Biotechnology: Principles and Processes

What to remember

• Biotechnology rests on two core techniques: genetic engineering (changing the genetic material and putting it into a host) and bioprocess engineering (keeping a sterile environment so that only the wanted cells grow in large quantity).
• The first recombinant DNA was made by Stanley Cohen and Herbert Boyer in 1972. They cut an antibiotic resistance gene from a plasmid of Salmonella typhimurium and joined it to a plasmid vector.
• Restriction endonucleases cut DNA at palindromic recognition sequences, ligase joins the pieces, and a vector with ori, a selectable marker and a cloning site carries the gene into a competent host.
• PCR makes many copies of a DNA segment in three repeated steps: denaturation, annealing of primers, and extension by Taq polymerase. Bioreactors then grow the host in large volumes, followed by downstream processing.

Principles of biotechnology

The European Federation of Biotechnology (EFB) defines biotechnology as the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.

Modern biotechnology depends on two basic techniques.

Technique	What it does	Why it matters
Genetic engineering	Alters the chemistry of genetic material (DNA or RNA), introduces it into a host organism, and so changes the phenotype of the host	Lets us give an organism a new useful trait
Bioprocess engineering	Maintains a sterile (contamination-free) environment in chemical engineering processes so that only the desired microbe or eukaryotic cell grows in large quantity	Needed for making antibiotics, vaccines, enzymes and other products on a large scale

If a piece of alien DNA is simply introduced into a cell, it usually cannot multiply by itself. It is lost when the cell divides. The alien DNA must be linked to a sequence called the origin of replication (ori). Only then can it replicate and be inherited.

The first recombinant DNA was built in 1972 by Stanley Cohen and Herbert Boyer. They isolated an antibiotic resistance gene from a plasmid of the bacterium Salmonella typhimurium. They linked it to a native plasmid vector of Escherichia coli. The two key tools were the cutting enzyme (restriction enzyme) and the joining enzyme (DNA ligase).

Three basic steps are needed to make a genetically modified organism:

• 1. Identification of DNA with the desirable genes.
• 2. Introduction of the identified DNA into a host.
• 3. Maintenance of the introduced DNA in the host and its transfer to the progeny.

The origin of replication is the specific base sequence from which replication starts. Any piece of DNA linked to ori can replicate within the host cell. The ori also controls the copy number of the linked DNA.

Tools of recombinant DNA technology

The main tools are restriction enzymes, polymerases and other enzymes, vectors, and the competent host.

Restriction enzymes. The first restriction endonuclease to be isolated was Hind II. It always cut DNA at a specific point by recognising a particular sequence of six base pairs. This sequence is the recognition sequence. More than 900 restriction enzymes have been isolated from over 230 strains of bacteria.

Naming rule for restriction enzymes (example: EcoRI):

Part of name	Meaning in EcoRI
E	First letter of the genus: Escherichia
co	First two letters of the species: coli
R	Name of the strain: RY 13
I	Roman numeral for the order in which the enzyme was isolated from that strain

Restriction enzymes belong to a larger class called nucleases. Nucleases are of two kinds.

Type	Action
Exonuclease	Removes nucleotides from the ends of the DNA
Endonuclease	Cuts DNA at a specific position within the DNA

Each restriction endonuclease inspects the length of a DNA sequence. Once it finds its specific recognition sequence, it binds to the DNA and cuts both strands at specific points in their sugar-phosphate backbones.

The recognition sequence is a palindrome. A palindromic sequence in DNA is a sequence of base pairs that reads the same on the two strands when the reading direction (5′ → 3′) is kept the same. Example for EcoRI:

• 5′ — GAATTC — 3′
• 3′ — CTTAAG — 5′

EcoRI cuts each strand between G and A. This leaves single-stranded overhanging stretches at the cut ends. These overhangs are called sticky ends, because they form hydrogen bonds with their complementary cut counterparts. The stickiness makes the joining easy. DNA ligase then joins the ends and seals the joint.

The same restriction enzyme must cut both the source DNA and the vector DNA. Then both have the same sticky ends and can be joined by ligase. The cut fragments joined to the vector form recombinant DNA.

