Recombinant DNA Technology : Introduction, History, Procedure and Applications

achaurasiya

5 years ago

Recombinant DNA Technology: Recombinant DNA Technology is DNA molecules formed by laboratory methods of genetic recombination to bring together genetic material from multiple sources, creating sequences that would not otherwise normally occur together. The DNA sequences used in the construction, can originate from any species. Some terms and their features in Recombinant DNA Technology All organisms use DNAas their molecule of heredity. At the chemical level, DNA is the same for various living organism. Thus, DNA from different organisms can be “cut and pasted” together which is termed as “Recombinant DNA”. It is also known as Chimeric DNA. Cloning: The generation of a large number of genetically identical DNA molecules Biotechnology: Science in which living organisms are manipulated, particularly at the molecular genetic level, to form useful products.

Introduction of Recombinant DNA Technology

Recombinant DNA Technology is DNA molecules formed by laboratory methods of genetic recombination to bring together genetic material from multiple sources, creating sequences that would not otherwise normally occur together. The DNA sequences used in the construction can originate from any species.

Some terms and their features in Recombinant DNA Technology

All organisms use DNA as their molecule of heredity.
At the chemical level, DNA is the same for various living organisms.
Thus, DNA from different organisms can be “cut and pasted” together which is termed as “Recombinant DNA”.
It is also known as Chimeric DNA.
Cloning: The generation of a large number of genetically identical DNA molecules
Biotechnology: Science in which living organisms are manipulated, particularly at the molecular genetic level, to form useful products.

History on Recombinant DNA Technology

In the late 1960s – Werner Arbor and Hamilton smith discovered the Restriction endonuclease enzyme.
In 1969- Herber Boyer isolated the restriction enzyme EcoRI from Escherichia coli.
In 1972- David Jackson, Robert Symons, and Paul Berg reported that they had successfully generated recombinant DNA molecules
In 1973- Herbert Boyer and Stanley Cohen created the first recombinant plasmid capable of being replicated within a bacterial host which was called pSC 101 (SC in plasmid stand for Stanley Cohen).
In 1974- The first eukaryotic gene was cloned
In 1979

-Insulin synthesized using recombinant DNA

– First human viral antigen (hepatitis B) cloned

Restriction Endonuclease

Molecular scissors
Naturally found in different bacteria
Protects bacteria from foreign/ phage DNA
It recognizes specific DNA sequences called Recognition sites
Typically recognize specific 4 to 8 bp sequences and then cleave DNA strands
Restriction sites commonly are in short palindromic sequences the restriction-site sequence is the same on each DNA strand when read in the 5→3 direction

Nomenclature of Enzymes used in Recombinant DNA Technology

Smith and Nathans (1973) proposed an enzyme naming scheme
Three-letter acronym for each enzyme derived from the source organism
The first letter from Genus
The next two letters represent species
Additional letters or numbers represents the strain or serotypes

Derivation of the EcoRI name
Abbreviation	Meaning	Description
E	Escherichia	Genus
co	coli	species
R	RY13	strain
I	First identified	order of identification in the bacterium

Restriction enzyme cut DNA producing either

1. Sticky End

Most restriction enzymes make staggered cuts in the recognition sequence
Generating ends with a short overhang of usually 4 bases
Produce “sticky-ends/cohesive end”

2. Blunt End

Some restriction enzymes cut DNA at opposite base
They leave blunt-ended DNA fragments

Vector

DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed

Features of vector

All engineered vectors share three main feature

1. An origin of replication

2. A region of DNA that bears unique restriction sites called Multicloning site or Polylinker

(Recognition sequences of several different restriction enzymes)

3. A selectable marker:

Discriminate between those cells that successfully obtained vector from those that did not

Transcription vector

Capable of being transcribed but not translated
Used to amplify their insert

Expression vector

Expression of the transgene in the target cell
Generally have a promoter sequence that drives expression

Shuttle vector

Have two origins of replication, recognized by two different host cells e.g. YEp24

Types of vector

There are four major types of vectors:

Plasmids,
Bacteriophages and other viruses,
Cosmids, and
Artificial chromosomes

Each type has its own advantages, so the selection of the proper cloning vector is critical to the success of any cloning experiment.

Plasmid vectors

Plasmids are naturally occurring extrachromosomal double-stranded circular DNA molecules that carry an origin of replication and replicate autonomously within bacterial cells
Plasmid vectors are engineered from bacterial plasmids for use in cloning
pBR322- “p” stands for “plasmid”,

– BR (Bolivar and Rodriquez)

– 322,the identification number

e.g. pBR322, pUC19
Replicates independently, many copies may be maintained in a single cell

Insert size-10 kb

Phage vectors

An engineered version of bacteriophage( l phage vector)
They infect cells much more efficiently than plasmids, so the yield of clones is usually higher
The advantage over plasmid vectors- can accommodate much more foreign DNA 10-25 kb long
Clones are not colonies of cells, but plaques
All the phages in the plaque derive from one original phage, they are all genetically identical—a clone.
Suitable for library construction

