENZYME
•Each enzyme is named after the bacterium from which it was isolated
using a naming system based on bacterial genus, species and strain.
◼For e.g EcoRI
Derivation of the EcoRI name
Abbreviation Meaning Description
E Escherichia genus
co coli species
R RY13 strain
I First identified order of identification in the
bacterium

Dr. Amulya. G, The Oxford College of Engineering
TYPES OF RESTRICTION ENZYMES
•Restriction endonucleases are categorized into three general groups.
•Type I
•Type II
•Type III
These types are categorization based on:
•Their composition.
•Enzyme co-factor requirement.
•the nature of their target sequence.
•position of their DNA cleavage site relative to the target sequence.

Dr. Amulya. G, The Oxford College of Engineering
Type I
•First to be discovered, they were found in two different strains of E. coli.
•Capable of both restriction and modification activities.
•The co factors S-Adenosyl Methionine(AdoMet), ATP, and mg+ are
required for their full activity.
•Contain:
two R(restriction) subunits two M(methylation) subunits one S(specifity)
subunits
•Cleave DNA at random length from recognition sites.
•Type I RE has three subunits.

Dr. Amulya. G, The Oxford College of Engineering
Type II
•These are the most commonly available and
used restriction enzymes. There are over
3000 different kinds of type II RE enzymes
•They are composed of only one subunit.
•Their recognition sites are usually undivided
and palindromic and 4-8 nucleotides in length,
•they recognize and cleave DNA at the same
site.
•They do not use ATP for their activity
•they usually require only Mg2+ as a cofactor.
HIND III
ECOR I

Dr. Amulya. G, The Oxford College of Engineering
Type III
•Type III restriction enzymes recognize two separate non-
palindromic sequences that are inversely oriented.
•They cut DNA about 20-30 base pairs after the
recognition site.
•It works by recognizing two different nucleotide
sequences as recognition sites.
•These enzymes contain more than one subunit.
•And require AdoMet and ATP cofactors for their roles in
DNA methylation and restriction

Dr. Amulya. G, The Oxford College of Engineering
Type IV
•Cleave only normal and modified DNA
(methylated, hydroxymethylated and glucosyl-
hydroxymethylated bases).
•Recognition sequences have not been well
defined
•Cleavage takes place ~30 bp away from one of
the sites

Dr. Amulya. G, The Oxford College of Engineering
Comparison of REs
Features Type I Type II Type III
Abundance Less common than
type II
Most common rare
Nature of the
enzyme
Occurs as a single
multifunctional
enzyme
Separate nuclease and
methylase
Occurs as a single
multifunctional
enzyme
Molecular weight 450kDa 20-30kDa 200kDa
Protein
conformation
3 different subunits2 proteins 2 different subunits
Location and
number of genes
Chromosomal; Three
genes
Either chromosomal
or plasmid
Either chromosomal
or plasmid, Two genes
Requirement of
cofactors
Ado-met, ATP, Mg2+Mg2+ Ado-met, Mg2+, ATP
Recognition site Cut both strands at a
non-specific location
away from recognition
site
Cut both strands at a
specific usually
palindromic
recognition site
Cut only one strand
away from the
recognition site
Cleavage site 1000bp from
recognition site
Within recognition site24-26bp to 3' of
recognition site
Site of methylationRecognition site Recognition site Recognition site

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
◼Applications
•Isolated restriction enzymes are used to manipulate DNA for different
scientific applications.
•used to assist insertion of genes intoplasmidvectorsduringgene
cloningandprotein expression experiments.
•used to distinguish geneallelesby specifically recognizing single base
changes in DNA known assingle nucleotide polymorphisms (SNPs)
•digestgenomicDNA for gene analysis bySouthern blot
•used in restriction fragment length polymorphism(RFLP) for identifying
individual/ strains of a particular species.

Dr. Amulya. G, The Oxford College of Engineering
◼They are used in gene cloning and protein expression experiments.
◼Restriction enzymes are used in biotechnology to cut DNA into
smaller strands in order to study fragment length differences among
individuals (Restriction Fragment Length Polymorphism – RFLP).
◼Each of these methods depends on the use of agarose gel
electrophoresis for separation of the DNA fragments.

Dr. Amulya. G, The Oxford College of Engineering
Exonucleases
◼Exonuclease are enzymes that work by cleaving nucleotides one at time
from the end (exo) of polynucleotide chain.
◼A hydrolyzing reaction that breaks phosphodiester bonds at either the 3’
or the 5’ end occurs.
◼It is close relative is endonucleases that cleaves phosphodiester bonds
in the middle (endo) of polynucleotide chain.

Dr. Amulya. G, The Oxford College of Engineering
◼Types of Exonucleases
◼Lambda exonucleases (ds 5’ 3’ exonucleases) – this is purified
from E. coli cells and have been infected with bacteriophage. It
catalyzes the stepwise and processive hydrolysis of duplex DNA from
5’- phosphoryl termini liberating 5’ mononucleotides. λ exo will not
degrade 5’- hydroxyl termini.
◼Exonuclease III (ds 3’ 5’ exonucleases) – the product of E. coli
xthA gene , is made from E. coli cells overproducing the protein. It us
a multifunctional enzyme that catalyses hydrolysis of several types of
phosphodiester bonds in ds DNA. The application of exo III is as 3’
5’ ds specific exonuclease that catalyses release of 5’ nucleotides
from 3’ hydroxy end of ds DNA.
◼Exonuclease VII (ss 5’ 3’ and 3’ 5’ exonucleases) – this enzyme
from E.coli consists two subunits, the products of xseA and xseB
genes. It is processive single stranded exonuclease that acts from
both 3’ and 5’ ends of ss DNA. The products of exo VII are small
oligonucleotides. Exo VII is unique among nucleases, it does not
require Mg2+ and retains full activity in presence of 10 mM EDTA.
(write diagram for each)

Dr. Amulya. G, The Oxford College of Engineering
Applications of exonucleases
◼Removal of 5’ to 3’ overhangs
◼Removal of oligonucleotides post PCR
◼Removal of chromosomal DNA in plasmid preparations
◼Removal of DNA in RNA preparations
◼Generating ssDNA from linear ds DNA
◼If 5’ 3’ polarity required (require one 5’ end with phosphate)
◼If 3’ 5’ polarity required (require one end with 5’ overhang and one
end with 3’ overhang)

Dr. Amulya. G, The Oxford College of Engineering
◼METHYLASES
•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 S-adenosyl methionine (SAM) which acts as the methyl donor.
•It normally occurs on cytosine (C) residue in DNA sequence. In
protein, methylation occurs on nitrogen atom either on N-terminus or
on side chain of protein.
◼Types of methylases
◼Methyltransferase can be classified in three groups:
◼a) m6A-generates N6 methyladenosine,
◼b) m4C-generates N4 methylcytosine,
◼c) m5C-generatesN5 methylcytosine.

