ABDUL WALI KHAN UNIVERSITY

MARDAN

Abdul Wali Khan University Mardan Pakistan. http://www.awkum.edu.pk/

Recombinant DNA Technology

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Recombinant DNA Technology

Contents:
❖Transgene
❖Transgenesis
❖Basic principles of rDNA Technology
❖Methods of Gene transfer
❖Applications of rDNA technology

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Transgene

TRANSGENE: It is a foreign gene or genetic material that has

been transferred naturally or by any of a number of genetic
engineering techniques from one organism to another.

TRANSGENESIS: The phenomenon of introduction of

exogenous DNA into the genome to create and maintain a stable
and heritable character.

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

 Recombinant DNA Technology

❖Based on the concept of gene recombination.

❖Encompasses a number of experimental protocols leading to the transfer of genetic information(DNA)

from one organism to another.

❖Involves the manipulation of genetic material(DNA) to achieve the desired goal in a pre-determined
way.

❖The present day rDNA technology has its roots in the experiments performed by Boyer & Cohen

❖ In 1973 Herbert Boyer, of the University of California at San Francisco, and Stanley Cohen, at
Stanford University, reported the construction of functional organisms that combined and replicated
genetic information from different species.

❖Their experiments dramatically demonstrated the potential impact of DNA recombinant engineering
on medicine and pharmacology, industry and agriculture.

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Goals of recombinant DNA technology

❖To isolate and characterize a gene

❖To make desired alterations in one or more isolated genes
❖To return altered genes to living cells
❖Artificially synthesize new gene
❖Alternating the genome of an organism
❖Understanding the hereditary diseases and their cure
❖ Improving human genome

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

 Procedure of making rDNA

❖Isolating of DNA
❖Cutting of DNA
❖Joining of DNA
❖Amplifying of DNA

DNA can be cut into large fragments by mechanical shearing.

Restriction enzymes are the scissors of molecular genetics.

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Restriction enzyme

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

 Restriction Endonucleases

❖Recognition sequence is the site where the DNA is cut by a restriction

endonuclease.
❖Restriction endonucleases can specifically recognise DNA with a particular
sequence of 4-8 nucleotides & cleave.
❖Cleavage patterns: The cut DNA fragments by R.endonucleases may have
mostly sticky ends or blunt ends.
❖DNA fragments with sticky ends are particularly useful for rDNA experiments,
since single stranded sticky DNA ends can easily pair with any other DNA fragment
having complementary

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Restriction Endonucleases

❖Enzymes for the manipulation of DNA.

❖Are bacterial enzymes that can cut/split DNA at specific sites.

❖These were first discovered in E.coli restricting the replication of

bacteriophages, by cutting the viral DNA(The host E.coli DNA is
protected from cleavage by addition of methyl groups).

❖Thus,the enzymes that restrict the viral replication are known as

restriction enzymes or restriction endonucleases.

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
 DNA ligases

❖These were originally isolated from viruses, also occur in E.coli & eukaryotic cells.

❖The cut DNA fragments are covalently joined together by DNA ligases.

❖DNA ligase joins the DNA fragments by forming a phosphodiester bond b/n the phosphate
group of 5’-carbon of one deoxyribose with the hydroxyl group of 3’-carbon of another
deoxyribose.

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Amplifying the recombinant DNA

❖Transforming the recombinant DNA into a bacterial host strain.

❖The cells are treated with CaCl2
❖DNA is added
❖ Cells are heat shocked at 42 C
❖DNA goes into cell by a somewhat unknown mechanism
❖Once in a cell, the recombinant DNA will be replicated
❖When the cell divides, the replicated recombinant molecules go to both
daughter cells which themselves will divide later.
❖Thus, the DNA is amplified

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
 Basic principles of rDNA Technology:

❖Generation of DNA fragments & selection of the desired piece of DNA.

❖Insertion of the selected DNA into a cloning vector to create a rDNA or

chimeric DNA.

❖Introduction of the recombinant vectors into host cells.

❖ Multiplication & selection of clones containing the recombinant

molecules.

❖ Expression of the gene to produce the desired product

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
 Host cells

❖The hosts are the living systems or cells in which the

carrier of rDNA molecule or vector can be propagated.

❖Host cells can be prokaryotic or eukaryotic.

❖Microorganisms are preferred as host cells, since they

multiply faster compared to cells of higher organisms

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

E.coli

❖This was the first organism used in the DNA technology

experiments.

❖The major drawback is that it cannot perform post

translational modifications.

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Eukaryotic Hosts

oThese are preferred to produce human proteins, since these

have complex structure suitable to synthesis complex
proteins.

oMammalian cells possess the machinery to modify the

protein to the active form. (post translational modifications)
E.g., Tissue plasminogen activator

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Vectors

oAre the DNA molecules, which can carry a foreign DNA

fragment to be cloned.

oThese are self replicating in an appropriate host cell.

oMost important vectors are plasmids, bacteriophages,

cosmids & artificial chromosome vectors.

