Biology 2 Marks Questions

The vector in which the DNA input is integrated and cloned in a suitable host or its copies are produced is called cloning vector.
Origin of Replication or Ori : Ori is a specific sequence from where replication starts and when any DNA gets attached to this sequence then it can replicate inside the host cells. The sequence is also responsible for controlling the number of copies of DNA added. If we want more copies of a target DNA, then it should be cloned into a vector whose origin helps in making more copies.

The features required for cloning in a vector are as follows :
Selectable Market : Along with 'Ori' the vector also requires selectable markers, which are helpful in identifying and eliminating non-transformants and in selective selection of transformants. Transformation is a process by which a segment of DNA is introduced into the host bacteria. Generally, genes coding resistance to some antibiotics like Ampicillin, Chloramphanicol, Tetracycline or Cannamycin etc. is not found in E. coli bacteria because the genes showing resistance are absent in them. Therefore, E. coli is considered a good selectable marker.

grobacterium tumifaciens is used in gene cloning. However, Agrobacterium tumifaciens is a pathogenic pathogen of many dicotyledonous plants. It transforms normal plant cells into tumors by transferring a segment of DNA called 'T-DNA' and these tumor cells produce chemicals necessary for the pathogen. On this basis, the Ti plasmid of Agrobacterium tumifaciens has now been converted into a cloning vector, which is not pathogenic for plants, but is used to transfer genes of interest to many plants.

Those enzymes which cut DNA at certain points and break it into small pieces of fixed size are called restriction enzymes.
Separation and isolation of DNA fragments can be done by gel electrophoresis. DNA fragments are negatively charged molecules, hence they can be separated by forcefully sending them towards the anode in an electric field. Agarose medium is used in this. DNA fragments are separated according to their size by the sieving effect of agarose gel. For this reason, the smaller the size of the fragments, the farther they will travel. When ethidium bromide stained gel is exposed to ultraviolet light, a bright orange band of DNA becomes visible. The separated strips of DNA are cut from the agarose gel and extracted from the gel pieces. By joining cloning vector to purified DNA it is used to create recombinant DNA.

Restriction enzyme used in genetic engineering is famous by the name of molecular scissors. These are of two types :
1. Restriction Exonuclease : Function : They separate nucleotides from the end of DNA.
2. Restriction Endonuclease : Function : Cuts at specific sites within the DNA. Each restriction endonuclease is dependent on the length of the DNA sequence. Each restriction endonuclease functions by ‘inspecting’ the length of a DNA sequence. When it finds its specific recognition sequence, it binds to the DNA and cuts both the strands of the double helix at specific centers of the sugar-phosphate bases.

This plasmid is used as a cloning vector. In this p = Plasmid, BR = the first letter of the name of the scientists Bolivar and Rodrigan who created it and 322 of their experiments. Their size can range from 1.5 Kb to 1500 Kb. It can contain anywhere from three genes to several thousand genes. Only DNA fragments smaller than 15 Kb can be cloned in this plasmid (see Fig. 9.5).
Two antibiotic resistance genes are found in it. One gene confers resistance to ampicillin and the other to tetracycline. Recognition sites of many specific restriction enzymes are found in it. 4361 base pairs are found in it and it is obtained in pure form.

Following are the properties of a good vector :
(1) Its isolation and purification should be simple and convenient.
(2) It can be successfully inserted into the host cell, that is, its transformation should be efficient and simple.
(3) It has to express the desired DNA, hence it is necessary to have regulatory elements like promoter, operator and other necessary sequences present in the vector.
(4) For gene transformation, the vector must have the ability to integrate either itself or its DNA input into the host chromosome.
(5) It should contain suitable reporter genes so that transformed host cells can be easily selected.

E. coli bacteria is considered a suitable host for cloning due to the following properties :
(1) It is easily transformed.
(2) It lacks functional restriction enzymes.
(3) This recombinant DNA helps in replication.
(4) Its DNA structure and other biochemical activities are completely known.
(5) Its plasmid and bacteriophages have been clearly characterized.

For entry of foreign DNA into host cells there are other methods also. For example, in the microinjection method, recombinant DNA is injected directly into the nucleus of the animal cell. Another method that is useful for plants is to bombard cells with high-velocity particles of gold or tungsten coated with DNA, called biolistics or gene gun. The last method in which harmless pathogen vectors are used. When these vectors are allowed to infect cells, they transfer recombinant DNA into the host.

DNA is a hydrophobic molecule, hence it is not able to enter through the cell membrane. Hence the bacteria are made capable of accepting the plasmid. For this, it is treated with a specific concentration of calcium which helps the DNA to enter through the pores located in the cell wall of the bacteria. These cells are first placed on ice with recombinant DNA and then the recombinant DNA is forcefully introduced into those cells. After this, the cells are kept at 42° Celsius (heat shock) for some time and then kept on ice again. Due to this the recombinant DNA enters the bacteria.

In the naming of these enzymes, according to tradition, the first letter of the name is genus and the second and third letters are taken from the species of prokaryotic cells from which they were isolated. Like EcoRI, Eco is derived from Escherichia coli and the letter 'R' is taken from the name of the strain. Roman numbers after the name indicate the order from which the enzyme was isolated from the bacterial strain

In biotechnology we know that plasmid acts like a DNA vector which transfers the DNA attached to it. This idea originated from mosquitoes and insect vectors. Generally mosquitoes, as insect vectors, transfer the malaria parasite to the human body. In the same way, by using a plasmid as a vector, a fragment of foreign DNA is delivered to the host organism.