Basic Concepts and Scope of

Recombinant DNA Technology

Dr Ravi Kant Agrawal, MVSc, PhD

Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India
 What is recombination?
• The exchange of
corresponding DNA segments
between adjacent
chromosomes during the
special type of cell division
that results in the production
of new genetic make up...
 Recombinant DNA technology
 A series of procedures used to recombine DNA segments.
 Under certain conditions, a recombinant DNA molecule can
enter a cell and replicate.
 Recombinant DNA technology is one of the recent advances in
biotechnology, which was developed by two scientists named
Boyer and Cohen in 1973.
 The DNA is inserted into another DNA molecule called ‘vector’.
 The recombinant vector is then introduced into a host cell
where it replicates itself, the gene is then produced

In vitro recombination
Genetic engineering
Genetic surgery
Basic principle of recombinant DNA technology
 Gentic Engineering
 GE: The technology entailing all
processes of altering the genetic
material of a cell to make it capable
of performing the desired functions,
such as producing novel substances.
 In other words: Genetic engineering
is the deliberate, controlled
manipulation of genes in an organism
in order to upgrade that organism.
 In genetic engineering,
recombination can also refer to
artificial and deliberate
recombination of pieces of DNA, from
different organisms, creating what is
called recombinant DNA.
 Gene
Gene: A gene is a basic unit of heredity in a living organism.
It is "a locatable region of genomic sequence, corresponding to a
unit of inheritance, which is associated with regulatory regions,
transcribed regions, and or other functional sequence regions “. [1]

Coding for proteins

3 classes of genes Coding for RNAs
Specific functions

Genes hold the information to build and maintain an organism's

cells and pass genetic traits to offspring. [2]

1. Pearson H (2006). "Genetics: what is a gene?". Nature 441 (7092): 398–401

2. http://en.wikipedia.org/wiki/Gene
 Allele: Each gene can have different alleles.

 An allele (from the Greek allelos, meaning each other) is one

of two or more forms of the DNA sequence of a particular gene.
E.g. Diploid , Triploid etc.

 The vast majority of living organisms encode their genes in

long strands of DNA.

 The most common form of DNA in a cell is in a double helix

structure

 RNA is common as genetic storage material in viruses, in

mammals in particular RNA inheritance has been observed
very rarely.
Central Dogma of Molecular Biology:
 The flow of genetic information in the cell starts at DNA, which
replicates to form more DNA.
 Information is then ‘transcribed” into RNA, and then it is
“translated” into protein.
 The proteins do most of the work in the cell. Once information
gets into protein, it can't flow back to nucleic acid.
 A Brief History of Genetic Engineering
Some major steps in the development of GE

Identification of DNA as the genetic material

Theoretic DNA double helix
basis Central dogma

DNA manipulative enzymes

DNA sequencing
Tools & Tech PCR
breakthroughs Plasmid Vector
Libraries
Bioinformatics
Animal Cloning w/t Nuclear Transfer
……
 Steps marked the beginning of a new age in biology

Working Draft of
the human
Rediscovery of
genome
Mendel's laws helps Huntington
establish the DNA recombination disease gene Genetic and
sequencing
complete
science of genetics & delivery method mapped to physical
chromosome 4 mapping

1900 1972 1983 1994-98 2000

2005
1953 1980 1990 1998 (or earlier)

DNA markers DNA markers Gene map

Watson and used to map used to map expected to
Crick identify human disease
Human Genome
human disease be
genes to genes to complete
DNA chromosomal Projects (HPG) chromosomal
(the double regions begins-an regions
international
helix) as the effort to map and
Chemical basis sequence all the
of heredity genes in the
human genome

Health Policy Research Bulletin, volume 1 issue2, September 2001

Some major steps in the development of GE

http://www.nature.com/milestones/miledna/timeline.html
2003 Finished the sequence of human genome
2005 Finished the sequence of chimpanzee genome
2006 Craig C. Mello and Andrew Fire's received a noble prize for RNAi (1998 discovered RNAi degrading
mRNA)
 Johan Friedrich Miescher Swiss Biologist Isolated nuclei of
white blood cells in 1869 and called it “Nuclein”. The major
component of “nuclein” is DNA. Protein is the other major
component of nuclein
 In 1889 Richard Altmann discovered that nuclein has acidic
properties, and it became called nucleic acid
 In 1938 Astbury and Bell published the first X-ray diffraction
pattern of DNA.[4]
 In 1953 Watson and Crick determined the structure of DNA.

