Cbse Class 12 Biology Revision Notes Chapter 11

Class 12 Biology Chapter 11 Notes: Biotechnology – Principles And Processes

 

Biology studies living organisms and includes their morphology, physiology, anatomy and origin. The concept of biotechnology is based on Biology. Biotechnology harnesses cellular processes to enhance the quality of products that help improve our lives. 

Biotechnology involves using living organisms, cells or enzymes in large-scale manufacturing. Promoting products and processes using modern biotechnology and Genetically Modified Organisms was made possible only when human beings mastered ways to alter DNA. This fundamental process is called Genetic engineering. 

Extramarks is the preferred online learning partner for lakhs of students preparing for Class 11 and Class 12 Biology subjects. Our team of academic experts have created study materials based on the latest CBSE syllabus and NCERT guidelines. Students can confidently rely on our study solutions. 

Extramarks Class 12 Biology Chapter 11 Notes covers all the fundamental points of Biotechnology to assist students in understanding the importance of genetic engineering in health sciences and technology. 

 

Key Topics In Class 12 Biology Chapter 11 Notes

Below are the important topics covered in our Class 12 Biology Chapter 11 Notes. 

  1. Principles of Biotechnology.
  2. Tools of Recombinant DNA Technology.
  3. Processes of Recombinant DNA Technology.

 

Principles Of Biotechnology

Class 12 Biology Chapter 11 Notes starts by explaining the two key strategies that introduced the creation of modern biotechnology:

 

(i) Genetic engineering:

The methods to alter the chemistry of the genetic material (DNA & RNA)  to introduce genes into host species and thus alter the phenotype of the host organism.

 

(ii) Bioprocess engineering: 

This process involves the maintenance of a sterile atmosphere in chemical engineering processes to allow the growth of only the desired microbes in significant quantities to manufacture biotechnological products like antibiotics, vaccines, etc. 

 

Recombinant DNA:

  • Traditional hybridisation procedures often lead to the multiplication of both unwanted and desired genes. At the same time, the new strategies of recombinant DNA technology have brought a breakthrough in this limitation with the help of gene cloning and gene transfer.
  • Recombinant DNA technology introduces only the desired genes without adding undesirable genes to the target organism.
  • DNA will only multiply in the progeny cells when incorporated into the recipient’s genome and inherited with the host DNA. The alien piece of DNA becomes part of a chromosome capable of replication. 
  • A specific DNA sequence in a chromosome responsible for starting replication is called the ‘Origin of Replication’.
  • To multiply a foreign piece of DNA in an organism, it needs to be a part of a chromosome with the ‘Origin of Replication’. The ori permits the foreign piece of DNA to replicate and multiply in the host organism by a process called cloning.

 

This section is further detailed in our Class 12 Biology Chapter 11 Notes. Students can access the chapter notes from Extramarks’ website.

The construction of the first recombinant DNA:

  • The first artificial recombinant DNA emerged from the potential of linking a gene encoded antibiotic resistance with a plasmid of Salmonella typhimurium. Stanley Cohen and Herbert Boyer achieved this by isolating the antibiotic resistance gene in 1972. They cut out a piece of DNA from a plasmid responsible for contributing to antibiotic resistance.
  • Restriction enzymes cut the DNA at specific locations and link the cut piece of DNA with the plasmid DNA. These plasmid DNA behave as vectors to transfer the piece of DNA adhered to it. 
  • With the aid of the enzyme DNA ligase, the antibiotic resistance gene was linked with the plasmid vector.
  • The new combination of circular autonomously replicating DNA created in vitro is called recombinant DNA. 
  • When this DNA is transferred into Escherichia coli, it could replicate using the new host’s DNA polymerase enzyme and produce multiple copies.
  • The cloning of antibiotic resistance genes in Escherichia coli was the ability to multiply copies of antibiotic resistance genes.

 

The three basic steps in genetically modifying an organism:

  1. Identification of DNA that comprises suitable genes
  2. Desirable DNA is introduced into the host organism.
  3. After maintaining introduced DNA in the host organism, the DNA is transferred to its progeny.

 

Students are recommended to register on Extramarks website and get full access to our Class 12 Biology Chapter 11 Notes. The study notes are specially prepared for students to gain a fundamental knowledge of the essential principles of Biotechnology. 