Dr. Amulya. G, The Oxford College of Engineering
Applications
•Used as research tool due to their ability to recognize and transfer methyl
group to target bases in specific DNA sequence.
• Construction of genomic DNA libraries
• To alter the cleavage specificity of certain restriction enzymes
•.Prokaryotic DNA MTase can be used in rDNA technology for alteration and
enhancing of cleavage specificity of RE.

Dr. Amulya. G, The Oxford College of Engineering
DNA LIGASES
•DNA ligases can join two pieces of DNA together.
•They catalyse the formation of phosphodiester bond between two
deoxynucleotide residues of two DNA strands.
•The reaction 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.

Dr. Amulya. G, The Oxford College of Engineering
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 phosphoamide 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.

Dr. Amulya. G, The Oxford College of Engineering
Homopolymer tailing
◼The complementary DNA strands can be joined together by annealing.
This principle is utilized in homopolymer tailing.
◼The technique involves addition of oligo (dA) to 3’-ends of some DNA
molecules and addition of oligo (dT) to 3’-ends of other molecules.
◼The homopolymer extensions by adding 10-40 residues can be
synthesized by using terminal deoxynucleotidyl transferase.
◼Homopolymer tailing can be achieved by annealing.
◼Suppose a vector has an oligo( dA) sequence at the 3 -OH end and the
insert has an oligo(dT) sequence at its 3 -OH end. Then when both the
molecules are mixed, the molecules are held by hydrogen bond or can
anneal until the ligase joins them by phosphodiester bond.

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
Linkers and adaptors
◼Linkers and adapters are chemically synthesized, short, ds-DNA
molecules.
◼Linkers posses 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
◼Adapters 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 adapters are finally ligated to vector
DNA molecules.

Dr. Amulya. G, The Oxford College of Engineering
T4 DNA ligase
◼Bacteriophage T4 DNA ligase is a single polypeptide with 68000 D
molecular weight.
◼The catalytic activity requires ATP and Mg2+. DNAs that lacks
phosphate residue can rendered capable of ligation by phosphorylation
with T4 polynucleotide kinase.
◼It also catalyses the addition reaction of phosphate between
pyrophosphate and ATP.
◼The maximal activity pH range is 7.5-8, requires presence of Mg2+ of
about 10mM and sulfhydryl reagents (DTT, 2-mercaptoethanol).

Dr. Amulya. G, The Oxford College of Engineering
E. coli DNA ligase
◼E. coli DNA ligase is a NAD+ dependent enzyme which catalyses the
formation of phosphodiester bonds between complementary 3’-
hydroxyl and 5’- phosphoryl termini of dsDNA.
◼The enzyme works best with cohesive dsDNA ends and is also active
on nicked DNA.
◼Blunt ends can be ligated in presence of condensing reagents like
polyethylene glycol.
◼The maximal activity pH range is 8, requires temperature of 16 ˚C.

Dr. Amulya. G, The Oxford College of Engineering
◼Applications
❑Cloning of restricted DNA to
vector to construct
recombinant vector.
❑Joining of adapters and linkers
to blunt end DNA molecule.
❑3’ end labelling of RNA, by
incorporating
32
P-labelled
mononucleotide-3’, 5’-
bisphosphate, which is added
to 3’-OH termini of RNA.
❑Synthesis of oligonucleotides.

Dr. Amulya. G, The Oxford College of Engineering
Enzymes In Nucleic Acid Modification
Phosphatases
•Phosphatases catalyse the cleavage of a phosphate (PO4) group from
substrate by using a water molecule (hydrolytic cleavage).
•On the basis of their activity there are two types of phosphatase, acid
phosphatase and alkaline phosphatase. Of the two, alkaline phosphatase
are most common.
◼Acid phosphatase - shows its optimal activity at pH between 3 and 6, a
lysosomal enzyme that hydrolyze organic phosphates liberating one or
more phosphate groups. It is found in prostatic epithelial cells, erythrocyte,
spleen, kidney etc.

Dr. Amulya. G, The Oxford College of Engineering
◼Alkaline phosphatase - Homodimeric metalloenzyme which catalyzes
reactions like hydrolysis and transphosphorylation of phosphate
monoester. They show their optimal activity at pH of about 10. It
hydrolyses or removes terminal monoesterified phosphate from
ribonucleotides, deoxyribonucleotides, alkaloids, proteins, etc.
Types of AP
Common alkaline phosphatases used in research include
◼Bacterial alkaline phosphatase (BAP) - Bacterial alkaline phosphate is
a phosphomonoester that hydrolyzes 3’ and 5’ phosphate from nucleic
acid (DNA/ RNA). BAP generally shows optimum activity at temperature
65°C. BAP is sensitive to inorganic phosphate so in presence of inorganic
phosphates activity may reduce.

Dr. Amulya. G, The Oxford College of Engineering
◼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.
◼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.

