Plant Biotechnology
Unit 2: Presentation 1
Semester 3rd
Dr. Amit Kumar Mishra
Assistant Professor
Department of Botany
School of Life Sciences
Mizoram University
Aizawl, India - 796004
Gene Cloning: Cutting and joining DNA Molecules
▪ The term gene cloning can be defined as the isolation and
amplification of an individual gene sequence by insertion of
that sequence into a bacterium where it can be replicated.
▪ The basic events in gene cloning have been described in the
following steps and shown in figure (right hand side):
1. Isolation of the gene of interest.
2. A fragment of DNA (gene of interest) to be cloned is
incorporated into a small replicating (usually circular) DNA
molecule called a vector. This can be an E. coli plasmid, a
virus, a cosmid, etc. The vector with an incorporated gene
is called a recombinant vector or molecule.
3. The recombinant vector is introduced into a host cell by
transformation.
4. Cells that have acquired recombinant DNA molecule are
selected.
5. Recombinant DNA molecule is multiplied within the host
cell to produce several identical copies of the cloned gene.
Cloning vectors
▪ One of the most important elements in recombinant DNA (rDNA) technology or in gene cloning is
the vector. A random DNA or cDNA segment or specific gene is linked into a vector to form rDNA
molecule, which can be propagated in suitable host cells to a large number, is a cloning vector.
▪ There are different types of cloning vectors for use with different types of host cells. The largest
number exists for E. coli and the best known of these is the plasmid vector. Besides, there are
cosmids, phages, phagemids, yeast artificial chromosomes, transposons, bacterial artificial
chromosomes, etc.
▪ A vector should have the following features:
1) It must contain a replicon that enables it to replicate in host cells.
2) It should have several marker genes, which help to differentiate the transformed cells from the
non-transformed cells and also from the transformed cells, which contain recombinant DNA
molecules, e.g., genes for ampicillin and tetracycline resistance.
3) It should have a unique cleavage site within one of the marker genes so that insertion of
foreign DNA into the marker gene leads to its inactivation and identification of recombinant
DNA molecule.
4) For the expression of cloned DNA, the vector DNA should contain suitable control elements,
such as promoters, terminators, and ribosome binding sites.
Plasmids
▪ Plasmids are double-stranded, closed circular DNA molecules, which exist in the cell as
extrachromosomal units. They are self replicating, and found in a variety of bacterial species,
where they behave as accessory genetic units.
▪ Plasmids replicate and are inherited independently.
▪ There are three general classes of plasmids: (i) virulence plasmids, which encode toxin genes,
(ii) drug resistance plasmids, which confer resistance to antibiotics, and (iii) plasmids, which
encode genes required for bacterial conjugation.
▪ Basically, all molecular cloning methods involve the manipulation of specific DNA fragments
that utilize some aspects of different plasmids.
▪ Plasmids range in size from 1 to 200 kb and depend on host proteins for maintenance and
replication functions.
▪ Most of the plasmid-encoded genes that have been characterized impart some growth
advantage to the bacterial host. These are present in a characteristic number of copies in a
bacterial cell, yeast cell or even in organelles (mitochondria) of eukaryotic cells.
▪ These plasmids can be single copy plasmids that are maintained as one plasmid DNA per cell
or multicopy plasmids that are maintained as 10–20 genomes per cell.
….continued
A plasmid vector used for cloning is specifically developed by adding certain features:
1. Reduction in size of vector to a minimum to expand the capacity of vector to clone large
fragments. Since the efficiency of transformation of bacterial cells drops drastically when
plasmids larger than 15 kb are used, the size of cloning vector should be small, preferably
3–4 kb. In this way foreign DNA fragments of 10–12 kb can be accommodated.
2. It should contain an origin of replication that operates in the organism into which the
cloned DNA is to be introduced.
3. Introduction of selectable markers.
4. Introduction of synthetic cloning sites termed polylinker, restriction site bank, or
polycloning sites that are recognized by restriction enzymes. This polycloning site is usually
present inside a marker gene so that with the insertion of foreign DNA it will inactivate
that marker gene and the recombinants can be selected.
5. Incorporation of axillary sequences such as visual identification of recombinant clones by
histochemical tests, generation of single stranded DNA templates for DNA sequencing,
transcription of foreign DNA sequences in vitro, direct selection of recombinant clones and
expression of large amounts of foreign proteins.
….continued
▪ pBR322 is one of the first vectors to be
developed by Boliver and Rodriguez in 1977.
▪ The name pBR322 denotes the following:
1. P- plasmid
2. B- Boliver
3. R- Rodriguez
▪ 322- Differentiate it from the other plasmid
produced in the same laboratory,
e.g., pBR325, pBR327, etc.
▪ It is 4361 base pair long. (GATE, ICAR-NET)
▪ It carries two sets of antibiotic resistance gene:
• Ampicillin
• Tetracycline
▪ It contains only single or unique recognition site
for 12 different restriction enzymes:
• Pst I
• Sca I A pBR322 plasmid vector
• Pvu I
….continued
pUC plasmids:
• pUC plasmids are small, high copy number plasmids of size
2686bp.
