Biotechnology Principles and Process Question Answers: NCERT Class 12 Biology

Plasmids are circular, extrachromosomal, self-replicating DNA which is present in many prokaryotic and eukaryotic organisms. Plasmid vectors provide a simple way of inserting small DNA fragments in bacterial cells and contains high copy number or have own origin of replication. R- DNA (recombinant DNA) can make their multiple copies as the plasmid vectors, therefore it determines the yield of recombinant protein. These vectors are engineered so that any foreign DNA can be transcribed in RNA and finally translated into the proteins.

Ti (tumour- inducing) plasmid which is present in Agrobacterium tumefaciens is used as vector for plants that has been considered as natural genetic engineers for transforming plants. It causes a crown gall disease in dicot plants by transferring a segment of Transfer- DNA. When the plant molecular biologists have started using Ti plasmid as vectors when it is disarmed, then it is no more harmful for the plants and they don’t cause any disease.

Note*:- Ti and Ri (root- inducing) are plasmid based vectors.  

A. Denaturation

B. Annealing

C. DNA synthesis or Extension of primers

PCR is a chain reaction because of newly synthesized DNA acts as templates for further DNA formation in the form of cycles. In each cycle, the duplex DNA strands are separated by the heat denaturation at 93- 95â—¦C and then it is cooled so that synthetic DNA primers can anneal to the complementary segments on each strand. The annealing temperature usually occurs at about 50- 70â—¦C; this annealing temperature should be low that enables the hybridization between primer and the template strand and should be high as to prevent the mismatched hybridization. Then DNA synthesis occurs at about 70-75â—¦C temperature and then this extension is obtained by DNA polymerase enzyme. The process is then repeated several times and the unit- length of the cycle doubles with every cycle.

1. Bam HI
2. Pst I
3. amp^R (ampicillin resistance gene)

This diagram is the representation of pBR 322 vector, in which p stands for plasmid and BR stands for their developers Bolivar and Rodriguez; and ‘322’ defines the plasmid identification number developed by other scientists in the same laboratory. This has tetracycline and ampicillin resistance gene on its host and have cleavage sites for Pst I, EcoRI, HindIII, Bam HI, SalI and ClaI, Pvu II. Ampicillin resistance gene inhibits bacterial cell wall synthesis by disrupting cross linkage of peptidoglycans.

As we all know that exonuclease enzyme cuts the nucleotides from the end of a nucleic acid one at a time or it particularly attacks either 5’- end or 3’- end but not both. When it cuts from the ends, it shortens the length or degrade complete DNA fragments. So, cloning vector will not get introduced into the host without any cut and this cut will only be cut down by the restriction endonuclease enzyme. Then recombinant DNA will form by joining of different DNA fragments by DNA ligase enzyme. Hence, this enzyme will not produce a recombinant DNA molecule.

Nomenclature of the restriction endonuclease contains three parts:-

1. Abbreviation of the genus and the species of the organism to the three letters
2. A letter, number or combination of the two to indicate the strain of the same species.
3. Last, Roman numeral to indicate the order of identification where modifications were found in the same organisms.

In the HindIII enzyme, ‘H’ stands for Haemophilus (Genus); ‘in’ stands for influenzae (species); and ‘d’ is the strain from R_d and lastly ‘III’ indicates the order of identification in which enzyme was isolated from the strain of the bacterium.

The restriction enzyme cuts double helical segments which contains particular nucleotide sequence which is cut down by the restriction endonuclease enzyme at specific recognition sequence of four to eight base pairs in length, is known as restriction or recognition site. Cloning vectors contain various features which allows foreign DNA to be inserted into the vectors that may be known as multiple cloning site or polylinker site which contains various unique restriction sites which are cleaved by restriction enzymes and then targeted gene is ligated into the vectors. If it will have more than one restriction site it will form various fragments by which no specific result will form.

The bacteria which are able to uptake of DNA is known as competent which is made by chemical treatment and the competency is the physiological state which changes the conformational structure and permeability of the cell membrane so that DNA can enter the cell.

Eukaryotic DNA that are intertwined (two things connected) with the proteins like histones, can be removed by the protease enzyme which converts proteins into the amino acids or to release the DNA i.e. to be isolated.

PCR chain reaction is carried out by the thermostability of the polymerase. PCR contains a series of cycles of three successive steps:- Denaturation, primer annealing and DNA synthesis; These three steps constitutes one complete cycle which is carried out repetitively just by the change in temperature.

Denaturation is the first step of the PCR reaction, in which the strands of duplex DNA are separated by heat at about 93- 95â—¦C. If this separation will not occur then primers will also not be able to anneal to complementary segments on each strand and extension will also not take place. Hence no DNA synthesis will occur and will have negative effect on the process.

The recombinant protein vaccine that is currently being used in humans is Hepatitis B vaccine against Hepatitis B virus. This vaccine contains viral envelope proteins. These surface antigens are produced by the yeast cells into which the gene is inserted. After that an immune system of the body produces the antibody against this antigen in the bloodstream. And, this antibody is called anti- HBs.   

All the biological activities related to DNA and proteins like transcription, translation, DNA replication, protein encoding, and inheritance etc. Anhydrous condition is when it contains no water. And, when the anhydrous condition occurs then biomolecules does not exhibit any biological activity because in this condition the toughness of biomolecules increase which occurs due to weakening of hydrogen bond strength. Apart from that all biological activities occurs in the denaturation process or in the presence of water and in anhydrous condition it becomes dysfunctional.