Dr. Amulya. G, The Oxford College of Engineering
SAP is used for 5’ dephosphorylation during
cloning experiments for various applications:
•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
application).
Fig: Action of alkaline phosphatase to prevent the recircularization
of vector plasmid

Dr. Amulya. G, The Oxford College of Engineering
Applications
•Removing 5' phosphates from fragments of DNA prior to labeling with
radioactive phosphate.Polynucleotide kinaseis much more effective in
phosphorylating DNA if the 5' phosphate has previously been removed.
•Removing 5' phosphates from plasmid and bacteriophage vectorsthat
have been cut with a restriction enzyme. In subsequent ligation reactions,
this treatment prevents self-ligation of the vector and thereby greatly
facilitatesligationof other DNA fragments into the vector (e.g. subcloning).

Dr. Amulya. G, The Oxford College of Engineering
POLYNUCLEOTIDE KINASE
•PNK is a homotetramer with phosphatase
activity at 3’ end and kinase activity at 5’ end
with a tunnel like active site.
• It catalyzes the transfer of phosphate group
from γ position of ATP to the 5' end of either
DNA/ RNA and nucleoside monophosphate.
• PNK can convert 3' PO
4/5' OH ends into 3'
PO
4/5' PO
4 ends which blocks further ligation
by ligase enzyme. Lys-15 and Ser-16 are
important for kinase activity of the enzyme.
•T4 polynucleotide kinase is the most widely
used PNK in molecular cloning experiments,
which was isolated from T4 bacteriophage
infected E.coli.

Dr. Amulya. G, The Oxford College of Engineering
◼PNK carries out two types of enzymatic activity:
•Forward reaction
•Exchange reaction
Applications
•used for radio labelling oligonucleotides, generally with
32
P for preparing hybridization
probes.
•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.

Dr. Amulya. G, The Oxford College of Engineering
Terminal transferase (terminal deoxyribonucleotidyl transferase)
◼Terminal transferase catalyses the addition of nucleotides to the 3’
terminus of DNA
◼Interestingly, it works on ssDNA including 3’ overhangs of ds DNA and is
thus an example of DNA polymerase that does not require a primer.
◼ It can also add homopolymers of ribonucleotides to 3’ end of DNA
◼ the much preferred substrate for this enzyme is protruding 3’ ends but it
will also less efficiently add nucleotides to blunt and 3’ recessed ends of
DNA fragements
◼Cobalt is necessary cofactor for activity of this enzyme
◼Terminal transferase is a mammalian enzyme, expressed in lymphocytes,
the enzyme purchased commercially is usually produced by expression of
bovine gene from E. coli.

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
Applications of terminal deoxyribonucleotidyl transferase
◼Adding complementary homopolymeric tails to DNA- this procedure was
used in past to clone into plasmid vectors but has largely been replaced
by other efficient techniques.
◼Basically, TT is used to tail a linearized plasmid vector with G’s and
cDNA with C’s.
◼When incubated together, the complementary G’s and C’s anneal to
‘insert’ the cDNA into vector which is then transformed into E.coli.
◼Labeling the 3’ ends of DNA – commonly the substrate for the reaction is
a fragement DNA generated by digestion with restriction enzyme that
leaves a 3’ overhang but oligonucleotide can also be used.
◼When such DNA is incubated with tagged nucleotides and TT a string of
tagged nucleotides will be added to the 3’ overhang or to 3’ end of
oligonucleotide .

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
DNase and RNase
◼Most of the time nucleases are the enemy of the molecular biologist
who tries to preserve the integrity of RNA or DNA samples.
◼However, DNase and RNase have certain indispensable roles in
molecular labs and so numerous types have been isolated and
characterized.
◼They differ in substrate specificity, cofactor requirements and
whether they cleave nucleic acids internally (endonucleases) or
chew in from the ends (exonucleases) or attack both of them.
◼In many cases the substrate specificity of nuclease depends upon
concentration of enzyme used in reaction, with high concentration
promoting less specific cleavages.

Dr. Amulya. G, The Oxford College of Engineering
RIBONUCLEASE (RNase)
•Ribonuclease(commonly abbreviatedRNase) is a type
ofnucleasethatcatalyzesthe degradation ofRNAinto smaller
components.
•It hydrolyses ribonucleotides from either single stranded or double stranded
RNA sequences.
•Ribonucleases can be divided into2 types depending on position of
cleavage - endoribonucleases(cleave internal bond) andexoribonucleases
(cleave terminal bond) and comprise several sub-classes.

Dr. Amulya. G, The Oxford College of Engineering
Major types of endoribonucleases
◼RNase A- commonly used in research. RNase A from bovine
pancreas is one of the hardiest enzymes in common laboratory usage.
It cleaves the 3'-end of unpaired C and U residues, ultimately forming a
3'-phosphorylated product via a 2',3'-cyclic monophosphate
intermediate. It does not require any cofactors for its activity.
◼RNase H - cleaves RNA in a DNA/RNA duplex to produce ssDNA. It is
a non-specific endonuclease and catalyzes the cleavage of RNA via a
hydrolytic mechanism, aided by an enzyme-bound divalent metal ion.
During cDNA library preparation from RNA sample, RNaseH enzyme is
used to cleave RNA strand of DNA-RNA duplex.

Dr. Amulya. G, The Oxford College of Engineering
◼RNase III- a type of ribonuclease that cleaves rRNA (16s rRNA and
23s rRNA) from transcribed polycistronic RNA operon in prokaryotes.
◼RNase L- an interferon-induced nuclease that, upon activation,
destroys all RNA within the cell.
◼RNase P- a type of ribonuclease that is unique in that it is
aribozyme– aribonucleic acidthat acts as a catalyst in the same way
as anenzyme. Its function is to cleave off an extra, or precursor,
sequence ontRNAmolecules.

Dr. Amulya. G, The Oxford College of Engineering
Major types of exoribonucleases
◼Polynucleotide Phosphorylase (PNPase) functions as
anexonucleaseas well as anucleotidyltransferase.
◼RNase Ris a close homolog of RNase II, but it can, unlike RNase II,
degrade RNA with secondary structures without help of accessory
factors.
◼RNase Dis involved in the 3'-to-5' processing of pre-tRNAs.
◼RNase Tis the major contributor for the 3'-to-5' maturation of many
stable RNAs.
◼Exoribonuclease Idegrades single-stranded RNA from 5'-to-3', exists
only in eukaryotes.

