Biotechnology is the integration of natural science and organisms, cells, parts thereof, and molecular analogs for product and services.
Genetic engineering is the process of genetic material by the man in vitro for the benefit of mankind.
Recombinant DNA Technology (RDT)
Restriction enzymes have the ability to recognize and cut specific nucleotide sequences.
Restriction enzymes are two types:-
- Exonuclease – Remove nucleotide from the ends of DNA.
- Endonuclease – Make cuts at the specific positions within DNA.
The first discovered restriction enzyme was Hind II. It was isolated from Haemophilus influenzae.
Types of cuts made by restriction enzymes:-
- Blunt end style – Alu I, Hae III, Hind II, Sma II, etc.
- Sticky end style or cohesive or Staggered cutting – Eco RI, Bgl II, Bam HI, Pst I, etc.
Types of restriction enzymes:-
- Type I – These cleave only one strand of DNA at an apparently random site.
- Type II – There cleavage action only at specific sites.
- Type III – They are intermediate between type I and type II, and require ATP and S-adenosyl methionine also along with Mg2+ for the functioning.
End modification enzymes used in Biotechnology
- Alkaline Phosphatase – These remove phosphate groups from the 5′ end of DNA molecules and prevents these molecules from being ligated to none another.
- T4 – polynucleotide kinase
DNA ligase (Molecular binders)
DNA ligase completes the DNA backbone by forming covalent bonds. They are used to join lengths of DNA from different sources.
Essential features of vectors
- Origin of replication (ori)
- It is the sequence from where the replication starts.
- Marker gene or Selectable markers
- Recognition sites
- Small size
- High copy number
Different types of vectors commonly used in RDT:-
- They are extrachromosomal, self-replicating, double-stranded, closed and circular DNA molecules.
- Artificial vector based on plasmids – pBR322, pUC8.
- Example – Lambda (λ) – phage.
- Cosmids (Plasmid + Lambda phage)
- Tumour-induced plasmids (Ti plasmids)
- They are found in Agrobacterium tumefaciens, which is bacteria infecting dicot plants.
- Yeast Artificial Chromosome (YAC)
- Bacterial Artificial Chromosomes (BAC)
Complementary DNA (cDNA) is made from mRNA by using the enzyme reverse transcriptase.
Synthetic DNA is synthesized with the help of DNA polymerase on DNA template or from free deoxyribonucleotide without a template.
DNA probes are used to identify and label DNA fragments that contain a specific sequence. Based on the type of label, DNA probe can be categorized into:
- Radioactively-labeled probe.
- Fluorescently-labeled probe.
The universal DNA probe is made up of repeated GATA sequence.
Genetic (Selectable) markers
These markers are used to identify host cells that have successfully taken up a cloning vector. R-plasmid (resistance plasmid for tetracycline, ampicillin etc.) is a common marker used in RDT. Only transformed cell with R-plasmid will survive in the antibiotic medium of tetracycline or ampicillin.
Techniques used in recombinant DNA technology
Electrophoresis is the technique used in biotechnology to separate different pieces of DNA on the basis of their size, shape, surface area and molecular weight. Agarose or polyacrylamide is used as the matrix for separation and ethidium bromide (EtBr) or azure A used for staining.
Polymerase Chain Reaction (PCR)
PCR is a technique to amplify specific DNA sequences. PCR is best defined as the ‘DNA replication in vitro’. The basic requirements of a PCR are – DNA template, Primers (tow primers are utilized), Enzyme (Thermus aquaticus – a thermophilic bacterium). PCR is used to synthesize DNA for fingerprinting assays.
It is used to detect the target DNA that has been transferred onto a blotting membrane with the help of a labeled probe.
In this technique mRNA, separated by gel electrophoresis cannot bind to the nitrocellulose membrane as in southern blotting. Then, RNA bands are blotted onto a DBM paper (Watman’s 540 paper). Theis paper is then hybridized to labeled probe and detected by autoradiography.
In this, proteins are electrophoresed in polyacrylamide gel and are then transferred onto a nitrocellulose membrane. These are later labeled with antibodies, lectins or some other compounds.
Steps involved in recombinant DNA technology:
- Isolation of genetic material (DNA).
- Cutting of DNA at a specific location.
- Amplification of gene of interest using PCR.
- Insertion of DNA fragments into vector DNA to form rDNA.
- Insertion of rDNA into hos cell.
- Selection and screening of transformed cell.
- Obtaining the foreign gene product.
The genetic material of retroviruses is the double-stranded RNA. On infection, this RNA is copied to DNA and the DNA is incorporated into the host’s chromosome. This means that the foreign genes are replicated in every daughter cell.
Physical method of gene transfer:-
- Heat shock
- The sudden heat shock (from 0oC to 42oC) causes some of the cell to take up the rDNA because the sudden heat develops pores in the cell membrane.
- Gene gun or Biolistics
- The foreign DNA is injected directly into the nucleus using an incredibly fine micropipette.
Chemical methods of direct gene transfer in biotechnology
- Polyethylene glycol-mediated transfer.
- Calcium-phosphate co-precipitation.
Other advanced technologies associated with genetic engineering
A genomic library or gene bank is the collection of the entire genome of an organism in the form of plasmid clones or phage lysates with recombinant DNA inserts.
Application of genomic library
- DNA fingerprinting
- Developed by Sir Alec Jefferys (1984)
- Used for forensic purpose
Cloning is the production of organisms, which are genetically identical to their parents.
Types of Cloning
- Gene Cloning
- Cell cloning
- Organismal cloning
RAPD – Random Amplification of Polymorphic DNA.
RFLP – Restriction Fragment Length Polymorphism.