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Which Vector to be used?

Must be compatible with host cell system (prokaryotic vectors for

prokaryotic cells, eukaryotic vectors for eukaryotic cells)

Needs a good combination of

❖Strong promoters
❖Ribosome binding sites
❖Termination sequences
❖Affinity tag or solubilization sequences
❖Multi-enzyme restriction site

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

 Key Parts to a Vector

Origin of replication (ORI) – DNA sequence for DNA polymerase to replicate the

plasmid.

Selectable marker (Amp or Tet) – a gene, when expressed on plasmid will allow host

cells to survive.

Inducible promoter – Short DNA sequence which enhances expression of adjacent gene.

Multi-cloning site (MCS) – Short DNA sequence that contains many restriction enzyme

sites

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Plasmids

oPlasmids Are extra chromosomal, double stranded, circular, self-

replicating DNA molecules.

oUsually plasmids contribute to about 0.5%- 5.0% of the total

DNA of bacteria. A few bacteria contain linear plasmids E.g.
streptomyces sp, Borelia burgdorferi. E.g., pBR322,pUC

oThe plasmids carries genes resistance for ampicillin &

tetracycline that serve as markers for the identification of clones
carrying plasmids.

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Bacteriophages

oBacteriophages Are the viruses that replicate within the bacteria.

o In case of certain phages, their DNA gets incorporated into the
bacterial chromosome & remains there permanently.
oCan take up larger DNA segments than plasmids, hence preferred
for working with genomes of human cells. E.g., phage λ,
phageM13.

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Cosmids

oCosmids Are the vectors possessing the characteristics of both

plasmid & bacteriophage λ.

oThese carry larger fragments of foreign DNA compared to

plasmids

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
 Artificial chromosome vectors

oE.g.,Human artificial chromosome, Yeast artificial chromosomes,

Bacterial artificial chromosome

oThese can accept large fragments of foreign DNA

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
 Methods of Gene transfer

1. Transformation

The uptake of plasmid DNA by E.coli is carried out in ice-cold

CaCl2(0-5C) & a subsequent heat shock (37-45C for about 90sec)

2. Conjugation Is a natural microbial recombination process.

Plasmid-insert DNA is transferred from one cell to another

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Electroporation

o Is based on the principle that high voltage electric pulses can

induce cell plasma membranes to fuse.

o Liposome-mediated gene transfer(Lipofection) are circular lipid

molecules, having aqueous interior that can carry nucleic acids.

o 4. Direct transfer of DNA

o DNA is directly transferred into the nucleus by microinjection &

particle bombardment

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

 Applications of rDNA technology

Manufacture of proteins/hormones Interferon, plasminogen activating

factor, blood clotting factors, insulin, growth hormone.

AIDS test: Has become simple & rapid

Diagnosis of molecular diseases: sickle cell anemia, thalassemia,

familial hypercholesterolemia, cystic fibrosis

Prenatal diagnosis: DNA from cells collected from amniotic fluid,

chorionic villi

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
 Gene Therapy:

❖This is achieved by cloning a gene into a vector that will readily be

taken up & incorporated into genome of a host cell.

❖Adenosine deaminase deficiency ADA deficiency has been

successfully treated

❖ Application in Agriculture:

❖Genetically engineered plants are developed to resist draught &

diseases.

❖Good quality of food & increased yield of crops is also possible

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

 Industrial Application:

❖Enzymes---use to produce sugars, cheese, detergents.

❖ Protein products---used as food additives, increases nutritive
value, besides imparting flavor.
❖Application in forensic medicine: The restriction analysis pattern
of DNA of one individual will be very specific
❖ (DNA fingerprinting),but the pattern will be different from person to
person.
❖Helps to identify criminals & to settle disputes of parenthood of
children.
❖ Transgenesis: Gene replacement therapy will not pass on to
offspring.
❖Therefore, genes are transferred into fertilised ovum which will be
found in somatic as well as germ cells

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

 Gene cloning

•The recombinant DNA molecule is transferred to a host cell.

•Within the host cell it replicates producing dozens of identical copies

i.e., it is cloned.

• The cloned DNA can be recovered from host cells purified,

analysed & transcribed.

•It’s mRNA translated.

•Gene product isolated & used for research or sold commercially.

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk
 Gene libraries

•Collections of cloned DNA sequences.

• Each cloned segment relatively small.

•Many separate clones are required.

•Libraries-----Genomic, chromosome specific c- DNA.

•IT is a resource that can be used to retrieve any

of the genes from our starting material.

Abdul Wali Khan University Mardan Pakistan. www.awkum.edu.pk