Dr. Amulya. G, The Oxford College of Engineering
RIBONUCLEASE A (RNASE A)
➢RNase A is a digestive enzyme whose function is to hydrolyze RNA
to its component nucleotides.
➢Bovine pancreatic RNase A is 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.

Dr. Amulya. G, The Oxford College of Engineering
Mechanism of action
◼RNaseA has two histidine residues in its active site (His12 and His119). In
the first step, His12 acts as a base; accepting proton forming a
nucleophile which then attacks positively charged phosphorus atom.
First is transphosphorylation followed by hydrolysis. A = His 12, B = His 119.

Dr. Amulya. G, The Oxford College of Engineering
Application
•It is used to remove RNA contamination from DNA sample.
•It is primarily used for removing unhybridized regions of RNA from
DNA-RNA hybrids
•Used for mapping single base mutations in DNA or RNA.
•To recognize and cleave single base mismatches in RNA-DNA or
RNA-RNA hybrid.

Dr. Amulya. G, The Oxford College of Engineering
◼RIBONUCLEASE H
•Ribonuclease H(RNase H)is a family of non-sequence
specificendonucleases thatcatalyze the cleavage ofRNAvia
ahydrolyticmechanism.
•It cleaves the3’-O-P bond ofRNAin aDNA/RNAduplexsubstrateto
produce 3’-hydroxyl and 5‘-phosphate terminated products.
• InDNA replication, RNase H is responsible for removing theRNA primer,
allowing completion of the newly synthesized DNA.
◼Applications- It is commonly used inmolecular biologyto destroy the RNA
template after first-strandcomplementary DNA(cDNA) synthesis
byreverse transcription.
◼The enzymeRNase Hcan selectively & repeatedly destroy only RNA probe
from DNA−RNA duplexes for signal amplification to detection limit
offemtomolelevel.

Dr. Amulya. G, The Oxford College of Engineering
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.

Dr. Amulya. G, The Oxford College of Engineering
DNase I
•DNase I is an endonuclease which cleaves ds or ss DNA and
chromatin. It is purified from bovine pancreas.
•It preferentially cuts from one end adjacent to C/T (pyrimidine)
residue and yields polynucleotides with 5’ phosphate terminal and
3’ free OH group.
•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.

Dr. Amulya. G, The Oxford College of Engineering
Applications
•Eliminating DNA contamination (e.g. plasmid) from
preparations of RNA.
•Clean-up of RNA prior to cDNA library preparation, RT-PCR,
Northern hybridization and in vitro transcription
•Analyzing the DNA-protein interactions via DNA foot printing.
•Nicking DNA prior to radio-labeling by nick translation.
•Creation of a fragmented library of DNA sequences for in vitro
recombination.

Dr. Amulya. G, The Oxford College of Engineering
◼Deoxyribonuclease II (DNase II)
•It is a non-specific endonuclease with optimal activity at acidic pH (4.5-5.5).
•It does not require any divalent cation for its activity.
•DNase II 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.
◼Applications
•DNA fragmentation
•Molecular weight marker
•Cell apoptosis assays

Dr. Amulya. G, The Oxford College of Engineering
POLYNUCLEOTIDE PHOSPHORYLASE (PNPASE)
•Polynucleotide Phosphorylase(PNPase) is a bifunctionalenzymewith
aphosphorolytic3' to 5'exoribonuclease activity and a 3'-
terminaloligonucleotidepolymeraseactivity.
•It dismantles the RNA chain starting at the 3' end and working toward the
5' end.
•It also synthesizes long chain polyribonucleotides (RNA) in 5’ to 3’
direction from nucleotide diphosphates as precursors.
•It is involved onmRNA processingand degradation in bacteria, plants,
and in humans and is present in bacteria, chloroplasts and mitochondria
of some eukaryotic cells.
•It catalyzes the synthesis of long chain polyribonucleotides (RNA) in 5’ to
3’ direction from nucleotide diphosphates as precursors.
•It also reversibly catalyzes phosphorolytic cleavage of polyribonucleotides
in 3’ to 5’ direction with a release of orthophosphate in presence of
inorganic phosphate.
•The function of PNPase depends upon inorganic phosphate (Pi)
concentration inside the cell.

Dr. Amulya. G, The Oxford College of Engineering
Applications
•Synthesis of radiolabeled polyribonucleotides as probes.
•Synthesis of homopolymers, heteropolymers and oligonucleotides with
defined sequence.
•Sequence analysis of oligonucleotides
•Used to probe structure and conformational aspects of duplex
polynucleotides
•Used to analyse size and composition of 3’-terminal sequence of RNA
molecules.

Dr. Amulya. G, The Oxford College of Engineering
Topoisomerase
◼Topoisomerase is also known as DNA gyrase inE. coli was discovered by
James C Wang.
◼The identical loops of DNA having different numbers of twists are
topoisomers.
◼This is the enzyme that solves the problem of the topological stress
caused during unwinding.
◼They cut one or both strands of the DNA allowing the strand to move
around each other to release tension before it rejoins the ends.
◼And therefore, the enzyme catalysts the reversible breakage it causes by
joining the broken strands and these transient breaks in DNA is unknotted
using a conserved tyrosine as catalytic residue.