 

Tools of Recombinant DNA Technology :

The critical tools that are involved in genetic engineering: 

  • Restriction enzymes
  • Polymerase enzymes 
  • Ligases 
  • Vectors
  • Host organism

 

Restriction Enzymes:

  • Two enzymes in Escherichia coli were isolated. They were responsible for restricting the growth of bacteriophages. One of these added methyl groups to DNA while the other cut DNA. This was known as restriction endonuclease.
  • Hind II was the first restriction of the endonuclease. Its functioning depended on a specific DNA nucleotide sequence that was isolated and characterised after five years. 
  • Hind II cut DNA molecules at specific points by recognising a specific sequence of six base pairs. This specific base sequence is termed the recognition sequence for Hind II. 
  • However, today, over 900 restriction enzymes have been secluded from more than 230 strains of bacteria, each of which recognises various recognition sequences.
  • While naming these enzymes, the first letter of the name is from the genus. The second two letters are derived from the species of the prokaryotic cell from which they were isolated. 

For Example, EcoRI (Escherichia coli RY 13).

  • The letter ‘R’ is derived from the strain’s name. Roman numbers that follow the names indicate the order of isolation of the enzymes from that strain of bacteria.
  • Restriction enzymes belong to a larger  class of enzymes called nucleases. 

 

These are of two kinds: 

  1. Exonucleases: get rid of nucleotides from the ends of the DNA.
  2. Endonucleases: make cuts at certain positions within the DNA.

 

  • A restriction endonuclease operates by ‘inspecting’ the length of a DNA sequence. Once the specific recognition sequence is found, it will combine with the DNA.
  • Then each of the two strands of the double helix is cut at distinct points in their sugar-phosphate backbones.
  • Each restriction endonuclease will distinguish specific palindromic nucleotide sequences in the DNA.

 

The process of Restriction Enzymes is further explained in our Class 12 Biology Chapter 11 Notes. Students who are looking for easy guided solutions for learning the chapter of Biotechnology should register on Extramarks and get our study notes.

 

What are Palindromes? 

Palindromes are groups of letters that form the exact words when read both forward and backward, e.g., MALAYALAM. The palindrome in DNA is a sequence of base pairs. It reads the same on the two strands when the orientation of the reading is kept the same. 

For Example, the following sequences are read-alike on the two strands in the 5′ to 3′ direction. This is also true if the arrangement is read in the 3′ to 5′ direction.

 

5′ —— GAATTC —— 3′

 

3′ —— CTTAAG —— 5′

 

Restriction enzymes cut up the DNA strand between the same two bases on the opposite strand and a little away from the centre of the palindrome sites. This leaves portions of single-stranded DNA at the ends. Overhanging stretches called sticky ends, present on each strand, form hydrogen bonds with complementary cut counterparts. This stickiness of the ends enables the action of the enzyme DNA ligase. 

In genetic engineering, ‘recombinant’ molecules of DNA are formed with the help of restriction endonucleases composed of different DNA genomes. When the same restriction enzyme cuts the DNA, the DNA fragments formed have the same kind of ‘sticky-ends’. These are joined together using DNA ligases.

Students will get more detailed information about the importance of restriction enzymes in genetic engineering in our Class 12 Biology Chapter 11 Notes.

 

Separation and isolation of DNA fragments:

 

After cutting DNA by a restriction endonuclease, the fragments get separated by gel electrophoresis. DNA fragments being negatively charged molecules are separated by moving towards the anode in the presence of an electric field through a medium. The most commonly used medium is agarose gel (a natural polymer) extracted from seaweeds. The separation of DNA fragments is based on their size through the sieving effect given by the agarose gel. Thus, the smaller fragments move farther on the agarose gel. 

The separated DNA fragments can be seen only after staining the DNA. A compound, ethidium bromide, is used for visualisation, followed by exposure to UV radiation. You will observe bright orange-coloured bands of DNA when exposed to UV light. The separated DNA bands are extracted from the gel piece after being cut. This process is called elution. The purified DNA fragments are used to construct recombinant DNA by joining them with cloning vectors.

 

Cloning Vectors:

 

  • Because of their more significant number per cell, Bacteriophages have large copy numbers of their genome within the bacterial cells. 
  • Some plasmids only consist of one or two copies per cell, while others may have 15-100 copies per cell. 
  • Multiplication of equal numbers to the copy number of the plasmid becomes easy with the ability to link an alien piece of DNA.  
  • Vectors are engineered to help the easy linking of foreign DNA and the selection of recombinants from non-recombinants.

