Biotechnology: Principles and Processes

Gel electrophoresis is a technique to separation of DNA fragments according to their size. DNA is negatively charged so in gel tank when electricity is passed, DNA move towards positive electrode.

The construction of the first recombinant DNA emerged from the possibility of linking a gene encoding antibiotic resistance with a native plasmid. The cutting of DNA at specific locations became possible with the discovery of the so-called ‘molecular scissors’ – restriction enzymes. The cut piece of DNA was then linked with the plasmid DNA. This plasmid DNA acts as vector to transfer the piece of DNA attached to it. The linking of antibiotic resistance gene with the plasmid vector became possible with the enzyme DNA ligase, which acts on cut DNA molecules and joins their ends. This makes a new combination of circular autonomously replicating DNA created in vitro and is known as recombinant DNA.

Escherichia coli is a bacterium found in human colon. On this bacterium scientists have made extensive genetic experiments to make some vital chemicals like insulin. Another bacterium is Agrobacterium tumefaciens which causes crown gall in plants is extensively used for genetic experiments.

Restriction endonucleases were found by Arber in 1962 in bacteria. They act as “molecular scissors” or chemical scalpels. They recognize the specific base sequence at palindrome sites in DNA duplex and cut its strands. For example, restriction endonuclease EcoRI found in the colon bacteria E. coli recognizes the base sequence GAATTC in DNA duplex and cuts its strands between G and A.

Restriction endonucleasses are enzymes that digest double stranded DNA following recognition of specific nucleotide sequences. This is achieved by cleaving the two phosphodiester bonds, one within each strand of the DNA duplex. They are found in bacteria and their function in bacteria is to cut up any invading virus as a part of its defense mechanism, thus restricting the multiplication of viruses in the bacterial cell. Different species of bacteria produce different restriction endonucleases.

The plants, in which a functional foreign gene has been incorporated by any biotechnological methods that generally is not present in plant, are called transgenic plants. When plant cell are transformed by any of the transformation methods it is necessary to isolate the transformed cells/tissue. There are certain selectable marker genes present in vectors that facilitate the selection process. In transformed cells the selectable marker genes are introduced through vector.

There is a number of marker genes which are commonly described as reporter genes screenable genes. Some of the reporter genes which are most commonly used in plant transformation are : cat, gus, lux, nptII., etc.

Agrobacterium tumefaciens has been extensively used in genetic engineering experiments. It is the causative agent of crown gall, an important disease of many commercial crops. This disease has come to be recognized in recent years as being caused by a DNA plasmid (Ti plasmid) carried by bacterium and transferred to the plant cells. Following the discovery of the relationship between crown gall and the Ti plasmid, this plasmid has come to be widely used in plant genetic engineering as a vector in order to inject a novel gene in host plant to form a transgenic plant.

Sequence of both mtDNA and Y chromosomes are considered for the study of human evolution because they are uniparental in origin. mtDNA is inherited along with the maternal cytoplasm and Y chromosome is inherited from father. So they do not take part in recombination. In addition, mtDNA has a higher mutation rate than nuclear DNA so that it is more useful for short term evolutionary studies.

DNA restriction endonuclease are important class of restriction exonucleases, class II, which cut double-stranded DNA molecules only at sites characterized by a specific nucleotide sequence. Restriction enzymes are isolated from bacterial cells and are tools for molecular biologists. Several hundred restriction enzymes are now known, each with a specific sequence requirement dictating where it will cut DNA. Some, such as

Hind III, make staggered cuts leaving ‘sticky ends’, three nucleotides long protruding on one strand from each severed terminus; others make clean cuts in both strands at the same place and thus generate ‘blunt ends’. Digesting DNA with a restriction enzyme therefore creates a characteristic set of fragments, which can be isolated by electrophoresis and subsequently analysed.

This combination allows to select only streptomycin resistant strains and non resistant strains would die on medium containing antibiotic streptomycin.

A mutant strain of T4 -bacteriophage, RII, fails to lyse the E.coli but when two strains R-IIX and R-IIY are mixed then they lyse the E.coli because both strains have different cistrons.

The enzyme that cuts DNA at specific places is known as a restriction endonuclease. Among the options provided, the one that fits this description is :

Option C : E.coli restriction endonuclease I

Restriction endonucleases, also known as restriction enzymes, are enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites. E.coli restriction endonuclease I is an example of such an enzyme, derived from the bacterium Escherichia coli (E. coli). These enzymes are essential tools in molecular biology, particularly in processes like gene cloning, as they allow for precise cutting and manipulation of DNA.

A plasmid is a DNA molecule separate from the chromosomal DNA and capable of autonomous replication. In many cases, it is typically circular and double-stranded. It usually occurs in bacteria and is sometimes found in eukaryotic organisms. The size of plasmids varies from 1 to over 400 kilobase pairs (kbp). There may be one copy, for large plasmids, to hundreds of copies of the same plasmid in a single cell. The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952.

Plasmid and bacteriophage are used as vectors in genetic engineering. Plasmid is an autonomously replicating circular extra chromosomal DNA found in bacteria. They can be transferred from cell to cell in a bacterial colony. This characteristic is being used in biotechnology for transferring desirable gene into target gene of the host. Bacteriophage is a bacterial virus which can infect it, quickly multiply within and destroy (lyse) their host (virus) cells. During infection bacteriophages inject their DNA into these cells.

The injected DNA selectively replicate and are expressed in the host that results in a multiplication of phages that ultimately burst out of the cell (by lysis). This ability of transferring DNA from the phage genome to specific host during infection process gave scientists the idea that specially designed phage vectors could be used for gene cloning.

The bacteria commonly used for genetic engineering is Agrobacterium. Agrobacterium tumefaciens, in particular, is known for its ability to transfer DNA to plants, and it is widely used in the creation of genetically modified plants. This capability is harnessed in biotechnology for the purpose of introducing new traits to plants, such as resistance to pests, diseases, or harsh environmental conditions. Therefore, the correct option is :

Option C : Agrobacterium