Dr. Amulya. G, The Oxford College of Engineering
Types of Topoisomerase
◼Type I Topoisomerase – Cuts the one strand of DNA double helix,
relaxation occurs and then the cut strand is reannealed.
◼ Type II Topoisomerase – Cuts both strands of one DNA double helix,
passes another unbroken DNA helix through it and then anneals the cut
strand. It is also split into two subclasses-
◼Type IIA and type IIB topoisomerases which share similar structure and
mechanisms.
◼Eg-
◼Type IIA- eukaryotic topo II, E. coli gyrase, E. coli topo IV
◼Type IIB- eukaryotic topo VI

Dr. Amulya. G, The Oxford College of Engineering
Applications of topoisomerase
◼Antibacterial compounds - the type II enzymes, DNA gyrase and
DNA topoisomerase IV, have enjoyed enormous success as targets
Eg - Fluoroquinolones (FQs), Aminocoumarins, Proteinaceous
inhibitors.
◼Anti-cancer compounds - Both human topo I and topo II (both α and
β isoforms) can be targeted in anticancer chemotherapy.
Eg- Camptothecin (CPT), Etoposide (VP-16), Doxorubicin, Merbarone

Dr. Amulya. G, The Oxford College of Engineering
Polymerases
◼The group of enzymes that catalyses the synthesis of nucleic acid
molecules are collectively referred to as polymerases.
◼It is customary to use the name of nucleic acid template on which
the polymerase acts.
◼The three important polymerases are-
◼DNA dependent DNA polymerases - that copies DNA from DNA.
◼RNA dependent DNA polymerases - (reverse transcriptase) that
synthesizes DNA from RNA
◼DNA dependent RNA polymerases - that produces RNA from DNA

Dr. Amulya. G, The Oxford College of Engineering
◼Most polymerases can function only if the template possesses a ds
region that acts as a primer for initiation of polymerization.
◼Four types of DNA polymerase are used routinely in genetic
engineering. The first is 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.
◼DNA polymerase I is therefore an example of an enzyme with a dual
activity—DNA polymerization and DNA degradation.

Dr. Amulya. G, The Oxford College of Engineering
◼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.

Dr. Amulya. G, The Oxford College of Engineering
DNA polymerase
◼DNA polymerases are enzymes used for the synthesis of DNA by adding
nucleotide one by one to the growing DNA chain. The enzyme incorporates
complementary amino acids to the template strand.
◼DNA polymerase is found in both prokaryotic and eukaryotic cells. They
both contain several different DNA polymerases responsible for different
functions in DNA replication and DNA repair mechanisms.
◼Three different prokaryotic DNA polymerasesareknown, of which DNA
polymerases I and II are meant for DNA repair and DNA polymerase III is
meant for actual DNA replication,
◼(i)DNA polymerase I(isolated in 1960 byArthur Kornberg)was the first
enzyme suggested to be involved in DNA replication. This enzyme is now
considered to be a DNA repair enzyme ratherthana replication enzyme.
◼DNA polymerase I consists of two fragments : a larger fragment,
calledKlenow fragment,which contains 3'-5' exonuclease activity with 5'-
3' polymerising activity and a smaller fragment, which contains 5'-3'
exonuclease activity.
DNA polymerase in prokaryotes

Dr. Amulya. G, The Oxford College of Engineering
(ii)DNA polymerase IIresembles DNA polymerase I in its activity to bring
about the growth in 5'→3' direction, using free 3'-OH groups, but mainly
uses duplexes with short gaps only.
◼Although it has 3'→5' exonuclease activity, it lacks 5’→3' exonuclease
activity
(iii)DNA polymerase IIIplays an essential role in DNA replication.
✓This holoenzyme is the main polymerase inE.coliDNA replication and is
one of the family C polymerases.
✓Polymerase III is made up of the clamp-loading complex, the beta sliding
clamp processivity factor and the Pol III core.
✓The core comprises three subunits – the α subunit which is the
polymerase activity hub, the δ subunit which is the exonucleolytic proof
reader, and the θ subunit which may stabilize δ.
Pol IV
◼This enzyme belongs to the Y family of DNA polymerases. Pol IV is an
error-prone polymerase that has no 3’ to 5’ proofreading activity and is
involved in mutagenesis or the altering of DNA to give rise to a mutation.
Pol V
◼Pol V also belongs to the Y family of polymerases and allows DNA
damage to be bypassed in order for replication to continue.

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
Enzymes in Eukaryotic DNA Replication
A.Eukaryotic DNA Polymerases DNA Polymerase α
◼Involved in initiation
◼Synthesizes an RNA primer then adds dNTPs
◼A complex of four subunits
◼50-kD and 60-kD are primase subunits;180-kD subunit DNA polymerase
◼Synthesizes 8-10 nt RNA primers, then adds DNA to the RNA primers
◼Low processivity of DNA synthesis (200 nt)
◼Has no 3’ -5’ exonuclease activity (proofreading), yet has high fidelity

Dr. Amulya. G, The Oxford College of Engineering
B. DNA Polymerase δ
◼The principal DNA polymerase in eukaryotic DNA replication
◼Has 3’-5’ exonuclease activity
◼Consists of a 125 kdal and a ~50 kdal subunit
◼The 50 kd subunit interacts with PCNA (Proliferating Cell Nuclear
Antigen)
◼Has high processivity when in association with PCNA.
C. DNA polymerase ɛ
◼ Required for replication, but its role is unclear
◼ May substitute for DNA polymerase d in lagging strand synthesis
◼DNA polymerase β
◼Helps in DNA repair (doesn’t participate in replication)

Dr. Amulya. G, The Oxford College of Engineering
RNA polymerase
◼Ribonucleic Acid (RNA)Polymerase (RNAP) enzyme is a multi-
subunit enzyme that applies its activity in the catalyzation of the
transcription process of RNA synthesized from a DNA template.
•And therefore, RNA polymerase enzyme is responsible for the copying of
DNA sequences into RNA sequences during transcription.
•The function of RNA polymerase is to control the process of transcription,
through which copying of information stored in DNA into a new molecule of
messenger RNA (mRNA.)
•During transcription, the RNA polymer is contemporary to the template
DNA that is synthesized in the direction of 5′ to 3′.
•The enzyme RNA polymerase interacts with proteins to enable it to
function in catalyzation of the synthesis of RNA.

Dr. Amulya. G, The Oxford College of Engineering
•The collaborator proteins assist in enabling the specific binding of RNA
polymerase, assist in the unwinding of the double chemical structure of
DNA, moderate the enzymatic activities of RNA polymerase and to
control the speed of transcription.
•The RNA polymerase enzyme has an interrupted mechanism whereby it
continuously synthesizes RNA polymers of over four thousand bases per
minute but they pause or stop occasionally to maintain fidelity.
•RNA polymerase is an enzyme that is responsible for copying a DNA
sequence into an RNA sequence, during the process of transcription. As
a complex molecule composed of protein subunits, RNA polymerase
controls the process of transcription, during which the information stored
in a molecule of DNA is copied into a new molecule of messenger RNA.