 

Following features are required to facilitate cloning into a vector:

  1. Origin of replication (ori): ori is a sequence from where replication begins. Any piece of DNA linked to this sequence, further replicates within the cells of a host. This sequence is also responsible for controlling the copy number of the linked DNA. Thus, if anyone wants to retrieve a large number of copies of the target DNA, it should be cloned in a vector whose origin supports a high copy number.
  2. Selectable marker: Apart from ‘ori’, the vector requires a selectable marker. The selectable marker assists in the identification and elimination of non-transformants. Also, it selectively allows the growth of the transformants. Transformation is the procedure through which a piece of DNA is introduced into a host bacterium. The genes encode antibiotic resistance. These include ampicillin, chloramphenicol, tetracycline or kanamycin, etc., which are useful selectable markers for E. coli. 
  3. Cloning sites: The vector requires very few single recognition sites for the restriction enzymes to link foreign DNA. The availability of recognition sites within the vector will produce numerous fragments, which will cause complications in gene cloning. 

 

The ligation of foreign DNA is carried out at a restriction site that occurs in one of the two antibiotic resistance genes. For instance, you can ligate an alien DNA at the BamH I site of the tetracycline resistance gene in the vector pBR322.

 

The transformants growing on an ampicillin containing matrix are then transported on a tetracycline matrix. The recombinants will multiply in an ampicillin containing matrix but not on that containing tetracycline. Non- recombinants will grow on the medium with both antibiotics. In this case, only one antibiotic resistance gene helps select the transformants. In contrast, the other antibiotic resistance gene gets ‘inactivated due to insertion’ of foreign DNA and helps choose recombinants. 

 

  1. Vectors for cloning genes in plants and animals: The methods of transferring genes into plants and animals extracted from bacteria and viruses have been known for ages. For instance, Agrobacterium tumefaciens is a pathogen of various dicot plants which can deliver a piece of DNA known as ‘T-DNA’. T-DNA converts normal plant cells into a tumour and directs these tumour cells to produce the chemicals required by the pathogen. In the same way, retroviruses in animals can transform normal cells into cancerous cells. 

The tumour-inducing (Ti) plasmid of Agrobacterium tumefacient has now been modified into a cloning vector that is not pathogenic to the plants but can still use the mechanisms to deliver desired genes into a variety of plants. 

These four features to facilitate the cloning of vectors are covered at length in our Class 12 Biology Chapter 11 Notes. Extramarks study notes are prepared by Biology teachers with decades of experience, hence making the  quality of content extremely good.

 

Competent Host:

  • DNA is a hydrophilic molecule. Thus, it cannot pass through cell membranes. In contrast, bacterial cells are made ‘competent’ to accept plasmid DNA.
  • The bacteria are treated with a specific divalent cation concentration, such as calcium. Calcium raises the efficiency with which DNA enters the bacterium through the pores of the cell wall.
  • Recombinant DNA is forced into such cells by incubating the cells with recombinant DNA on ice.
  • After that, the cells are placed shortly at 42 degrees  celsius to give heat shock and then put back on ice. This allows the bacteria to take up the recombinant DNA.
  • Another way to introduce foreign DNA into a host cell is through micro-injection. Recombinant DNA is injected directly into the nucleus of an animal cell.
  • Another method appropriate for plant cells is bombardment with high-velocity micro-particles of gold or tungsten coated with DNA. This procedure is known as biolistics or gene gun.
  • The last method uses ‘disarmed pathogen’ vectors. The vectors transport the recombinant DNA into the host cell when allowed to infect the cell.

 

Processes Of Recombinant DNA Technology

The process is explained in detail in our Class 12 Biology Chapter 11 Notes. Below are the summary points highlighting the key steps in Recombinant DA technology:

 

Isolation of DNA:

  • DNA and other macro_molecules such as RNA proteins and lipids are enclosed within the cell membrane. 
  • The cell is treated with enzymes such as lysozyme(bacteria), cellulase(plant cells) and Chitinase(fungus) that isolate the DNA.
  • The treatment with ribbon nucleus proteins can extract RNA through the treatment with proteins.
  • DNA finally precipitates upon adding chill ethanol in a suspension of fine threads.

