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Lec 2 DNA Cloning Techniques

The document discusses DNA cloning techniques and recombinant DNA technology, detailing the process of creating identical DNA copies using plasmids. It covers the classification of plasmids, their functions, and the essential components of cloning and expression vectors. Additionally, it outlines various types of cloning vectors, including plasmids, bacteriophages, cosmids, bacterial artificial chromosomes, yeast artificial chromosomes, and human artificial chromosomes.

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11 views29 pages

Lec 2 DNA Cloning Techniques

The document discusses DNA cloning techniques and recombinant DNA technology, detailing the process of creating identical DNA copies using plasmids. It covers the classification of plasmids, their functions, and the essential components of cloning and expression vectors. Additionally, it outlines various types of cloning vectors, including plasmids, bacteriophages, cosmids, bacterial artificial chromosomes, yeast artificial chromosomes, and human artificial chromosomes.

Uploaded by

Hiba Shakil
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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DNA cloning techniques and recombinant DNA

technology
DR. ANUM GUL
BT-506
DNA Cloning
 It is a molecular biology technique that makes many identical copies of piece
of DNA such as a gene.

 In a typical DNA cloning procedure, the gene or other DNA fragment of


interest is first inserted into a circular piece of DNA called a plasmid.

 The insertion is done using enzymes that “cut and paste” DNA.

 It produces a molecule of recombinant DNA, or DNA assembled out of


fragments from multiple sources.
DNA Cloning
The one of the first cloning process was described
by Boyer and Cohen in 1973.

It involved two plasmids that were linearized by


the restriction enzyme EcoRI and then ligated to
form one plasmid using ligase.

Plasmid pSC101
DNA Cloning
About 5 years later, the first recombinant plasmid vector was created
that fulfils all of the necessary conditions for DNA fragment cloning.

It consists of three segments:

Tetracycline resistance gene that occurs naturally in the Salmonella


plasmid pSC101,

Ampicillin resistance gene of the transposon Tn3

Replication region (ori), as well as neighboring sequences of the


Escherichia coli plasmid pMB1.
DNA Cloning
This vector is known as pBR322, named after the two researchers
who first described it, Bolivar and Rodriguez (1977).
Plasmid Classifications and Types
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 as Saccharomyces cerevisiae.

Plasmids can be broadly classified into

Conjugative plasmids

Non-conjugative plasmids
Conjugative plasmids
Contain a set of transfer or tra genes
which promote sexual conjugation
between different cells.

Plasmid may be transferred from one


bacterium to another via sex pili
encoded by some of the tra genes.
Non-conjugative plasmids
Non-conjugative plasmids are incapable of initiating conjugation;
hence they can be transferred only with the assistance of
conjugative plasmids.
Plasmid Classifications and Types
Plasmids can also be classified into incompatibility groups.

A microbe can harbour different types of plasmids, but different


plasmids can only exist in a single bacterial cell if they are
compatible.

If two plasmids are not compatible, one or the other will be rapidly
lost from the cell.
Plasmid Classifications and Types
Another way to classify plasmids is by function. There are five main
classes:

Fertility F-plasmids, which contain tra genes. They are capable of


conjugation and result in the expression of sex pili.

Resistance (R) plasmids, which contain genes that provide resistance


against antibiotics or poisons. Historically known as R-factors, before the
nature of plasmids was understood.
Plasmid Classifications and Types
Col plasmids, which contain genes that code for bacteriocins, proteins
that can kill other bacteria.

Degradative plasmids, which enable the digestion of unusual


substances, e.g., salicylic acid.

Virulence plasmids, which turn the bacterium into a pathogen.


Vectors for Gene Cloning
A DNA molecule needs to display several features to be able to act
as a vector for gene cloning.

It must be able to replicate within the host cell, so that numerous
copies of the recombinant DNA molecule can be produced and
passed to the daughter cells.

A cloning vector also needs to be relatively small, ideally less than 10


kb in size.
Essential Components of Vectors
Bacterial Origin of Replication (ori )

A bacterial replication origin is essential in a vector, as it is needed to


ensure its amplification in bacteria

Antibiotic Resistance

Antibiotic resistance is used to select the transformed bacteria.