Dr. Amulya. G, The Oxford College of Engineering
◼Types of RNA polymerase
◼Prokaryotic (Bacteria, viruses, archaea) organisms have a single type of
RNA polymerase that synthesizes all the subtypes of RNA, while eukaryotes
(multicellular organisms) have 5 different types of RNA polymerases which
perform different functions in the synthesis of different RNA molecules.
◼Prokaryotic RNA polymerase
•The prokaryotes have a single type of RNA polymerase (RNAP) which
synthesizes all the classes of RNA, i.e mRNA, tRNA, rRNA, sRNA.
•The RNA Polymerase molecule is made up of 2 domains and 5 subunits:
i.Core and holoenzyme
ii.Subunits (β, β’, α (αI and αII), ω,)
•The promoter is the sequence of DNA that is required for accurate and
specific initiation of transcription, and also, it is the sequence of DNA to which
RNA polymerase binds accurately to initiate transcription.

Dr. Amulya. G, The Oxford College of Engineering
Each of the subunit structure is as follows: Prokaryotic RNA Polymerase
Subunits

•The ‘a’ subunit is made up of two distinct domains. The N-terminal domain (a-NTD)
and the C-terminal.
•The N-terminal is involved in dimerization forming a2 and further assembly of the
RNA polymerase.
•The C-terminal domain functions such as binding to the Upstream Promoter (UP)
DNA sequence at promoters for rRNA and tRNA genes and in communication with
several transcriptional activators.
Subunit Size Function
β 150.4 kDa The β’ + β form the catalytic center, responsible for RNA synthesis.
β’ 155.0 kDa The β’ + β form the catalytic center, responsible for RNA synthesis.
α (αI and
αII)
36.5 kDa
It is made up of the enzyme assembly, and it also binds the UP
sequence in the promoter.
ω 155.0 kDa
It confers specificity for promoter; and binds to -10 and -35 sites in
the promoter.

Dr. Amulya. G, The Oxford College of Engineering
Eukaryotic RNA polymerase
•There are 5 known types of RNA polymerases each responsible for the synthesis of
specific subtypes of RNA. These include:
•RNA polymerase I that synthesizes a pre-rRNA 45S (35S in yeast), which matures
and forms the major RNA sections of the ribosome.
•RNA polymerase IIsynthesizes precursors of mRNAs and mostsnRNAand
microRNAs.
•RNA polymerase III synthesizes tRNAs, rRNA 5S, and othersmall RNAsfound in
thenucleusandcytosol.
•RNA polymerase IV and V found in plants are not well understood, however, they
make siRNA. The plant chloroplast encodes the ssRNAPs and uses bacteria-like RNA
Polymerase.
•Each of the nuclear RNA polymerases is a large protein molecule with about 8 to 14
subunits and the molecular weight is approximately 500,000 for each.
•They commonly have 3 subunits, a, b and b’. The largest subunits being b and b’.
•These subunits are used as catalytic promoters and for assembly of proteins.
•Each of these polymerases has a different function:

Dr. Amulya. G, The Oxford College of Engineering
◼RNA polymerase I
•This enzyme is located in the nucleolus of the cell.
•It is a specialized nuclear substructure where the ribosomal RNA (rRNA) is
synthesized by transcription and assembled into ribosomes.
•The rRNA are component elements of the ribosomes and are important in the
process of translation.
•Therefore, RNA polymerase I synthesize almost all rRNAs except 5S rRNA.
•In yeast, the enzyme has a mass of 600kDa and 13 subunits.

Dr. Amulya. G, The Oxford College of Engineering
◼RNA polymerase II
•This enzyme is located in the nucleus.
•Most organisms that possess RNA polymerase IIhave a 12-subunit RNAP II
(with a mass of about 550 kDa)
•It is structurally made up of holoenzyme and mediators, with General
Transcriptional factors (GTFs).
•They contain transcription factors and transcriptional regulators.
•It functions by synthesizing all proteins that code for the nuclear pre-
mRNAs in eukaryotic cells (mRNAs in prokaryotic cells).
•It is responsible for transcribing most of the eukaryotic genes and especially
found in human genes.

Dr. Amulya. G, The Oxford College of Engineering
RNA polymeraseIII
•It is located in the nucleus.
•The RNA polymerase III has 14 or more distinct subunits with a mass of
approximately 700 kDa.
•Its function is to transcribe transfer RNA (tRNA), ribosomal RNA (rRNA), and
other small RNAs.
•Some of its target points are important for the normal functioning of the cell

Dr. Amulya. G, The Oxford College of Engineering
◼RNA polymerases IV and V
•They are exclusively found in plants, and they perform combined action in the
formation of small interfering RNA and heterochromatin in the cell nucleus.
•In Plants, the RNA polymerase is found in the chloroplast (plastids) and
mitochondria, encoded by the mitochondrial DNA.
•These enzymes are much more related to bacterial RNA polymerase than to the
nuclear RNA polymerase.
•Their function is to catalyze specific transcription of organelle genes.

Dr. Amulya. G, The Oxford College of Engineering
Functions of RNA Polymerase
•Generally, the RNA molecule is a messenger molecule that is used to
export information that is coded in DNA out of the cell nucleus, to
synthesize proteins in the cell cytoplasm.
•RNA polymerase is used in the production of molecules that play a wide
range of roles, of which one of its functions is to regulate the number and
type of RNA transcript that is formed in response to the requirements of
the cell.
•The RNA polymerase enzyme interacts with different molecular
proteins, transcription factors, and signaling molecules on the
carboxyl-terminal, which regulates its mechanisms, which play a major
role in gene expression and gene specialization in multicellular
(eukaryotic) organisms.