 

Cutting of DNA at Specific Locations:

  • Restriction enzyme digestion is performed by incubating purified DNA molecules with the restriction enzyme.
  • The specific enzymes require optimum conditions. 
  • DNA is negatively charged and moves towards the anode when the Agarose gel electrophoresis is employed.
  • The process is repeated with vector DNA as well. 
  • The cut source of DNA, the vector DNA with a specific restriction enzyme, the cut-out gene of interest and the cut vector with space are mixed, followed by the addition of ligase. 
  • By this Recombinant DNA is prepared.

 

Amplification of Gene of Interest using PCR:

  • In Polymerase  Chain  Reaction(PCR), multiple copies of DNA of interest are synthesised in vitro with the help of two sets of primers; chemically synthesied oligonucleaotides and theDNA polymerase enzyme.
  • DNA polymerase enzymes extend the primers utilising nucleotides provided in the reaction and the genomic DNA as template.
  • When the DNA replication is repeated many times, this segment of DNA can be amplified to construct a billion copies.
  • Thermostable DNA polymerase achieves repeated amplification and remains active during high temperature-induced denaturation of double standard DNA.
  • Amplified fragments can be used for ligation with a vector to continue cloning.

 

Each cycle has three steps: 

(i) Denaturation;

(ii) Primer annealing

(iii) Extension of primers

 

Insertion of Recombinant DNA into the Host Cell:

  • Competent recipient cells take up DNA present in their surroundings.
  • If recombinant DNA consists of a gene for resistance to an antibiotic is transferred into E.coli cells , the host cells transform into ampicillin-resistant cells.
  • Only transformants will grow if they  are spread on agar plates containing ampicillin, while untransformed recipient cells will die.
  • The presence of the ampicillin resistance gene allows the selection of the desired transformed cell.
  • The ampicillin resistance gene is called a selectable marker.

 

Obtaining the Foreign Gene Product:

  • The ultimate aim is to produce a desirable protein.
  • Knowledge of many technical details is essential when the expression of foreign genes in host cells is involved.
  • The cloned gene of interest is optimised to induce the expense expression of the protein. This production is taken on a large scale.
  • A recombinant protein is expressed in a heterologous host.
  • Cells with the cloned gene of interest are grown in a lab for small-scale production.
  • The cultures extract the required protein and then purify it using different separation techniques.
  • With the help of a continuous culture system, the cells are multiplied where the used medium is drained out, and the fresh medium is added to maintain the exponential phase. This culture method produces larger biomass, which yields a more significant number of desired proteins.

 

What are Bioreactors? 

As defined in our Class 12 Biology Chapter 11 Notes, Bioreactors are vessels where raw materials are biologically converted into specific products using microbes. A bioreactor processes large volumes of culture. The advantage of bioreactors is that it provides suitable growth conditions for achieving the desired product, such as optimum temperature, PH vitamins, etc.

Class 12 Biology Notes Chapter 11 emphasises the need for bioreactors for large scale production. A commonly used  stirred tank bioreactor is in the shape of a cylinder with a curve base. It facilitates mixing with the help of a stirrer and allows the availability of oxygen throughout the bioreactor. The air can be bubbled into the reactor. Stirred tank reactor consists of an oxygen delivery system, a foam control system, a temperature control system, a pH control system, and sampling ports for small volumes of culture to be withdrawn.

 

 (b) Sparged stirred-tank bioreactor through which sterile air bubbles are sparged

 

 

Downstream Processing:

 

Before marketing a finished product, it must be subjected to several processes. The processes are:

  • Separation and Purification(Collectively referred to as Downstream processing). 
  • Formulation with suitable preservatives. In the case of drugs, the Formulation has to undergo a full clinical trial.
  • Proper quality control testing for each product is mandatory. 
  • The downstream processing and quality control are different for every product.

 

Class 12 Biology Chapter 11 Notes: Exercises & Solutions

 

Extramarks website provides excellent study materials in the form of  exercise and solutions. Class 12 Biology Chapter 11 Notes covers all the major principles in the Chapter, ‘Biotechnology: processes and principles’. Experts in Biology prepare the solution guide to provide comprehensive solutions to students for their examinations. To gain access to Class 12 Biology Chapter 11 Notes students can register on the Extramarks’ website.

Students can click on the links provided below to access the solutions for various exercises they will need to prepare for the examination.