Separation and isolation of DNA fragments

The cutting of DNA by restriction endonucleases gives fragments of different lengths. These are separated by gel electrophoresis.

Feature	Detail
Gel material	Agarose, a natural polymer extracted from sea weeds
Charge on DNA	DNA is negatively charged, so it moves towards the anode (positive electrode)
Basis of separation	The gel acts as a sieve. Smaller fragments move farther. Fragments are separated by size
Staining	Ethidium bromide, followed by exposure to UV radiation, gives bright orange-coloured bands
Elution	The separated bands are cut out of the agarose gel and the DNA is extracted from the gel piece. This step is elution

The purified DNA fragments are then used to construct recombinant DNA by joining them with cloning vectors.

Cloning vectors

Plasmids and bacteriophages multiply at very high copy numbers within bacterial cells. They are the commonly used vectors. A cloning vector must have the following features.

Feature	Role
Origin of replication (ori)	Starts replication and controls the copy number of the linked DNA
Selectable marker	Helps to identify and eliminate non-transformants and allows only transformants to grow. Common markers are genes for resistance to ampicillin, chloramphenicol, tetracycline or kanamycin
Cloning sites	Unique recognition sites for restriction enzymes. Only one site for the chosen enzyme should be present in the vector, so that the vector is cut once

Normal E. coli cells do not carry resistance to these antibiotics. So the resistance gene is a good selectable marker.

pBR322. It is a well-known E. coli cloning vector. Its main parts are:

Part	Function
ori	Origin of replication
ampR	Gene for ampicillin resistance
tetR	Gene for tetracycline resistance
rop	Codes for proteins involved in the replication of the plasmid
Restriction sites	HindIII, EcoRI, BamHI, SalI, PvuII, PstI and ClaI. BamHI, SalI and HindIII lie within tetR, and PstI lies within ampR

Insertional inactivation. A foreign DNA is ligated at the BamHI site of tetR. The recombinant plasmid then loses tetracycline resistance because the gene is interrupted. The non-recombinant plasmid keeps both resistance genes. Selection works in two steps:

• 1. Plate the transformed cells on ampicillin. Both recombinants and non-recombinants that carry a plasmid grow.
• 2. Transfer the colonies to tetracycline medium. Non-recombinants grow. Recombinants do not grow, because tetR is inactivated.

Blue-white selection. This is an alternative marker system based on insertional inactivation of an enzyme. The vector carries a gene for β-galactosidase. A foreign DNA is inserted into the coding sequence of β-galactosidase. This inactivates the enzyme. The bacteria are grown on a medium with a chromogenic substrate.

Colony	Plasmid	Enzyme	Colour
Non-recombinant	Intact β-galactosidase gene	Active	Blue
Recombinant	Gene interrupted by insert	Inactive	White (no colour)

Vectors for plants and animals.

• Agrobacterium tumefaciens is a pathogen of many dicot plants. It delivers a piece of its DNA, the T-DNA, into plant cells and makes them tumour-like. The tumour-inducing (Ti) plasmid has been modified into a cloning vector which is no longer pathogenic. It is still able to deliver genes into plant cells. This is a disarmed vector.
• Retroviruses in animals can transform normal cells into cancerous cells. They have been disarmed and used to deliver desirable genes into animal cells.

Competent host and getting DNA into it

DNA is a hydrophilic molecule. It cannot pass through cell membranes easily. The host cell must be made competent, which means capable of taking up DNA.

Method	Host	How it works
Calcium chloride treatment and heat shock	Bacterial cells	Cells are treated with a specific concentration of a divalent cation such as calcium. This increases the efficiency of DNA entering through pores in the cell wall. Cells are incubated with recombinant DNA on ice, then given a heat shock at 42°C for a short time, then put back on ice. The bacteria then take up the recombinant DNA
Microinjection	Animal cells	Recombinant DNA is injected directly into the nucleus of an animal cell
Biolistics (gene gun)	Plant cells	Cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA
Disarmed pathogen vectors	Plant and animal cells	The disarmed pathogen infects the cell and transfers the recombinant DNA into the host

Processes of recombinant DNA technology

The order of steps is: isolation of genetic material → cutting with restriction enzymes → amplification of the gene by PCR → ligation into a vector → insertion into host → culturing the host → obtaining the foreign gene product → downstream processing.

