Jai Chelani Biology Class 12 Main File

AIM:-
TO STUDY ABOUT -RECOMBINANT DNA

TECHNNOLOGY IN TODAY’S MEDICINE
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INTRODUCTION
Recombinant DNA (rDNA) molecules are DNA molecules formed by

laboratory methods of genetic recombination (such as molecular cloning) that
bring together genetic material from multiple sources, creating sequences that
would not otherwise be found in the genome.
Recombinant DNA is the general name for a piece of DNA that has been
created by combining at least two fragments from two different sources.
Recombinant DNA is possible because DNA molecules from all organisms
share the same chemical structure, and differ only in the nucleotide sequence
within that identical overall structure. Recombinant DNA molecules are
sometimes called chimeric DNA, because they can be made of material from
two different species, like the mythical chimera. R-DNA technology
uses palindromic sequences and leads to the production of sticky and blunt
ends.
The DNA sequences used in the construction of recombinant DNA molecules

can originate from any species. For example, plant DNA may be joined to
bacterial DNA, or human DNA may be joined with fungal DNA. In addition, DNA
sequences that do not occur anywhere in nature may be created by
the chemical synthesis of DNA, and incorporated into recombinant molecules.
Using recombinant DNA technology and synthetic DNA, literally any DNA
sequence may be created and introduced into any of a very wide range of
living organisms.
Proteins that can result from the expression of recombinant DNA within living
cells are termed recombinant proteins. When recombinant DNA encoding a
protein is introduced into a host organism, the recombinant protein is not
necessarily produced. Expression of foreign proteins requires the use of
specialized expression vectors and often necessitates significant restructuring
by foreign coding sequences.
HISTORY
The idea of recombinant DNA was first proposed by Peter Lobban, a

graduate student of Prof. Dale Kaiser in the Biochemistry
Department at Stanford University Medical School. The first
publications describing the successful production and intracellular
replication of recombinant DNA appeared in 1972 and 1973,
from Stanford and UCSF. In 1980 Paul Berg, a professor in the
Biochemistry Department at Stanford and an author on one of the
first papers was awarded the Nobel Prize in Chemistry for his work
on nucleic acids "with particular regard to recombinant
DNA". Werner Arber, Hamilton Smith, and Daniel Nathans shared the
1978 Nobel Prize in Physiology or Medicine for the discovery
of restriction endonucleases which enhanced the techniques of rDNA
technology.
Stanford University applied for a US patent on recombinant DNA in
1974, listing the inventors as Herbert W. Boyer (professor at
the University of California, San Francisco) and Stanley N.
Cohen (professor at Stanford University); this patent was awarded in
1980. The first licensed drug generated using recombinant DNA
technology was human insulin, developed by Genentech and licensed
by Eli Lilly and Company.
GENETIC ENGINEERING
Genetic Engineering plays a very important role, not only in scientific research,
but also in the diagnosis and treatment of disease. Recombinant DNA is a tool
in understanding the structure, function, and regulation of genes and their
products.
Objectives of Recombinant DNA Technology Include:

 Identifying The genes
 Isolating genes
 Modifying genes
 Re-expressing genes in other hosts or organisms
These steps permit scientists and clinicians to:
Identify new genes and the proteins they encode
 To correct endogenous genetic defects

 To manufacture large quantities of specific gene products such as
hormones, vaccines, and other biological agents of medical
interest
Genetic engineering produces proteins that offer advantages over proteins

isolated from other biological sources. These advantages include:
 High purity
 High specific activity
 Steady supply
 Batch-to-batch consistency
STEPS IN SYNTHESIZING A RECOMBINANT PROTEIN
 Recombinant technology begins with the isolation of a gene of interest.