  • Chapter 11: Exercise 11.1 Solutions
  • Chapter 11: Exercise 11.2 Solutions 
  • Chapter 11: Exercise 11.3 Solutions
  • Chapter 11: Exercise 11.4 Solutions
  • Chapter 11: Exercise 11.5 Solutions
  • Chapter 11: Exercise 11.6 Solutions
  • Chapter 11: Exercise 11.7 Solutions
  • Chapter 11: Exercise 11.8 Solutions
  • Chapter 11: Exercise 11.9 Solutions
  • Chapter 11: Exercise 11.10 Solutions
  • Chapter 11: Exercise 11.11 Solutions
  • Chapter 11: Exercise 11.12 Solutions
  • Chapter 11: Exercise 11.13 Solutions
  • Chapter 11: Exercise 11.14 Solutions
  • Chapter 11: Exercise 11.15 Solutions
  • Chapter 11: Exercise 11.16 Solutions

 

NCERT Exemplar for Class 12 Biology 

NCERT Exemplar book has a repository of resources for students to help them with tricky and complex topics besides providing revision notes which come in handy during tests and exams. The Exemplar includes a lot of questions from the chapter including short questions, long format questions, MCQs, etc. 

By solving the questions from Exemplar students will be able to revise the chapter and also understand their weak sections. So they can revise these sections to gain more confidence in their final exams.

After referring to the Class 12 Biology Chapter 11 Notes and NCERT Exemplar, the students will have a comprehensive understanding of the Biotechnology chapter. Our study notes also contain solutions for all the questions covered in NCERT Textbook and NCERT Exemplar books.

Key features of Class 12 Biology Chapter 11 Notes

Biotechnology is a field of technology that creates valuable products and opportunities with the help of genetic engineering. For students aiming for careers in biotechnology, Class 12 Biology Chapter 11 Notes provide well-explained concepts. 

 

Let us look at the Key features of Class 12 Biology Chapter 11 Notes:

  • It covers the entire CBSE syllabus
  • It is based on the NCERT Textbooks.
  • It comprises biological concepts prepared in a pointwise manner by academic experts. 

Apart from Biology Chapter 11 Class 12 Notes,  students can visit the Extramarks website for more information on CBSE Revision Notes, CBSE Sample Papers, CBSE past years’  Question Papers, CBSE additional questions and essential Formulas. 

Q.1 What happens to a DNA fragment that is transferred into an alien organism?

Ans

Two situations arise:
a. The alien DNA doesn’t integrate to the host’s genome and is lost after the cell containing the alien DNA dies.
b. The alien DNA integrates to the host’s genome and multiplies as the cell reproduces.

Q.2 What is insertional inactivation? Give an example.

Ans

The inactivation of a gene by inserting a fragment of DNA into the middle of its coding sequence is called insertional inactivation. Any products from the inactivated gene will not work because of the codes added to it. Cromogenic selectable markers show insertional inactivation. A recombinant DNA is inserted within the coding sequence of an enzyme, -galactosidase inactivating the enzyme. The presence of a substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the -galactosidase and the colonies do not produce any colour.

Q.3 Define Biotechnology.

Ans

Biotechnology is defined as the industrial use of living organisms (or their parts) to produce various products and services. It is the fusion of biology and technology.

Q.4 Expand EFB. What is the definition of Biotechnology according to EFB?

Ans

EFB stands for European Federation of Biotechnology The definition of Biotechnology as given by EFB: ‘The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services’.

Q.5 What are the two core techniques that enabled birth of modern biotechnology?

Ans

The two core techniques are:
a. Genetic engineering: The ability to alter genetic material of an organism according to ones need and get the desired phenotypic expression
b. Sterile microbial culturing techniques: The ability to grow desired organism in a contamination free environment

Q.6 What do you understand by the term genetic engineering? What are the basic three steps involved in genetic modification of an organism?

Ans

Genetic engineering is the scientific alteration of genes or genetic material to produce desirable new traits in organisms or to eliminate undesirable ones. It aims at introducing new characteristics or attributes physiologically or physically, such as introducing a novel trait, enhancing existing ones, or producing a new protein. It involves:
a. Isolation of DNA
b. Manipulation of the DNA
c. Reintroduction of DNA into cells/organisms.

Q.7 What is the difference between modern biotechnology and biotechnology in general?

Ans

Modern biotechnology involves highly sophisticated techniques such as genetic engineering, protein and enzyme engineering and in vitro fertilisation and reproduction techniques whereas biotechnology in general also includes non-sophisticated techniques like agriculture, genetic breeding, curd and bread making etc. In brief, modern biotechnology is biotechnology at the molecular level.