Isolation of the genetic material (DNA). DNA is present inside the cell enclosed by membranes. To release it, the cell wall and membranes must be broken. Different enzymes are used.

Cell type	Enzyme used
Bacterial cell	Lysozyme
Plant cell	Cellulase
Fungal cell	Chitinase

Along with DNA, the cell has RNA, proteins, polysaccharides and lipids. RNA is removed by treatment with ribonuclease (RNase). Proteins are removed by treatment with protease. Other molecules are removed by appropriate treatments. When chilled ethanol is added, purified DNA precipitates out as fine threads in the suspension. The threads can be spooled out.

Cutting of DNA at specific locations. Purified DNA and the vector are incubated with the chosen restriction enzyme under optimal conditions. The progress of the digestion is checked by agarose gel electrophoresis. DNA is a negatively charged molecule, so the bands move towards the anode. The cut vector and the cut gene of interest are then joined with ligase.

Amplification of the gene of interest by PCR. PCR means polymerase chain reaction. Multiple copies of the gene are made in vitro, using two sets of primers and the enzyme DNA polymerase. Primers are small, chemically synthesised oligonucleotides. They are complementary to the regions at the two ends of the target DNA. The enzyme extends the primers using the nucleotides in the reaction and the target DNA as a template. If the cycle is repeated many times, the DNA segment is amplified about a billion times after about 30 cycles.

Step	What happens	Approximate temperature
Denaturation	Double-stranded DNA is heated and the two strands separate	About 94°C
Annealing	The two primers pair with their complementary sequences on the single strands	About 50–60°C
Extension	DNA polymerase adds nucleotides to the primers and extends them	About 72°C

The enzyme used is Taq polymerase. It is isolated from the bacterium Thermus aquaticus. It remains active during the high temperature used for denaturation of double-stranded DNA. In each cycle the number of DNA copies doubles.

Insertion of recombinant DNA into the host cell. The recombinant DNA is mixed with competent host cells. It is introduced by the methods in the table above (heat shock, microinjection, gene gun or disarmed vectors).

Obtaining the foreign gene product. If a gene encoding a useful protein is expressed in a heterologous host, the protein is called a recombinant protein. The cells with the foreign gene can be grown on a small scale in the laboratory. For production on a large scale, the cells are grown in bioreactors. Bioreactors are vessels in which raw materials are biologically converted into specific products under controlled conditions. Large volumes of 100–1000 litres of culture can be processed.

Two common bioreactor types:

Reactor	Features
Simple stirred-tank bioreactor	A cylindrical vessel with a stirrer. The stirrer mixes the contents evenly and also helps oxygen availability throughout the culture
Sparged stirred-tank bioreactor	Has a stirrer and, in addition, sterile air is bubbled (sparged) through the culture. This gives better oxygen transfer

Parts of a bioreactor: an agitator system, an oxygen delivery system, a foam control system, a temperature control system, a pH control system and sampling ports. Sampling ports allow small volumes of the culture to be withdrawn periodically.

Downstream processing. After the synthesis of the product, it must be separated and purified before it is marketed as a finished product. The steps together are called downstream processing. The product is formulated with suitable preservatives. Clinical trials and strict quality control testing are needed for drugs.

NEET traps

• 1. Exonuclease removes nucleotides from the ends of DNA; endonuclease cuts within the DNA. Restriction enzymes are endonucleases.
• 2. In EcoRI, "R" stands for the strain (RY 13), not for "restriction". "I" is the order of isolation, not the number of cuts.
• 3. A palindromic sequence reads the same on the two strands when both are read in the same 5′ → 3′ direction. It is not simply the same word read forwards and backwards on one strand.
• 4. EcoRI cuts the two strands at different points (between G and A on each strand), so sticky ends result and not blunt ends.
• 5. DNA moves to the anode (positive electrode) because it is negatively charged. Smaller fragments move farther.
• 6. Ethidium bromide with UV gives orange bands. It is a stain and does not make DNA move.
• 7. In blue-white selection, recombinants are white and non-recombinants are blue. Insertional inactivation of β-galactosidase gives the white colour.
• 8. In pBR322, cloning at BamHI inactivates tetR, so recombinants are ampicillin-resistant but tetracycline-sensitive.
• 9. Lysozyme acts on bacteria, cellulase on plant cells and chitinase on fungi. RNase removes RNA and protease removes proteins.
• 10. The Ti plasmid is for plants (Agrobacterium tumefaciens); disarmed retroviruses are for animal cells. Biolistics is used for plant cells; microinjection for animal cells.