The gene is then inserted into a vector and cloned. A vector is a piece of
DNA that is capable of independent growth; commonly used vectors are
bacterial plasmids and viral phages. The gene of interest (foreign DNA)
is integrated into the plasmid or phage, and this is referred to as
recombinant DNA.
 Before introducing the vector containing the foreign DNA into host cells
to express the protein, it must be cloned. Cloning is necessary to
produce numerous copies of the DNA since the initial supply is
inadequate to insert into host cells.
 Once the vector is isolated in large quantities, it can be introduced into

the desired host cells such as mammalian, yeast, or special bacterial
cells. The host cells will then synthesize the foreign protein from the
recombinant DNA. When the cells are grown in vast quantities, the
foreign or recombinant protein can be isolated and purified in large
amounts.
 Recombinant DNA technology is not only an important tool in scientific

research, but has also resulted in enormous progress in the diagnosis
and treatment of certain diseases and genetic disorders in many areas of
medicine.
GENETIC ENGINEERING HAS PERMITTED
Identification of mutations:
 People may be tested for the presence of mutated proteins that may
be involved in the progression of breast cancer, retino-blastoma, and
neurofibromatosis
Diagnosis of affected and carrier states for hereditary diseases:

 Tests exist to determine if people are carriers of the cystic fibrosis gene,
the Huntington’s disease gene, the Tay-Sachs disease gene, or the
Duchenne muscular dystrophy gene.
Mapping of human genes on chromosomes:

 Scientists are able to link mutations and disease states to specific sites on
chromosomes.
Transferring genes from one organism to another:

 People suffering from cystic fibrosis, rheumatoid arthritis, vascular
disease, and certain cancers may now benefit from the progress made
in gene therapy.
Isolation and alteration of genes:

 Once gene modification becomes successful, alteration of genes to
produce a more functional protein than the endogenous protein
may become possible, opening up the route of gene therapy.
Performing structure and function analyses on proteins:

 Researchers may now employ rational drug design to synthesize
drug compounds that will be efficacious and selective in treating
disease.
Isolation of large quantities of pure protein:

 Insulin, growth hormone, follicle-stimulating hormone, as well as other
proteins, are now available as recombinant products. Physicians will no
longer have to rely on biological products of low purity and specific
activity from inconsistent batch preparations to treat their patients
RECOMBINANT DNA TECHNIQUE
 Restriction enzymes used to cut out insulin gene and to cut a bacterial
(E. coli) plasmid at the same “sticky ends”
 Mutant strains of E. coli used to avoid bacteria attacking “foreign” genes
 Insert insulin gene next to E. coli
 B-galactosidase gene which controls transcription
 Bacterial cells replicate and make copies of insulin gene
 Insulin protein is purified (B-galactosidase removed)
 Chains are mixed and disulfide bridges form
 Cells provide a sterile growth medium
 Final product is Humulin - chemically identical to human insulin

PLASMID POLYLINKERS AND MARKER GENES
FOR BLUE-WHITE SCREENING
 A vector usually contains a sequence (polylinker) which can
recognize several restriction enzymes so that the vector can be used
for cloning a variety of DNA samples.
 Colonies with recombinant plasmids are white, and colonies

with nonrecombinant plasmids are blue.
 Example: pUC19
 Resistant to ampicillin, has (amprgene)
 Contains portion of the lac operon which codes for beta-galactosidase.
 X-gal is a substrate of beta-galactosidase and turns blue in the presence

of functional beta-galactosidase is added to the medium.
 Insertion of foreign DNA into the polylinker disrupts the lac operon, beta-
galactosidase becomes non-functional and the colonies fail to turn blue,
but appear white.
.
BACTERIAL ARTIFICIAL CHROMOSOMES(BACS)
 BACs can hold up to 300 kbs.
 The F factor of E.coli is capable of handling large segments of DNA.
 Recombinant BACs are introduced into E.coli by electroportation( a brief

high-voltage current). Once in the cell, the rBAC replicates like an F
factor.
 Example: pBAC108L
 Has a set of regulatory genes, OriS, and repE which control F-factor
replication, and parA and parB which limit the number of copies to one
or two.
 A chloramphenicol resistance gene, and a cloning segment.
YEAST ARTIFICIAL CHROMOSOMES(YACS)
 YACs can hold up to 500 kbs.
 YACs are designed to replicate as plasmids in bacteria when no

foreign DNA is present. Once a fragment is inserted, YACs are
transferred to cells, they then replicate as eukaryotic chromosomes.
 YACs contain: a yeast centromere, two yeast telomeres, a bacterial

origin of replication, and bacterial selectable markers.
 YAC plasmid----> Yeast chromosome