Dr. Amulya. G, The Oxford College of Engineering
•The RNA enzyme also ensures irregularities and errors during the
conversion of DNA to RNA (transcription). Such as ensuring that the right
nucleotide is added to the newly synthesized RNA strand, inserting the right
amino acid-base which is complementary to the template of the DNA strand.
•When the right nucleotides have been inserted, the RNA polymerase can
then catalyze and elongate the RNA strand, at the same time, proofread
the new strand and remove incorrect bases.
•RNA polymerase is also involved in the post-transcription modification
of RNAs, converting them into functional molecules that facilitate the
transportation of molecules from the nucleus to their site of action.

Dr. Amulya. G, The Oxford College of Engineering
•Besides its role in the synthesis of proteins, RNA performs other
functions such as
•Protein coding
•Regulation of gene expression
•Act as enzymes
•Formation of gametes by the non-coding RNA (ncRNA)
•Production of regulatory molecules.

Dr. Amulya. G, The Oxford College of Engineering
Reverse transcriptase
◼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.
◼Retroviruses (possessing RNA) contain RNA dependent DNA polymerase
which is called reverse transcriptase. This produces single stranded DNA,
which in turn functions as template for complementary long chain of DNA.
◼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.
◼The enzyme is very useful for the synthesis of cDNA and construction of
cDNA clone bank and to make short labelled probes.

Dr. Amulya. G, The Oxford College of Engineering
◼Reverse Transcriptase also known as RNA directed DNA Polymerase • -
DNA Nucleotidyl transferase (RNA directed) • - Revertase •
◼Reverse Transcriptase was discovered by Howard Temin and Baltimore
in 1970 independently. He shared Nobel Prize in Physiology or Medicine
in 1975 for their discovery.
◼Reverse transcriptase common in Retrovirus. Eg • - HIV • M-MLV
(Moloney Murine Leukemia Virus) • AMV (Avian Myeloblastosis Virus)
◼Reverse Transcriptase enzyme includes two activity: DNA polymerase
and RNase H.

Dr. Amulya. G, The Oxford College of Engineering
Mechanism of Retrovirus Replication
1. A Retrovirus specific cellular tRNA hybridizes with a complementary
region called PBS (Primer Binding Sites)
2. Reverse Transcriptase (RT) starts at this binding site and copies RNA into
a single strand of complementary DNA. A DNA segment is extended from
tRNA based on the sequence of the retroviral genomic RNA.
◼Typical retrovirus has three or four genes.
◼Retrovirus are called + strand because viral RNA itself code for protein
products. Reverse transcriptase enzyme code for proteins are called –
strand
◼Like DNA polymerase, Reverse transcriptase requires primers.
◼tRNA of the host is Primer

Dr. Amulya. G, The Oxford College of Engineering
3. The viral R and U5 sequences are removed by RNase H.
4. First jump: DNA hybridizes with the remaining R sequence at the 3' end.
5. A DNA strand is extended from the 3' end.
6. Most viral RNA is removed by RNase H.
7. A second DNA strand is extended from the viral RNA.
8. Both tRNA and the remaining viral RNA are removed by RNase H.
9. Second jump: The PBS region of the second strand hybridizes with the
PBS region of the first strand.
10. Extension on both DNA strands.

Dr. Amulya. G, The Oxford College of Engineering
Application
◼Antiviral drugs - As HIV uses reverse transcriptase to copy its genetic
material and generate new viruses (part of a retrovirus proliferation circle),
specific drugs have been designed to disrupt the process and thereby
suppress its growth. include the nucleoside and nucleotide analogues
zidovudine, lamivudine and tenofovir, as well as non-nucleoside
inhibitors, such as nevirapine.
◼Molecular biology- Reverse transcriptase is commonly used in research to
apply the polymerase chain reaction technique to RNA in a technique
called reverse transcription polymerase chain reaction (RT-PCR).
◼Reverse transcriptase is used also to create cDNA libraries from mRNA.

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
Gene cloning- Steps involved in gene cloning
◼Gene cloning
▪Gene cloning involves separation of specific gene or DNA fragments
from a donor cell, attaching it to small carrier molecule called vector
and then replicating this recombinant vector into a host cell.
◼Steps involved in gene cloning
◼Isolation of donor DNA fragment or gene
◼Selection of suitable vector
◼Incorporation of donor DNA fragment into the vector
◼Transformation of recombinant vector into a suitable host cell
◼Isolation of recombinant host cell

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
1. Isolation of donor DNA fragment or gene
▪At first a donor DNA fragment should be isolated. There are two method
for isolation of desired gene or DNA fragment.
▪Using restriction endonuclease enzyme: the enzyme restriction
endonuclease is a key enzyme in molecular gene cloning. It has specific
restriction site for its action. The enzyme RE generates a DNA fragment
either with blunt end or with sticky end
▪Using reverse transcriptase enzyme: reverse transcriptase enzyme
Synthesizes complementary DNA strand of the desired gene using its
mRNA.

Dr. Amulya. G, The Oxford College of Engineering
2. Selection of suitable cloning vector
▪When donor DNA fragment is incorporated into a host cell, it will not
replicates because the isolated gene do not have the capacity to
replicated itself. So before introduction of donor fragment into host, a
suitable vector should be selected.
▪Cloning vector is the DNA molecule capable of self-replication inside the
host cell. the main function of cloning vector is to replicates the inserted
DNA fragment inside the host cell.
▪Examples of cloning vectors: Plasmid, BAC, YAC, Λ-bacteriophase,
expression vectors etc.
◼Characteristics of a cloning vectors
◼It must be self-replicating inside host cell
◼It must possess restriction site for RE enzymes
◼Introduction of donor DNA fragment must not interfere with replication
property of the vector
◼It must possess some marker gene such that it can be used for later
identification of recombinant cell.