One-liners

• 1. EFB defines biotechnology as the integration of natural science and organisms, cells, parts thereof and molecular analogues for products and services.
• 2. The two core techniques are genetic engineering and bioprocess engineering.
• 3. Cohen and Boyer made the first recombinant DNA in 1972 using a plasmid of Salmonella typhimurium.
• 4. The origin of replication (ori) starts replication and controls the copy number.
• 5. Hind II was the first restriction endonuclease isolated; it recognises a specific six base pair sequence.
• 6. EcoRI recognises GAATTC and cuts between G and A on each strand.
• 7. Sticky ends are single-stranded overhangs that pair with complementary ends.
• 8. DNA ligase joins the cut DNA fragments.
• 9. Agarose is extracted from sea weeds and is used for gel electrophoresis of DNA.
• 10. pBR322 has ori, ampR, tetR and rop.
• 11. White colonies contain recombinant plasmids; blue colonies contain non-recombinant plasmids.
• 12. Heat shock at 42°C after calcium chloride treatment makes bacteria take up DNA.
• 13. Taq polymerase comes from Thermus aquaticus and is stable at high temperature.
• 14. Chilled ethanol precipitates DNA as fine threads.
• 15. Downstream processing means separation and purification of the product before marketing.

Practice questions

1. Which enzyme is used to join two DNA fragments after they are cut with the same restriction enzyme?

   1. Exonuclease
   2. Lysozyme
   3. DNA ligase
   4. Taq polymerase
   Answer

   C. DNA ligase

   DNA ligase joins the sticky ends and seals the joint.

2. The recognition sequence of EcoRI is

   1. 5′-GAATTC-3′
   2. 5′-GTTAAC-3′
   3. 5′-GAGGTC-3′
   4. 5′-GAATCC-3′
   Answer

   A. 5′-GAATTC-3′

   EcoRI recognises the palindrome GAATTC and cuts between G and A.

3. Taq polymerase used in PCR is obtained from

   1. Salmonella typhimurium
   2. Agrobacterium tumefaciens
   3. Escherichia coli
   4. Thermus aquaticus
   Answer

   D. Thermus aquaticus

   Taq polymerase comes from the thermophilic bacterium Thermus aquaticus and stays active at high temperature.

4. To isolate DNA from plant cells, the cell wall is broken down using

   1. protease
   2. cellulase
   3. chitinase
   4. lysozyme
   Answer

   B. cellulase

   Cellulase digests the plant cell wall. Lysozyme is for bacteria and chitinase for fungi.

5. Agarose, used for the separation of DNA fragments, is

   1. a polysaccharide extracted from plant roots
   2. a synthetic polymer made from ethidium bromide
   3. a natural polymer extracted from sea weeds
   4. a protein extracted from bacteria
   Answer

   C. a natural polymer extracted from sea weeds

   Agarose is a natural polymer obtained from sea weeds and forms a sieving gel.

6. In the biolistic method, cells are bombarded with high-velocity micro-particles of

   1. silver or copper coated with RNA
   2. iron or zinc coated with lipids
   3. calcium coated with protein
   4. gold or tungsten coated with DNA
   Answer

   D. gold or tungsten coated with DNA

   The gene gun uses gold or tungsten particles coated with DNA, mainly for plant cells.

7. In the name EcoRI, the letter R indicates

   1. the name of the strain of the bacterium
   2. the species of the bacterium
   3. the type of cut made by the enzyme
   4. the order of isolation of the enzyme
   Answer

   A. the name of the strain of the bacterium

   R is from the strain RY 13. The numeral I shows the order of isolation.