Cosmids

Hybrid molecules containing components of both lambda and plasmid
Lambda components: cos sites, or cohesive(allow the DNA to be packaged into l phage heads)
Plasmid DNA components: ori site, replicate as plasmids in bacteria
They can carry about 40 to 50 kb inserts
After infection of a host, DNA molecules replicate as plasmids

Artificial chromosomes

Special cloning vectors for large fragments of DNA
Constructing a genomic library/ sequencing an organism’s entire genome
Like natural chromosomes replicate only once per cell cycle

Yeast artificial chromosome (YAC)

Are genetically engineered chromosomes derived from the DNA of the yeast, ligated into a bacterial plasmid
Can accommodate >1 Mb (1000 kbp)
YAC tend to be unstable

Bacterial artificial chromosome

Based on F factor- derived from an E. coli plasmid
Useful for cloning up to 300 kb
Are more stable

DNA Ligase

The relatively weak hydrogen bonds hold the strands together only temporarily.
DNA ligase forms a covalent bond between the sugar-phosphate residues of adjacent nucleotides, joining the two molecules.
DNA ligase is the glue that pastes the fragments together.
The first DNA ligase was purified and characterized in 1967.
The common commercially available DNA ligases were originally discovered in bacteriophages T4, E. coli, and other bacteria.
The DNA ligase from bacteriophage T4 is most commonly used in the laboratory.

Host cell

A good host organism-easy to transform and easy replication of rec DNA.
No interfering element against the replication of rec DNA in the host cells(engineered to lack restriction enzymes)
E. coli is the most frequent prokaryotic host.
Saccharomyces cerevisiae is the most common among eukaryotes.

Steps in Recombinant DNA technology

Step 1- Isolation and identification of gene of interest

Step 2- Selection of suitable cloning vector

Step 3- Isolated genes fused with the cloning vector -Formation of recombinant DNA molecules

Step 4- Insertion into a suitable host cell (May is not in the same domain as the original gene donor)

Step 5- Selection of transformed host cells

Finally expression or multiplication of DNA-insert in the host

Step 1- Isolation of Gene of interest

The DNA to be incorporated/cloned into the vector is called an insert
Viral, bacterial, plant, or animal origin
Process:

Cells are first broken open
Mechanical disruption (Grinding frozen material)
Chemicals(lysozyme, EDTA, the detergent-sodium dodecyl sulphate)
DNA purified from the cell extract-specific enzymes (Protease/RNase)
Purified DNA cut by restriction enzyme to smaller fragments
DNA fragments are separated electrophoretically
The desired fragment is located by the Southern blotting technique

Other ways

Genomic library
Complementary DNA library (cloning the eukaryotic genes)
Synthetic DNA/oligonucleotides
Polymerase chain reaction (PCR) products

Step 2-Selection of suitable cloning vector

Step 3-Production of Recombinant DNA

(i) rDNA formation by the use of restriction endonuclease creating sticky ends

The two DNAs acted separately by the same restriction endonuclease (BamHI) giving staggered two stranded cut
Are heated, mixed and cooled, so that the sticky ends will base-pair

(ii) rDNA formation by use of restriction endonuclease creating blunt ends

Step 4- Insertion into Host Cell

Transformation

The cells are treated with CaCl2–DNA is added –Cells are heat-shocked at 42°C -DNA goes into the cell
Calcium phosphate
DEAE (diethylaminoethyl )-dextran
Polyethylene glycol

Transduction

Viruses increasingly are used to insert desired genes into eukaryotic cells. e.g. retrovirus, adenoviruses, recombinant baculoviruses

Electroporation
Gene gun: A blast of compressed gas shoots a spray of DNA-coated metallic microprojectiles into the cells

Microinjection-

Genetic material is micro pipetted into the host cell

Step 5- Selection of the Transformed Cells:

Distinguishing the transformed cells from the non-transformed cells

Several methods

Antibiotic resistance
Visible characters
Assay for biological activity
Colony hybridization
Blotting

Applications of Recombinant DNA Technology

Application in genetic engineering e.g. Pharmaceutical proteins
Vaccine, DNA production, research gene function and regulation, transgenic plants
The Molecular Analysis of Disease-sickle-cell disease T-to-A DNA substitution- valine rather than glutamic acid
Gene Therapy –“Replacing” defective genes to cure human disease-Transfer and expression of a gene into the patients’ cell
Applications in forensic science: DNA fingerprinting- parentage/identifying criminals
Environment Protection e.g. Degradation of toxic pollutants and different microbes used for sewage treatment, wastewater treatment, industrial effluent treatment.

Disadvantages of Recombinant DNA Technology

Hazardous toxins(botulinum toxin, neurotoxins, aflatoxins) can be produced easily to be used as biological weapons.
The genetically engineered microbes causing severe diseases.
Superior genetically engineered varieties may replace the wild type, causing a considerable loss to biodiversity.
During the gene transfer process, the antibiotic resistance marker genes might get introduced to the bacteria which are pathogenic to humans.
Insertion of foreign genes into the incorrect sites in the host genome may result in progeny with deformities.
We people are also worried about the safety of modifying food and medicines using recombinant DNA technology.