Recombinant proteins are obtained from the recombinant DNA technology. This technology involves the transfer of specific genes from an organism into another organism using vectors and restriction enzymes as molecular tools.

Ten recombinant proteins used in medical practice are −

(a) Origin of replication −Origin of replication is defined as the DNA sequence in a genome from where replication initiates. The initiation of replication can be either uni-directional or bi-directional. A protein complex recognizes the ‘on’ site, unwinds the two strands, and initiates the copying of the DNA.

(b) Bioreactors − Bioreactors are large vessels used for the large-scale production of biotechnology products from raw materials. They provide optimal conditions to obtain the desired product by providing the optimum temperature, pH, vitamin, oxygen, etc. Bioreactors have an oxygen delivery system, a foam control system, a PH, a temperature control system, and a sampling port to obtain a small volume of culture for sampling.

(c) Downstream processing − Downstream processing is a method of separation and purification of foreign gene products after the completion of the biosynthetic stage. The product is subjected to various processes in order to separate and purify the product. After downstream processing, the product is formulated and is passed through various clinical trials for quality control and other tests.

 (a) PCR: - Polymerase chain reaction (PCR) is a technique in molecular biology to amplify a gene or a piece of DNA to obtain its several copies. It is extensively used in the process of gene manipulation. The process involves in-vitro synthesis of sequences using a primer, a template strand, and a thermostable DNA polymerase enzyme obtained from a bacterium, called Thermus aquaticus. The enzyme utilizes building blocks dNTPs (deoxynucleotides) to extend the primer. In the first step, the double stranded DNA molecules are heated to a high temperature so that the two strands separate into a single stranded DNA molecule. This process is called denaturation. Then, this ssDNA molecule is used as a template strand for the synthesis of a new strand by the DNA polymerase enzyme and this process is called annealing, which results in the duplication of the original DNA molecule. This process is repeated over several cycles to obtain multiple copies of the rDNA fragment.

(b) Restriction enzymes are molecular scissors used in molecular biology for cutting DNA sequences from a specific site. It plays an important role in gene manipulation. The enzymes recognize a specific six-box pair sequence known as the recognition sequence and cut the sequence at a specific site. For example, the recognition site for enzyme ECORI is as follows:

Restriction enzyme are categorized into two types −

(i) Exonuclease − It is a type of restriction enzyme that removes the nucleotide from either 5^’ or 3^’ ends of the DNA molecule.

(ii) Endonuclease − It is a type of restriction enzyme that makes a cut within the DNA at a specific site. This enzyme acts as an important tool in genetic engineering. It is commonly used to make a cut in the sequence to obtain DNA fragments with sticky ends, which are later joined by enzyme DNA ligase.

(c) Chitinase − Chitinase is a class of enzymes used for the degradation of chitin, which forms a major component of the fungal cell wall. Therefore, to isolate the DNA enclosed within the cell membrane of the fungus, enzyme chitinase is used to break the cell for releasing its genetic material.

(a) Plasmid DNA and chromosomal DNA

(b) RNA and DNA

(c) Exonuclease and Endonuclease

The name of the restriction enzyme is Bam H 1.

Enzymes are smaller in size than DNA molecules. This is because DNA contains genetic information for the development and functioning of all living organisms. It contains instructions for the synthesis of proteins and DNA molecules. On the other hand, enzymes are proteins which are synthesised from a small stretch of DNA known as ‘genes’, which are involved in the production of the polypeptide chain.

The molar concentration of human DNA in a human diploid cell is as follows:

⇒ Total number of chromosomes × 6.023 × 10²³

⇒ 46 × 6.023 × 10_­­­²³

⇒ 2.77 ×10¹⁸ Moles

Hence, the molar concentration of DNA in each diploid cell in humans is 2.77 × 10 ²³ moles.

No, eukaryotic cells do not have restriction endonucleases. This is because the DNA of eukaryotes is highly methylated by a modification enzyme, called methylase. Methylation protects the DNA from the activity of restriction enzymes .These enzymes are present in prokaryotic cells where they help prevent the invasion of DNA by virus.

The shake flask method is used for a small-scale production of biotechnological products in a laboratory. On the other hand, stirred tank bioreactors are used for a large-scale production of biotechnology products.

Stirred tank bioreactors have several advantages over shake flasks:

(1) Small volumes of culture can be taken out from the reactor for sampling or testing.

(2) It has a foam breaker for regulating the foam.

(3) It has a control system that regulates the temperature and pH.

The palindromic sequence is a certain sequence of the DNA that reads the same whether read from 5^’ → 3^’ direction or from 3^’→ 5^’direction. They are the site for the action of restriction enzymes. Most restriction enzymes are palindromic sequences.

Five examples of palindromic sequences are:

Meiosis is a process that involves the reduction in the amount of genetic material. It is two types, namely meiosis I and meiosis II. During the pachytene stage of prophase I, crossing over of chromosomes takes place where the exchange of segments between non-sister chromatids of homlogous chromosomes takes place. This results in the formation of recombinant DNA.

A reporter gene can be used to monitor the transformation of host cells by foreign DNA. They act as a selectable marker to determine whether the host cell has taken up the foreign DNA or the foreign gene gets expressed in the cell. The researchers place the reporter gene and the foreign gene in the same DNA construct. Then, this combined DNA construct is inserted in the cell. Here, the reporter gene is used as a selectable marker to find out the successful uptake of genes of interest (foreign genes). An example of reporter genes includes lac Z gene, which encodes a green fluorescent protein in a jelly fish.