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Biotechnology - I

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Study of cloning vectors,
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restriction end nucleases and

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DNA ligase.
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Chimeara
 Molecular Cloning
• Molecular cloning is a process of creating recombinant DNA and

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generally involves the following steps:

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1. Selection and Preparation of foreign DNA to be cloned and vector

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2. Selection of a host organism
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3. Creation of recombinant DNA vector having foreign DNA

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4. Introduction of recombinant vector into host organism

5. Selection of clones having recombinant vector.

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DNA cloning
 Cloning Vectors
Vector is an autonomously replicating DNA molecule designed from a plasmid or phage DNA to

carry a foreign DNA inside the host cell. In simple words it can be summarized as the given

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points.

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1. Vectors are autonomously self replicating DNA molecules that can be used to accomodate

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foreign DNA fragments.

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2. Vectors are DNA sequences that are found naturally, modified to serve particular functions.
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3. Selection of a vector depends mainly on its capability to accommodate desired size of DNA

molecules that have to be carried.

4. Different cloning vectors have been engineered for specific purposes, including sequencing,

expression of proteins and expression of RNA etc

 Characteristics of a good cloning
vector
1. It should autonomously replicate that means it should have ori region (origin of
replication).

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2. A vector should contain at least one selectable marker like gene for antibiotic resistance

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and it may contain a reporter marker (β -galactosidase, green fluorescent protein etc.)

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3. Presence of unique restriction enzyme site, this region is also known as multiple cloning

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sites.

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4. Preferably small in size and easy to handle.
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5. Relaxed control of replication to obtain multiple copies of recombinant vector.

6. Presence of appropriate regulatory elements for expression of foreign gene.

7. The selection of a suitable vector system depends mainly on the size limit of insert
DNA and the type of host intended for cloning or expression of foreign DNA
 Type of manipulated vectors
Allowing the exogenous DNA to be inserted, stored, and manipulated vectors can be
classified as following

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1. Plasmid Vectors- Plasmids are basically designed for cloning of small DNA

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fragments in a bacterial host.

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Example- pUC18, pUC19, pMB322 etc.

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2. Phagemids- Phagemids are single stranded circular DNA which is widely used
for sequencing purposes. ha
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Example-M13
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3. Cosmids – Cosmids are hybrid vectors which have both the oris of Bacterial
and viral origins, the cos site present in cosmids enable them to be packed into a
viral coat and thus these can easily get absorbed by host bacterium.
 Artificial chromosomes
• There are some artificial chromosomes, capable to carry mega bases of
DNA fragments.
1. Bacterial Artificial Chromosomes

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2. Yeast artificial Chromosomes

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3. Mammalian artificial chromosomes.

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• Cosmids, BAC, YAC and MACs are known as high cloning capacity vectors

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having

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capability to accommodate huge size DNA molecules.
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 m
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Biotechnology -III

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Study of cloning vectors,
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restriction end nucleases and

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DNA ligase.
 Origins of replication
The replication of DNA does not initiates at any random site in the
genome, but rather it starts at specific points known as origin of
replication. With the start of DNA synthesis, two replication forks
in opposite direction proceed to complete replication of the
whole genome. E. coli like bacteria, have only single origin of
replication (called OriC). While Eukaryotic organisms cells have

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multiple origins of replication and these are different from OriC.

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Saccharomyces cerevisiae a yeast has been recorded to have

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about 300 replication origins, where as DNA replication in human

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cells initiates over 2,000 origins for replication of the genome.
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OriC is a 245bp DNA sequence, it acts as the binding site to start
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replication for a number of proteins (namely DnaA, B and C). The
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melting (opening) of DNA helix starts after binding of these

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proteins promotes, this process is essential for further action of

DNA replicating enzymes. In both prokaryotes and eukaryotes
replication origins serve the same overall function, but both the
replication origins of prokaryotic cells and eukaryotic are
functional in their cell system only.
 Restriction-modification
• There are two component of Bacterial restriction–modification
systems, a restriction endonuclease and a DNA methylase. The
restriction enzyme (or restriction endonuclease) cut DNA at specific

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sequences known as recognition site. The term ‘endonuclease’ used

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for the sequence specific nucleases that cleaves nucleic acid chains

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anywhere within the DNA, not at the ends of the molecule.

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• The first restriction enzyme to be isolated was that from E. coli K
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laboratory strains in 1968 (Meselson and Yuan). This enzyme was

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able to cleave DNA, but the precise site of DNA cleavage remained
unclear. HindII was the first endonuclease (1970), isolated from
Haemophilus influenzae strain Rd, which cuts DNA at specific
recognition site.
 Restriction endonucleases
Werner Arber, Daniel Nathans and Hamilton O. Smith got
the Nobel Prize for this discovery in 1978.

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 Type I restriction enzymes
• These enzymes have both restriction and modification activities.
Restriction depends upon the methylation, whether the target DNA
is methylated or not. In case of type I restriction endonuceases, the

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cleavage takes place approximately 1000bp away from the site of

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recognition.

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• In case of type I restriction endonucleases, the recognition site is

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asymmetrical, composed of two specific portions of 3-4 nucleotides
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first and 4-5 nucleotides second one. Both the parts of the type I RE
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are separated by a nonspecific spacer of approximatly 6-8

nucleotides
 Type II restriction enzymes
• In type II restriction enzymes the restriction-modification are performed
by different enzymes so it is possible to cleave DNA in the absence of
modification. Both the enzymes are responsible for recognizing the

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same target sequence, but they can be purified separately.

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• The digestion of nucleotide sequence occurs at the restriction site.

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These enzymes are used to identify rotationally symmetrical sequence

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which is often called as palindromic sequence. Type II endonucleases

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need Mg2+ as a cofactor and ATP is not required for their activity. Type II

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endonucleases are widely utilized for mapping and reconstructing DNA
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in vitro due to their ability to recognize specific cleavage sites and they
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cleave within these sites

 Type III restriction enzymes
• The type III restriction enzymes recognize and methylate the same
DNA sequence but digest DNA about 24-26 bp away. These RE have
two different subunits, Subunit(M) is responsible for recognition as

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well as modification of DNA sequence while other subunit (R) works

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as nuclease.

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• Mg+2 ions, ATP are needed for DNA cleavage and process of

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cleavage is stimulated by SAM. Cleave only one strand. Two
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recognition sites in opposite orientation are necessary to break the

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DNA duplex.
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Types of restriction enzymes
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Common restriction enzymes
 DNA Ligase
After cutting DNA molecules, we now need to consider how to join them together
to
form recombinant DNA Molecule. The formation of phosphodiester bond
between
two DNA chains are catalized by DNA ligases For the formation of

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phosphodiester bonds DNA ligase enzymes need a free hydroxyl group at the 3’-

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end of one DNA chain and a phosphate group at the 5’-end of the other chain.

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Energy is required in form of NAD+ or ATP for the formation of a phosphodiester

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bond between these groups. In E. coli and other bacteria the energy is supplied

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in the form of NAD+, whereas in bacteriophages and animal cells, ATP helps the

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reaction.
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(Weiss and Richardson, 1967;

Zimmerman et al., 1967).
Any DNA ligases can join DNA molecules that are part of a
double helix, it means that these ligases are unable to join
two chains of single stranded DNA molecules. The ligase
enzyme from bacteriophage T4 will ligate blunt-ended DNA
fragments.

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Marker genes