Q.8 What is the benefit of sexual reproduction over asexual reproduction?

Ans

Sexual reproduction provides opportunities for variations in the genetic makeup of an organism, which may be beneficial to the organism as well as the population, especially under environmental stress.

Q.9 Traditional hybridization procedures used in plants and animal breeding, very often lead to inclusion and multiplication of undesirable genes. How can you overcome this problem?

Ans

We can overcome the problem by the use of genetic engineering where we can isolate and introduce a single or a set of specific genes without introducing undesired genes into the target organism.

Q.10 What is origin of replication?

Ans

The origin of replication is a particular DNA sequence in the chromosome or a plasmid at which DNA replication is initiated.

Q.11 What is cloning?

Ans

Cloning collectively refers to the process used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. The term also encompasses situations whereby organisms reproduce asexually.

Q.12 What are ‘molecular scissors’?

Ans

Molecular scissors are enzymes that cut molecules at a specific point.
– Restriction enzymes are molecular scissors that cut double-stranded DNA.
– Ribozymes are molecular scissors that cut RNA.
Molecular scissors are used as tools of modern biotechnology for cutting the desired fragment of DNA, RNA etc.

Q.13 What are vectors? Write a short note on cloning vectors.

Ans

A vector is an organism that does not cause disease itself but which acts as a vehicle for the disease causing pathogens. A cloning vector is a small DNA vehicle that carries a foreign DNA fragment. It is used to transfer the desired gene into the host during genetic engineering experiments. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with the same restriction enzyme, then ligating the fragments together. There are many different types of cloning vectors.
Eg.: Plasmids, Bacteriophages (λ phage), Cosmid, Bacterial Artificial Chromosome (BAC), Yeast Artificial Chromosome (YAC) etc.


Plasmid pBR322 is an E. coli cloning vector, showing restriction sites (HindIII, EcoRI, BamHI, SalI, PvuII, PstI, ClaI), ori and antibiotic resistance genes (ampR and tetR).

Q.14 Write a short note on DNA ligase.

Ans

DNA ligase is a type of enzyme that joins double stranded DNA fragments. DNA ligase is used in vivo in DNA repair and DNA replication and in vitro in recombinant DNA technologies. DNA ligase forms covalent phosphodiester bonds between 3′ hydroxyl ends of one nucleotide and the 5′ phosphate end of the another.

Q.15 Why are plasmids and bacteriophages used as cloning vectors?

Ans

It is so because:

  • Plasmids and bacteriophages have the ability to replicate inside bacterial cells without being controlled by the chromosomal DNA.
  • Bacteriophages and many plasmids are present in high number of copies per cell and thus high number of the engineered gene can be obtained.

Q.16 What are the key tools required for genetic engineering?

Ans

The key tools required for genetic engineering are:
a. Enzymes: Restriction endonuclease, DNA ligase, DNA polymerase etc.
b. Vectors: Plasmids, phasemids, cosmids, BACs, YACs etc.
c. Transformation tools like gene gun, microinjection etc.
Other tools required are those used for gene isolation, separation and screening of recombinants.

Q.17 Write a short note on the followings:
a. Ori
b. Selectable marker
c. Cloning sites

Ans

a. Ori: Ori or origin of replication is a particular DNA sequence at which DNA replication is initiated. The ori binds the pre-replication complex, a protein complex that recognizes, unwinds, and begins to copy DNA. Prokaryotes have single origin of replication per circular chromosome while eukaryotes have multiple origin of replication per chromosome (to help speedup the replication of the usually large genetic content).
b. Selectable marker: Selectable markers are genes coding for a certain known phenotypic characteristics that helps in identifying and eliminating non-transformed organisms and selectively permitting the growth of the transformed (having the recombinant gene) organisms. Genes that code for resistance to antibiotic such as ampicillin, chloramphenicol, tetracycline or kanamycin or impart specific colours to the organism/ microbial colonies are used as selectable markers.
c. Cloning sites: Cloning site is that region of a vector in which the gene to be transferred to the host is inserted. The site contains recognition sequences specific to various restriction endonuclease so that the gene-containing DNA fragment can be inserted into the vector. Multiple recognition sequences may be present but generally one is preferred to keep thing simple.

Q.18 From which organism restriction endonuclease was first extracted?

Ans

Escherichia coli

Q.19 Write a short note on Agrobacterium tumifaciens.