The most frequently used resistance-conveying genes are Ampr and Kanr.
Essential Components of Vectors
Essential Components of Vectors
Polylinkers or (multiple cloning sites, MCSs)

 Every vector contains a defined number of recognition sites for


restriction enzymes that cut the vector sequence only once (single
cutters) to enable the cloning of a DNA fragment.

 These sites usually lie closely together, and these sections are
referred to as polylinkers.

 50–100 bp on average and may contain up to 25 restriction sites for


single-cutting restriction enzymes.
Essential Components of Vectors
There are also many vectors that carry several sequence elements
from bacteriophagal genomes right next to the polylinkers.

These phage components can act as promoters that enable the in


vitro transcription of the cloned genes (e.g., the SP6 or T7
promoters derived from the phages).
Size and Copy Number
 The copy number refers to the number of molecules of an individual
plasmid that are normally found in a single bacterial cell.

 Some plasmids, especially the larger ones, are stringent and have a
low copy number of perhaps just one or two per cell; others, called
relaxed plasmids, are present in multiple copies of 50 or more per
cell.
What are the Different types of Cloning
Vectors?
Plasmid – The size of a plasmid is 4361 bp, and the cloning limit is 0.1-15 kb.
Ampicillin and the tetracycline resistant gene whereas it remains isolated
from the E.coli. Example: pBR322

A Bacteriophage – Its size is 48502 bp and 1/3rd of this is not essential.

It can recombine about 5-20 kb of the donor DNA. An example is lambda


genome.
What are the Different Kinds of Cloning
Vectors?
Cosmid – The size of cosmid is 7900 bp and the cloning limit is 30-50 kb.

It has features similar to both phage and plasmid and an example of it is


super COS1.

Bacterial Artificial Chromosome – The size of BAC is 11827 bp and the cloning
limit is 35-300 kb.

The marker gene is chloramphenicol and lactose metabolizing gene. It is a


modification of f-plasmid and is artificially synthesized. Example: pUvBBAC.
What are the Different Kinds of Cloning
Vectors?
Yeast Artificial Chromosome – The size of YAC is 11400 bp and the
cloning limit 100-1000 kb. The marker is similar to that of yeast.

It is artificial and has yeast centromere, which is isolated from the


Saccharomyces cerevisiae.

Human Artificial Chromosome – It is an artificial chromosome that is


used to transfer human gene and has no limit on cloning as it can carry a
large segment of the DNA.
Vector Classification
Vectors are classified into the following two types depending upon
their function;

Cloning vectors

Expression vectors
Expression vectors
Components of Expression vectors
Promoter. The promotor initiates transcription and is positioned 10-100
nucleotides upstream of the ribosome binding site.

The ideal promoter exhibits several desirable features:

It is strong enough to allow product accumulation up to 50% of the total


cellular protein.

It has a low basal expression level (i.e., it is tightly regulated to prevent
product toxicity).

It is easy to induce.


Components of Expression vectors
Shine-Dalgarno sequence. The Shine-Dalgarno (SD) sequence is
required for translation initiation and is complementary to the 3'-end of
the 16S ribosomal RNA. It is positioned 4-14 nucleotides upstream the
start codon.

Start codon. Initiation point of translation. In E. coli the most used


start codon is AUG. GUG is used in 8% of the cases. UUG is hardly used.
Components of Expression vectors
Regulatory gene (repressor). Many promoters show leakiness in their
expression i.e., gene products are expressed at low level before the
addition of the inducer.

This becomes a problem when the gene product is toxic for the host.
This can be prevented by the constitutive expression of a repressor
protein.
Components of Expression vectors
Tags: N- or C-terminal fusions of heterologous proteins to short
peptides (tags) or to other proteins (fusion partners) offer several
potential advantages:

Improved expression.

Improved solubility.

Improved detection.

Improved purification.
Components of Expression vectors
Protease cleavage site

 Protease cleavage sites are often added to be able to remove a tag or


fusion partner from the fusion protein after expression.

 However, cleavage is rarely complete and often additional purification


steps are required.

Transcription terminator. The transcription terminator reduces unwanted


transcription and increases plasmid and mRNA stability.
Components of Expression vectors
Stop codon. Termination of translation. There are 3 possible stop
codons, but UAA is preferred because it is less prone to read-through
than UAG and UGA.

The efficiency of termination is increased by using 2 or 3 stop codons


in series.

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