 Wilhelm Roux: in 1883 speculated that chromosomes are the

carriers of inheritance.
 Walter Sutton: Determined in 1903 that chromosomes carried
units of heredity identified by Mendel
 William Bateson in 1905 coined the term genetics ...
 Wilhelm Johannsen Danish botanist coined the word "gene"
("gen" in Danish and German) in 1909 to describe these
fundamental physical and functional units of heredity.
The Chromosome Theory of Inheritance
 Genes are arranged in linear fashion on chromosome.
 The reason that certain traits tend to be inheritated together is
that the genes governing these traits are on the same
chromosome.
 Every gene has its place (locus)

 Diploid organism (human) normally have two copies of all

chromosomes (except sex chromosomes)

 DNA recombination occurs in nature

Thomas Hunt Morgan
Studied genetics of fruit flies in early 1900’s

 Experimented with eye color

 Eye color phenotype was sex-linked
 His work contributed to the knowledge of X and Y
chromosomes
 Nobel Peace Prize in 1933 for research in gene theory
 Griffith’s Transformation Experiment
• The discovery of the genetic role of DNA in 1928
• Two strains of a bacterium, a pathogenic “S” and a harmless
“R”
• Mixed heat-killed remains of the pathogenic strain with
living cells of the harmless strain, some living cells became
pathogenic
• He called this phenomenon transformation, now defined as a
change in genotype and phenotype due to assimilation of
foreign DNA
• Was is the Transforming principle ???
 Living S cells Living R cells Heat-killed Mixture of heat-killed
(control) (control) S cells (control) S cells and living R cells

RESULTS

Mouse dies Mouse healthy Mouse healthy Mouse dies

Living S cells
are found in
blood sample

Challenges: “principle “ transform the R into S with smooth coat?

 Evidence that Transforming principle is DNA
 In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod
announced that the transforming substance was DNA

 Their conclusion was based on experimental evidence that

only DNA worked in transforming harmless bacteria into
pathogenic bacteria

 Many biologists remained skeptical, mainly because little

was known about DNA

 Led by the earlier experiment of transfer genetic trait from

one strain of bacteria to another
Avery’s Transformation Experiment

Identity the “principle”

Animation: http://www.dnaftb.org/dnaftb/17/animation/animation.html
 Hershey-Chase Bacteriophage Experiment
• In 1952, Alfred Hershey and Martha Chase performed
experiments showing that DNA IS THE GENETIC MATERIAL of
a phage known as T2
• To determine the source of genetic material in the phage, they
designed an experiment showing that only one of the two
components of T2 (DNA or protein) enters an E. coli cell during
infection
• 32P is discovered within the bacteria and progeny phages,
whereas 35S is not found within the bacteria but released with
phage ghosts.
• They concluded that the injected DNA of the phage provides
the genetic information

http://glencoe.mcgraw-
hill.com/sites/9834092339/student_view0/chapter14/hershey_and_chase_experiment.html
 Empty
Radioactive protein shell
Radioactivity
Phage protein (phage protein)
in liquid

Bacterial cell

Batch 1: DNA
Sulfur (35S)

Phage
Labeled DNA

Pr shell
Centrifuge

Pellet (bacterial
Radioactive
cells and contents)
DNA

Batch 2:
Phosphorus (32P)

Labeled
DNA Core
Centrifuge

Radioactivity
(phage DNA)
Pellet in pellet
 Additional Evidence That DNA Is the Genetic Material
 1947: Erwin Chargaff- DNA composition varies from one species
to the next
 By 1950s: DNA is a polymer of nucleotides, G=C, A=T
 Franklin’s X-ray crystallographic images of DNA enabled
Watson and Crick to deduce that DNA was helical
 The X-ray images also enabled Watson and Crick to deduce the
width of the helix and the spacing of the nitrogenous bases

• The width suggested that the DNA molecule was made up of

two strands, forming a double helix

This evidence of diversity made DNA a more credible candidate

for the genetic material
 Double Helix Model of DNA Structure
James Watson and Francis Crick
• Collaborated at Cambridge University and presented the
double helix model of DNA structure in 1953 based on Franklin
and Wilkin’s X-ray structure.

