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
- 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|
|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
- 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
- Capable of being transcribed but not translated
- Used to amplify their insert
- Expression of the transgene in the target cell
- Generally have a promoter sequence that drives expression
Have two origins of replication, recognized by two different host cells e.g. YEp24
Types of vector
There are four major types of vectors:
- 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.
- 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
- 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
- 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
- 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
- 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.
- 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
- 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
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
- 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
Viruses increasingly are used to insert desired genes into eukaryotic cells. e.g. retrovirus, adenoviruses, recombinant baculoviruses
- Gene gun: A blast of compressed gas shoots a spray of DNA-coated metallic microprojectiles into the cells
Step 5- Selection of the Transformed Cells:
- Distinguishing the transformed cells from the non-transformed cells
- Antibiotic resistance
- Visible characters
- Assay for biological activity
- Colony hybridization
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.
- Molecular cloning a laboratory manual, Joseph Sambrook, David W Russel,3rd edition
- Prescott, Harley and Klein Microbiology-7th edition
- Current Protocols in Molecular Biology,2009, John Wiley and Sons
- Microbiology, A Human Perspective by Nester, Anderson, Roberts- 6th edition
- Molecular cell biology, Lordish,5th edition