Dr. Amulya. G, The Oxford College of Engineering
3. Incorporation of donor DNA fragment with Plasmid vector
▪The plasmid vector is cut open by the same RE enzyme used for isolation
of donor DNA fragment
▪The mixture of donor DNA fragment and plasmid vector are mixed
together.
▪In the presence of DNA ligase, base pairing of donor DNA fragment and
plasmid vector occurs forming recombinant vector in the mixture

Dr. Amulya. G, The Oxford College of Engineering
4. Transformation of recombinant vector into suitable host
▪The recombinant vector is transformed into suitable host cell. ie
bacterial cell
▪Some bacteria are naturally transformable, they take up the
recombinant vector automatically. For examples:Bacillus,
Haemophillus, Helicobacter pylori, are naturally competent
▪Some other bacteria are not naturally competent, in those bacteria
recombinant vector are incorporated by artificial method such as Ca++
ion treatment, electroporation etc

Dr. Amulya. G, The Oxford College of Engineering
5. Isolation of recombinant cell
▪The recombinant host cell is then grown in culture media but the culture
may contains colonies both recombinant cell and non-recombinant cell.
▪For isolation of recombinant cell from non-recombinant cell, marker gene
of plasmid vector is employed.
▪For examples, PBR322 plasmid vector contains different marker gene
(Ampicillin resistant gene and Tetracycline resistant gene. When pst1 RE
is used it knock out Ampicillin resistant gene from the plasmid, so that the
recombinant cell become sensitive to Ampicillin.

Dr. Amulya. G, The Oxford College of Engineering
1. USING RESTRICTION ENZYMES
◼This is the traditional cloning technique where a gene of interest is
inserted in to a vector by a cut and paste method. Restriction digestion
of both gene of interest and vector was performed by using restriction
enzymes that cut the gene at a specific sequence. Several enzymes
like EcoR I, BamH I, Nco I, Nde I, Xho I etc.,are used.
◼Digestion with some of these enzymes result in a sticky end and some
in blunt ends. After the digested fragments arecleaned up, the
digested gene and vector are ligated to form a recombinant plasmid
using DNA ligase enzyme. There are so many standardized protocols
for this technique, but in some cases optimization of buffer is required
and time of restriction digestion also plays a major role.
◼Procedure – follow cloning techniques

Dr. Amulya. G, The Oxford College of Engineering
2.TA CLONING OR TA/TOPO CLONING
◼TA cloning is an easy, rapid cloning technique that consumes less time
compared to traditional cloning method. It relies on the hybridization of the
complementary base pairs adenine (A) and thymine (T).
◼Here a single enzyme is used for both digestion and ligation called
Topoisomerase I that digest DNA at specific site 5’-(C/T)CCTT-3’ and
ligates at 3’ phosphate group of thymidine base. This can be performed
within 5 to 10 minutes. TOPO cloning thus does not need restriction
enzymes.
◼Polymerase enzyme used is also very important in this technique, like Taq
DNA polymerase. Taq enzyme add poly A tail to the PCR product which is
complementary to the thymidine residues and thus can easily be ligated.
◼Procedure – follow cloning techniques

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
3.LIGATION INDEPENDENT CLONING
◼Ligation independent cloning is cost effective simple cloning technique.
◼The gene was amplified with specific gene sequences of 12 nt length
complementary to modified LIC vectors.
◼These vectors have to be linearized by PCR or by restriction digestion.
Both enzyme and vector were then incubated with T4 DNA polymerase.
◼This polymerase has 3’ to 5’ exonuclease activity resulting in overhangs
that are complementary to both gene and vector.
◼Later dCTP was added to get back its polymerase activity.
◼The resulting clone will have nicks which are later repaired by E.coli cells.
◼Procedure – follow cloning techniques

Dr. Amulya. G, The Oxford College of Engineering

Dr. Amulya. G, The Oxford College of Engineering
Linker and Adaptors
◼Linkers and adaptors are oligonucleotides that are useful in DNA ligation.
They are chemically synthesised molecules. In recombinant DNA
technology, DNA sequences are cut and need ligation to join back the
strands. The cleaved DNA often has blunt or sticky ends. Sticky ends are
easy to ligate, but DNA strands with blunt ends are hard to ligate. The
linker and adaptor molecules then come into play.
◼The linker and adapter molecules also have internal restriction sites, which
help in DNA ligation.
◼What is a Linker?
◼The linkers are short double-stranded sequences of DNA. They have blunt
ends. They are chemically synthesised oligonucleotides. They are ligated
with the blunt ends of a vector of foreign DNA.

Dr. Amulya. G, The Oxford College of Engineering
◼It contains restriction sites for the identification of restriction enzymes.
Therestriction enzymescleave the ligated linker and the DNA fragment to
produce cohesive or sticky ends.
◼One drawback of linkers is that the foreign DNA fragment sometimes
already possesses restriction sites for the enzymes that are used to cut the
linker, resulting in cleaving the DNA internally. This limits the use of linker
molecules.
◼Example: Eco-RI linkers and Sal-I linkers
◼What is an Adaptor?
◼An adaptor molecule is a double-stranded, chemically synthesised
oligonucleotide that is used for the ligation of DNA.
◼Uses of adaptor molecules include adding sticky ends to cDNA for easy
ligation into theplasmidsand similar vector ligations.

Dr. Amulya. G, The Oxford College of Engineering
◼The adaptors have one sticky and one blunt end. The blunt end is ligated
with the blunt end of the target DNA producing a DNA fragment with
sticky ends. The sticky end helps in the easy ligation of the DNA
fragments. The adaptors also have restriction sites that can be used to
create new protruding terminuses by the action of restriction enzymes.
◼One disadvantage of adaptor molecules is that two sticky ends can join to
form dimers resulting in the blunt end DNA molecule again. This can,
however, be prevented by treating the molecules with alkaline
phosphatases.
◼(For Diagram – refer DNA ligase)

Dr. Amulya. G, The Oxford College of Engineering
Linker Adaptor
Description
Linkers are chemically
synthesized
oligonucleotides that have
two blunt ends.
Adaptors are chemically
synthesized
oligonucleotides with one
blunt and one sticky end.
Tail
No single-stranded tail is
present.
A single-stranded tail is
present at the sticky end.
Disadvantage
The DNA fragment might
already have a restriction
site, limiting the use of
linker molecules.
The adaptor molecules
tend to join and make
dimers.

Thank You !

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