• Described DNA dimensions and spacing of base pairs

• Had major impact on genetic engineering carried out today

• 1958, 1970 Crick: Central Dogma

• 1988, Watson: Principle scientist of the HGP
Meselson-Stahl
DNA must replicate during each cell division
1958 DNA replication: semiconservative model

Nirenberg, Ochoa, Khorana

1966 genetic code elucidation
 Meselson-Stahl Experiments
• Labeled the nucleotides of old strands with a heavy isotope of
nitrogen (15N), new nucleotides were indicated by a lighter
isotope (14N).
• The first replication in the 14N medium produced a band of hybrid
(15N-14N) DNA, eliminating the conservative model.
• A second replication produced both light and hybrid DNA,
eliminating the dispersive model and supporting the
semiconservative model.

http://highered.mcgraw-hill.com/olc/dl/120076/bio22.swf
 Bacteria Bacteria
cultured in transferred to
medium medium
containing containing
15
N 14
N

Tech supports
DNA sample DNA sample Less
centrifuged centrifuged dense
after 20 min after 40 min
(after first (after second
replication) replication) Radio labelling
More
dense
Ultracentrifuge
First replication Second replication
Synchronization
Conservative
model

Semiconservative
model

Dispersive
model
 Molecular tools and Technological breakthroughs
• Enzymes- nucleic acid cleavage, ligation, ……
• Vector- molecular cloning
• Polymerase chain reaction
• DNA sequencing
• Electrophoretic separation
• Detection of genes: DNA-Southern blotting; in situ
hybridization;
FISH technique; RNA - Northern blotting; Protein-Western
blotting; inmmunohistochemistry
• Purification
• Transgenetic organisms
Discovery of DNA ligase - the dawn of DNA manipulation

 DNA recombination happens in the

cell — for example, when breaks
caused by UV are repaired

 Search for an enzyme that could join

DNA molecules

 1967: The first DNA ligase was purified

and characterized in different labs
(Gellert Lab)
In this illustration,
DNA ligase (in color) Enzymatic breakage and joining of deoxyribonucleic acid, I. Repair of
single-strand breaks in DNA by an enzyme system from Escherichia coli
encircles the DNA infected with T4 bacteriophage" , PNAS 57: 1021-1028. 1967
double helix.
 Making the Cut - discovery of the restriction enzymes
Hamilton Smith and Kent Wilcox
• 1970: Isolation/characterization of endonuclease R (HindII) from
extracts of Haemophilus influenzae strain Rd.

• The enzyme degraded foreign DNA, such as that of phage T7,

but did not affect native H. influenzae DNA.

• Proposed that the enzyme recognizes a specific sequence on

the foreign DNA,
The 'recombination' potential of restriction enzymes was first
demonstrated by Janet Mertz and Ronald Davis.

They showed that the R1 restriction endonuclease produces

‘STAGGERED’ BREAKS, generating ‘COHESIVE’ ENDS that are
identical and complementary.

Their findings suggested that any R1-generated ends can be joined

by incubation with DNA ligase to generate hybrid DNA molecules.

THUS, THE ERA OF RECOMBINANT DNA TECHNOLOGY WAS BORN.