Ans

Agrobacterium tumifaciens is a rod shaped, gram-negative bacteria that is able to deliver a piece of its DNA known as ‘T-DNA’ to transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by it. This tumor inducing (Ti) plasmid has been modified into a cloning vector which can deliver genes into a variety of plants without causing the disease.

Q.20 What is known as recognition sequence of a restriction endonuclease? Give an example.

Ans

Many restriction endonucleases exhibit binding specificity and function only after binding to a specific DNA sequence. This specific base sequence is known as the recognition sequence for that particular restriction endonuclease.

Enzyme
Source

Recognition Sequence
Cut
EcoRI
Escherichia coli
5’GAATTC

3’CTTAAG
5’-G AATTC-3’
3’-CTTAA G-5’
HindIII
Haemophillus
influenzae

5’AAGCTT

3’TTCGAA
5’-A AGCTT-3’
3’-TTCGA A-5’

Q.21 Describe the convention for naming restriction endonucleases.

Ans

Naming of restriction enzymes is based on the bacteria from which they are isolated in the following manner:-

  • The first letter of the name comes from the genus.
  • The second two letters come from the species.
  • The next letter comes from the strain.
  • Roman numbers following the names indicate the order in which the enzymes were isolated from that strain of bacteria.
    E.g., Naming of EcoRI
E Escherichia genus
co coli species
R RY 13 strain
I First identified order of identification
in the bacterium

 

Q.22 What are nucleases? What is the difference between endonucleases and exonucleases?

Ans

A nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Restriction endonuclease cleaves the double-stranded DNA at a specific recognition site after binding to it at that point whereas restriction exonuclease removes nucleotides sequentially from the end of the DNA.

Q.23 Explain the working of a restriction endonuclease.

Ans

A restriction endonuclease functions by “scanning” the length of a DNA molecule. Once it finds its specific recognition sequence it binds to the DNA molecule and makes one cut in each of the two sugar-phosphate backbones of the double helix at the specific points.

Q.24 What is a palindromic sequence? What is its significance in genetic engineering?

Ans

A palindromic sequence is a sequence that has the property of reading the same in either direction. Restriction endonucleases used in genetic engineering to cut double-stranded DNA have various palindromic sequences as their recognition sequence. The endonucleases identify these specific palindromic sequences, binds and cleaves the DNA at that point.

5’-GAATTC-3’
3’-CTTAAG-5’
 
5'-G     AATTC-3'
3'-CTTAA     G-5'

Action of EcoRI

Q.25 What are sticky ends? What is its significance?

Ans

A sticky end also called overhangs which is a stretch of unpaired nucleotides at the end of a double-stranded DNA molecule. These unpaired nucleotides can be in either strand, creating either 3′ or 5′ overhangs.

Sticky ends have the ability to form hydrogen bonds with their complementary cut counterparts and facilitate the action of DNA ligases.

5′—G AATTC—3′
3′—CTTAA G—5′

Q.26 How is a DNA fragment amplified?

Ans

A DNA fragment is amplified either by polymerase chain reaction or by microbial cloning method.

Q.27 What is the sole purpose of recombinant DNA technology?

Ans

The sole purpose of the recombinant DNA technology is to express a certain phenotypic character or a protein in an organism in which it is not naturally found. This, in turn, has many applications like in development of better varieties of animals; pest resistant, drought resistant, high yielding plants; drug production and gene therapy etc.

Q.28 With a diagram explain the steps involved in recombinant DNA technology.

Ans

The steps involved in recombinant DNA technology are:

  1. Identification and isolation of desired gene.
  2. Selection of a vector and inserting the DNA fragment containing the desired gene into it. This is done with the help of restriction endonuclease and DNA ligase enzymes.
  3. Inserting the engineered vector into the host cell/organism. This is done by the use of transformation techniques like biolistic (use of gene gun), microinjection or disarmed pathogens.
  4. Screening of recombinant organisms by exploiting the selectable marker introduced along with the desired gene.
  5. Maintenance and multiplication of the recombinants obtained.

Steps involved in recombinant DNA technology

Q.29 How do you separate DNA fragments?

Ans

DNA is negatively charged and can be separated using gel electrophoresis. Here, the negatively charged DNA fragments are forced to move towards the positively charged anode under an electric field through a matrix, generally agarose gel. The fragments separate according to their size, the smallest moving the fastest and reaching the fastest from the point of loading.

Q.30 What is agarose?