 DNA is inserted to a unique restriction site, and cleaves the plasmid
with another restriction endonuclease that removes a fragment of DNA
and causes the YAC to become linear. Once in the cell, the rYAC
replicates as a chromosome, also replicating the foreign DNA.
WHY IS A LENTIVIRUS NECESSARY?
Lentiviruses can introduce a gene of interest into cells that do not divide –
simple retroviruses cannot.
This ability makes them ideal for a delivery system because most of our cells,
like hemopoietic stem cells, do not divide.
Why use HIV?

A genetically stripped down amalgam of HIV components can be fashioned
with a molecular switch system that turns them off in response to a
common antibiotic.
This type of control allows doctors to control gene expression in people who are
treated with gene therapy - If something goes wrong, the expression can be
turned off.
Adenoviruses are often used as a vector in gene therapy research but they do not
have the capacity to integrate their genome into the hosts genome.
The advantage to using a retrovirus is that you don’t lose the genomic sequence
that is incorporated into the host DNA following cell division.
BIBLIOGRAPHY
 http://www.rvc.ac.uk/Extranet/DNA_1/DNA_1_intro.htm
 http://library.thinkquest.org/24355/data/light/details/media/
recombinantanim.html
 http://www.organoninc.com/products/consumer/
 NCERT TEXTBOOK
 GOOGLE
 PRICIPLES OF GENETICS BY SNUTSON AND BOVERY

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AIM:-

TO STUDY ABOUT -RECOMBINANT DNA

Recombinant DNA (rDNA) molecules are DNA molecules formed by

The DNA sequences used in the construction of recombinant DNA molecules

The idea of recombinant DNA was first proposed by Peter Lobban, a

Objectives of Recombinant DNA Technology Include:

These steps permit scientists and clinicians to:

Identify new genes and the proteins they encode

 To correct endogenous genetic defects

Genetic engineering produces proteins that offer advantages over proteins

 Recombinant technology begins with the isolation of a gene of interest.

 Once the vector is isolated in large quantities, it can be introduced into

 Recombinant DNA technology is not only an important tool in scientific

Diagnosis of affected and carrier states for hereditary diseases:

Mapping of human genes on chromosomes:

Transferring genes from one organism to another:

Isolation and alteration of genes:

Performing structure and function analyses on proteins:

Isolation of large quantities of pure protein:

 Mutant strains of E. coli used to avoid bacteria attacking “foreign” genes

 Insert insulin gene next to E. coli

 B-galactosidase gene which controls transcription

 Bacterial cells replicate and make copies of insulin gene

 Insulin protein is purified (B-galactosidase removed)

 Chains are mixed and disulfide bridges form

 Cells provide a sterile growth medium

 Final product is Humulin - chemically identical to human insulin

 Colonies with recombinant plasmids are white, and colonies

 Resistant to ampicillin, has (amprgene)

 Contains portion of the lac operon which codes for beta-galactosidase.

 X-gal is a substrate of beta-galactosidase and turns blue in the presence

 BACs can hold up to 300 kbs.

 The F factor of E.coli is capable of handling large segments of DNA.

 Recombinant BACs are introduced into E.coli by electroportation( a brief

 A chloramphenicol resistance gene, and a cloning segment.

YEAST ARTIFICIAL CHROMOSOMES(YACS)

 YACs can hold up to 500 kbs.

 YACs are designed to replicate as plasmids in bacteria when no

 YACs contain: a yeast centromere, two yeast telomeres, a bacterial

 YAC plasmid----> Yeast chromosome

Why use HIV?

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