 Key concept
Use of plasmid as vector for shuttling DNA into bacteria
 1973, Stanley Cohen and his Stanford colleague Annie Chang, in
collaboration with Herbert Boyer and Robert Helling at the
University of California in San Francisco, reported the first in
vitro construction of a bacterial plasmid.
 Using EcoR I, they generated fragments from two plasmids
(each conferring resistance to one antibiotic), joined them using
DNA ligase and applied the mixture to transform E. coli. As they
had hoped, a fraction of the transformed bacteria became
resistant to both antibiotics while carrying a single hybrid
plasmid.
 Not only had they demonstrated that bacterial plasmids
constructed in vitro were functional in bacteria, but they had
also described the first plasmid vector.
 Paul Berg had devised a similar experiment to transfer foreign
DNA into mammalian cells, using the tumour virus SV40 as a
vector.
 In 1972, he made a hybrid molecule in vitro by inserting phage
sequences into SV40.

 These reports immediately raised concerns, as E. coli, which is a

natural habitant of the human gut, could now carry hybrid DNA
molecules containing SV40 oncogenes or other potentially
harmful sequences.

 These fears led the community to a self-imposed moratorium on

recombinant DNA experiments.

 However, the foundation had been laid and progress soon

resumed.
 Discovery of reverse transcriptase –
Full-length cDNA technologies
Puzzle: the ability of RNA tumour viruses to stably transform cells
without incorporation of a DNA copy of viral genes into the host
genome

Baltimore, Temin and Mizutani, looked for DNA polymerase

activity in purified preparations of such viruses.

DNA was being synthesized in RNA-dependent way.

Baltimore, D. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature 226, 1209–1211
(1970)
Reverse transcriptase could be used in vitro to synthesize cDNA
from mammalian mRNAs.

Verma et al. and Kacian et al. both added preparations of glob in

mRNAs to reverse transcriptase from avian myeloblastosis virus.
They correctly hypothesized that the reaction would only work
efficiently if they also added oligo (dT)

Reverse transcription has become hugely important in molecular

biology. Its influence extends from cloning to the development of
microarrays to the annotation of genomes.
 DNA Libraries: YACs and BACs 1987, 1992

 A vector carrying a 50-kb insert was far too small to also

contain all regulatory regions.
 Constructing comprehensive libraries covering the whole
genomes of higher organisms.
 Maynard Olson and colleagues exchanged the E. coli plasmid
for a yeast artificial chromosome (YAC): a linear DNA molecule
that mimics a yeast chromosome, complete with centromere
and telomeres.

YAC problems: chimaeras of non-contiguous DNA fragments; inserts

unstable; purification of YACs proved challenging
 Challenge: combination of the plasmid simplicity and stability
with the aim of adapting it for large-fragment cloning.

 A group led by Melvin Simon modified an endogenous circular

plasmid in E. coli, the fertility (F) factor present at one or two
copies per cell, to create a cloning vector.

 In reference to its yeast cousin, they called it BACTERIAL

ARTIFICIAL CHROMOSOMES (BACs).

 With a cloning capacity of 300 kb, BACs are not as potent as

YACs, but they have all the advantages of a bacterial vector:
stability, and ease of manipulation and purification.

 Today YACs are still in use; however, BACs have become the
workhorses in genomic research for any application that
requires large DNA inserts.
Safety Issues in relation to Recombinant DNA Technology
 As bacteria is commonly used in recombinant DNA work, there
has always been a concern among scientists and a worry among
people that there is a possibility that a clone of highly
pathogenic recombinant bacteria were made by accident, then
escaped from the laboratory and caused an epidemic for which
no drugs were available.
 It is always possible that an antibiotic-resistant plasmid could
be accidentally incorporated into a dangerous pathogen with
serious medical consequences.

 Recombinant DNA Advisory Committee (RAC) was established

in 1974 in the United States, which responds to public concerns
regarding the safety of manipulation of genetic material
through the use of recombinant DNA techniques.
2 types of control: Physical containment and
biological containment
Physical Containment:
 Effective biological safety programs were operated in a variety
of laboratories, which include a set of standard practices
generally used in microbiological laboratories, and special
procedures, equipment and laboratory installations that provide
physical barriers of varying degrees.

Biological Containment:
In considering biological containment, the vector (plasmid,
organelle, or virus) for the recombinant DNA and the host
(bacterial, plant, or animal cell) in which the vector is
propagated in the laboratory will be considered together.