• This series of cloning vectors were developed by Messing and co-
workers in the University of California.
• pUC vectors contain a lacZ sequence and multiple cloning site
(MCS) within lacZ. This helps in use of broad spectrum of restriction
endonucleases and permits rapid visual detection of an insert.
• pUC18 and pUC19 vectors are identical apart from the fact that
the MCS is arranged in opposite orientation.
• pUC vectors consists of following elements:
➢ pMB1 “rep” replicon region derived from plasmid pBR322 with
single point mutation (to increase copy number). A pUC plasmid vector
➢ “bla” gene encoding β lactamase which provide ampicillin
resistance which is derived from pBR322. This site is different
from pBR322 by two-point mutations.
➢ E.coli lac operon system.
• “rop” gene is removed from this vector which leads to an
increase in copy number.
Cosmids
▪ A cosmid, first described by Collins and Hohn in 1978, is a type of hybrid plasmid
with a bacterial “ori” sequence and a “cos” sequences derived from the lambda
phage.
▪ It is formed by joining ends of a linearized plasmid DNA with cos-site of lambda
DNA.
▪ It is a derived vector.
▪ The cosmid DNA can be packed in a capsid of lambda phage in vitro to form
recombinant phage particles.
▪ It is linear inside the phage capsid.
▪ The cosmid gets circularized and behaves like a plasmid.
▪ Cosmid has an origin of replication, selectable markers, and gene cloning sites of
plasmid DNA.
▪ They lack structural and regulatory genes of lambda DNA.
▪ Hence there is no lysis and integration of cosmid DNA in the host cell.
▪ Examples: Col EI cosmid, pHC 79, pJB8, pWE cosmid, etc.
….continued
Cosmid pJB8
▪ pJB8 is constructed from the plasmid
pBR322 and cos sites of lambda DNA.
▪ It is 5.4 kbp in size.
▪ It has an origin of replication and ampicillin
resistance gene derived from pBR322 and
two cos-ends from lambda DNA.
▪ A foreign DNA of about 45 kbp is inserted
into BamHI or RcoRI or HindIII restriction
site of the cosmid.
▪ The recombinant cosmid is packaged into
lambda phage head to form an infective
phage particle.
▪ The phage delivers its rDNA into E.coli while
infecting the cell.
Bacteriophages
▪ Bacteriophages are viruses that infect bacteria. These are usually called phages.
▪ All bacteriophages are composed of a nucleic acid molecule that is surrounded by a protein
structure.
▪ Phages can be double stranded (T2, T4, T6, λ) or single stranded (ϕX174, M13). There are
both DNA (T2, T4, T6, λ, ϕX174) and RNA (MS2) phages.
▪ Phages contain an origin of replication, but unlike plasmids, phages lack the machinery
necessary to make proteins.
▪ Phages generally enter the host cell by injecting their DNA directly into the cell or by being
internalized by host cell processes. In most cases, once the phage genome is inside the host
cell, phage proteins and phage DNA are synthesized, and infectious particles are released
through lysis or membrane budding.
▪ The ability to transfer DNA from the phage genome to bacterial hosts during the bacterial
infection is known as transfection.
▪ In gene cloning, two specially designed phage vectors are used, which are constructed from
components of the temperate bacteriophage lambda and the production of single-strand
DNA using cells infected with vectors based on filamentous bacteriophage M13.
Genomic DNA cloning in
bacteriophage λ
Phagemids
▪ M13-based cloning vectors have disadvantages:
i. It is difficult to obtain enough double-stranded DNA required for recombinant DNA
reactions;
ii. the rolling circle mechanism of M13 replication can be affected by insert sequence
and size, causing under representation of certain recombinant molecules following
infection, and
iii. since the intergenic region is very small, the cloning range is very less.
▪ To overcome these problems, hybrid M13 phage/plasmid cloning vectors called
phagemids were constructed in the early 1990s. The original phagemid vectors were
derived from the high copy pUC18/pUC19 plasmids first developed by Messing.
▪ Phagemids are plasmid vectors having both bacterial as well as phage ori of
replication, for example, pBluescript II KS.
▪ pBluescript II KS is a pUC-based plasmid and is 2958 bp long with lacZ gene precedes
from polycloning site.
pBluescript SK+ consists of the
following:
1. ColE1 ori
2. Phage f1 (M13) origin of
replication.
3. A small portion of lacZ gene.
4. MCS within lacZ Gene from Lac
promoter.
5. Phage T7 and T3 promoter
sequence.
6. Ampicillin gene for Ampicillin
resistance.
Suggested readings
1. Genomes 4 by T.A. Brown, Garland Science Publishing, New York.
2. Plant Biotechnology and Genetics by C. Neal Stewart Jr.
Thank you