Ans

Agarose is a polysaccharide extracted from seaweeds and is a constituent of agar. It is widely used as a medium of gel-electrophoresis.

Q.31 Write a short note on gel-electrophoresis?

Ans

Gel-electrophoresis is a technique for separating a mixture of molecules on the basis of their size under an electric field. DNA, RNA and proteins can be separated using this procedure. The larger molecules takes more time to pass through the matrix, generally made of agarose, compared to the smaller molecules. It can be used both for analysis and purification (small scale) of samples.

Q.32 What is an antibiotic resistance gene? What is its significance in recombinant DNA technology?

Ans

Antibiotic resistance gene is a gene which is expressed in a microorganism. It is able to withstand the effect of that specific antibiotic. In recombinant DNA technology, antibiotic resistant genes are used in the vectors as selectable markers to identify transformed hosts.

Q.33 Define Transformation.

Ans

Transformation is defined as the genetic alteration of a cell resulting from the uptake and expression of foreign genetic material.

Q.34 What are the various methods available for inserting an engineered vector into the host?

Ans

The various methods are:
a. Use of competent cells: These have the ability to take up extracellular DNA. Competence can be induced by treating the cells with divalents like calcium and subjecting them to heat shocks.
b. Microinjection: Use of a micro-needle to insert the vector into the cell.
c. Gene Gun: Biolistic particle delivery system injects the vector (coated onto a gold particle) into the host cell, generally plant cells.
d. Use of disarmed pathogens: Pathogens which are devoid of the virulent gene but retain the ability to transfer the recombinant DNA into the host.

Q.35 What do you understand by the term competent host with reference to recombinant DNA technology?

Ans

A competent host is a cell that has the ability to take up extracellular DNA from the environment. In recombinant DNA technology, competent microbial cells are used in order to avoid expensive and complex transformation techniques like biolistics and microinjection.

Q.36 Write short note on the following:
a. Micro-injection
b. Biolistics
c. Disarmed pathogen

Ans

a. Micro-injection: Microinjection is a transformation technique where engineered vectors/genes are injected into the host cell using a micro-needle. The process is carried out under an optical microscope called the micromanipulator and the DNA molecules can not only be delivered inside the cell membrane but also the nuclear envelope when needed.
b. Biolistics: Biolistics is a transformation technique involving the use of a gene-gun or the Biolistic Particle Delivery System. Generally used to transform plant cells, the system uses a heavy metal particle coated with the engineered vector. This coated particle is fired into the host cells using a specialized airgun. Specialized biolistics can not only transform genetic material but also cell organelles.
c. Disarmed pathogen: Pathogens/viruses that are devoid of their virulent genes are called disarmed pathogens. These pathogen vectors when allowed to infect a host are able to transfer the recombinant DNA into the cell without causing any disease. Such vectors are however not completely safe as there are chances of such vectors reverting to virulent forms.

Q.37 Expand PCR. Explain the steps involved in the technique. What is the PCR machine called?

Ans

Polymerase Chain Reaction.
The steps involved are: – A PCR starts with a denaturing step. The DNA sample is heated to 94-960C to break the hydrogen bonds between the bases of the two strands.
– When the two strands separate out, the temperature is lowered to 50-560C, which allows the primers (short DNA fragments that acts as starting point for replication) to anneal (base pair) with the denatured strands.
– The temperature is raised to 720C at which the Taq polymerase (a thermostable polymerase) elongates the primer by adding nucleotides.

The PCR machine is called a thermocycler.

Q.38 What is a primer? Why is it required?

Ans

A primer is a short DNA sequence that pairs to a larger DNA fragment at a complementary sequence and acts as a starting point for DNA replication. DNA polymerase elongates a sequence by adding nucleotides to it and thus requires a primer strand.

Q.39 What is a recombinant protein?

Ans

A recombinant protein is a protein expressed artificially in an organism with the help of recombinant DNA technology, e.g., artificial human insulin expressed in E. coli.

Q.40 What is a bioreactor?

Ans

A bioreactor is a device, natural or artificial (bioreactor vessels) that supports biologically active environment and in which chemical reactions involving organisms and/or biologically active substances are carried out.

Q.41 Write a short note on pBR322.

Ans

pBR322 is a commonly used E. coli plasmid cloning vector. The molecule is a double-stranded circular DNA, 4361 base pairs in length. It was the first artificial plasmid created by Bolivar and Rodriguez and was named after them.

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