(i) survival of the vector in its host outside the laboratory, and (ii)
transmission of the vector from the propagation host to other
non-laboratory hosts.
 Applications (Micro-organisms)
Production of humulin
 Insulin is a hormone that controls sugar levels in an
organism. Diabetes occurs in people when there is too
little or too much insulin produced. To control diabetes,
sufferers usually inject insulin once or twice daily.
 Until the mid-1980's most insulin was produced by
extracting a human-equivalent insulin from the pancreas
of animals (usually pigs).
 GM insulin (Humilin) is a genetically modified form of
insulin.
 The genetic sequence for insulin production is removed Recombinant Human
from human DNA. This is then inserted into the DNA of a Growth Hormone
bacteria, E. coli. The gene inserted into the bacteria cell Used by diabetics
is inherited from cell to cell as the cells multiply. The
insulin protein is produced (expressed) by the cells. The
insulin is then extracted from the cells.
 Problems with BSE in Britain have made users wary of
products derived from animals. GM insulin carries none
of these concerns.
 GM insulin is also cheaper to produce than pig-based
extraction.
www.healthtap.com
 Bacteria make interferon which can fight virus Recombinant insulin
infections and some cancers (Humulin)
 Plant Application
• Golden Rice – a possible solution to
Vitamin A deficiency.
• Vitamin A in Rice
- The gene which produces vitamin A was
taken from daffodils and put into rice to
help prevent blindness

• Weed killer resistant crops

- Weeds die but the crops survive
 Applications of Genetic Engineering
• Pharming
• Gene pharming is a technology that
scientists use to alter an animal's own
DNA, or to splice in new DNA, called a
transgene, from another species.
• In pharming, these genetically modified
(transgenic) animals are mostly used to
make human proteins that have medicinal
value.
• The protein encoded by the transgene is
secreted into the animal's milk, eggs or
blood, and then collected and purified.
 Tracy the Sheep
• One of the first mammals engineered successfully for the
purpose of pharming was a sheep named Tracy, born in 1990
and created by scientists led by British developmental biologist
Ian Wilmut at Roslin Institute in Scotland.
• Tracy was created from a zygote genetically engineered
through DNA injection to produce milk containing large
quantities of the human enzyme alpha-1 antitrypsin, a
substance used to treat cystic fibrosis and emphysema
 Xenotransplantation
 Xenotransplantation is the transplantation of living cells,
tissues or organs from one species to another.
 However there are ethical issues and issues with rejection
 There are also issues with virus transmission from one species
to another
 Porcine islet transplants are being investigated for use in type 1
diabetes due to the shortage of human islet cells
 Gene Therapy
• It involves modifying human DNA either
to repair it or to replace a faulty gene.
• The idea of gene therapy is to overcome
the effects of a mutation which causes a
genetic disease.
• Cystic fibrosis is the best known disease
where gene therapy has been tried.
 Diagnostic Tests
 Genetic engineering can produce very specific and sensitive
diagnostic tests for many diseases, using engineered proteins.
 This new technology is also opening up novel ways of
delivering medicines to specific targets.
 Vaccines
• Genetically engineered microbes can be
used to produce the antigens needed in a
safe and controllable way.
• The use of genetically modified yeast
cells to produce a vaccine against the
hepatitis B virus has been a major
success story.
• Firstly, the gene in a pathogenic virus
that stimulates protective immunity
should be identified.
• That portion of DNA is then isolated and
incorporated into an established
harmless virus (e.g. vaccinia virus).
• This new recombinant virus is used as a
vaccine.
• These vaccines are much safer since they
do not expose the patients to the actual
virus and do not risk to infection.
• This method may be useful in vaccines
against malaria and schistosomiasis and
many viruses (e.g. HBV)
 Pharmacogenomics
 Deals with the
influence of
genetic variation
on drug response
in patients by
correlating gene
expression with a
drug's efficacy or
toxicity
 Design drugs
adapted to an
individual's genetic
make-up
 Questions???

Thanks

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