8. The copy number of the DNA linked to a cloning vector is controlled by

   1. the cloning site
   2. the origin of replication
   3. the recognition sequence
   4. the selectable marker
   Answer

   B. the origin of replication

   The ori starts replication and controls the copy number.

9. Which of the following serves as a selectable marker in a cloning vector?

   1. A gene for kanamycin resistance
   2. The rop gene
   3. The origin of replication
   4. A single EcoRI recognition site
   Answer

   A. A gene for kanamycin resistance

   Antibiotic resistance genes such as for kanamycin, ampicillin, tetracycline or chloramphenicol are selectable markers.

10. Bacterial cells treated with calcium chloride and recombinant DNA on ice are given a heat shock at

    1. about 94°C
    2. about 72°C
    3. about 42°C
    4. about 4°C
    Answer

    C. about 42°C

    The brief heat shock at 42°C makes the competent cells take up DNA.

11. Purified DNA is separated from a cell lysate as fine threads by adding

    1. lysozyme
    2. ribonuclease
    3. protease
    4. chilled ethanol
    Answer

    D. chilled ethanol

    Chilled ethanol precipitates DNA as fine threads.

12. An exonuclease differs from an endonuclease because an exonuclease

    1. cuts DNA only at palindromic sequences
    2. joins two DNA fragments
    3. adds nucleotides to the 3′ end of primers
    4. removes nucleotides from the ends of DNA
    Answer

    D. removes nucleotides from the ends of DNA

    Exonucleases remove nucleotides from the ends, while endonucleases cut within the DNA.

13. The first recombinant DNA was constructed by

    1. Watson and Crick
    2. Cohen and Boyer
    3. Hershey and Chase
    4. Meselson and Stahl
    Answer

    B. Cohen and Boyer

    Stanley Cohen and Herbert Boyer made it in 1972 using a plasmid of Salmonella typhimurium.

14. A sparged stirred-tank bioreactor differs from a simple stirred-tank bioreactor in that it

    1. has no stirrer
    2. uses only anaerobic cultures
    3. has sterile air bubbled through the culture
    4. is used only for animal cells
    Answer

    C. has sterile air bubbled through the culture

    Sparging passes sterile air bubbles through the culture, in addition to stirring, for better oxygen transfer.

15. If one double-stranded DNA molecule is amplified for 5 cycles of PCR, the number of copies of the target obtained is

    1. 10
    2. 32
    3. 64
    4. 16
    Answer

    B. 32

    Each cycle doubles the copies, so 2⁵ = 32.

16. The vector most commonly used to deliver genes into plant cells is

    1. the disarmed Ti plasmid of Agrobacterium tumefaciens
    2. a disarmed retrovirus
    3. pBR322 of Escherichia coli
    4. a bacteriophage
    Answer

    A. the disarmed Ti plasmid of Agrobacterium tumefaciens

    The Ti plasmid, made non-pathogenic, delivers genes into plant cells. Retroviruses are used for animal cells.

17. Given below are two statements: Statement I: EcoRI is an endonuclease. Statement II: Exonucleases cut DNA at specific positions within the DNA. Choose the correct answer.

    1. Both Statement I and Statement II are correct
    2. Both Statement I and Statement II are incorrect
    3. Statement I is correct but Statement II is incorrect
    4. Statement I is incorrect but Statement II is correct
    Answer

    C. Statement I is correct but Statement II is incorrect

    Statement I is correct. Exonucleases remove nucleotides from the ends, so Statement II is incorrect.

18. Given below are two statements: Statement I: Smaller DNA fragments move farther in an agarose gel. Statement II: The agarose gel acts as a sieve. Choose the correct answer.

    1. Both Statement I and Statement II are correct
    2. Both Statement I and Statement II are incorrect
    3. Statement I is correct but Statement II is incorrect
    4. Statement I is incorrect but Statement II is correct
    Answer

    A. Both Statement I and Statement II are correct

    Both are correct. The gel sieves DNA by size and smaller fragments move farther towards the anode.

19. Given below are two statements: Statement I: The gene gun is used only for introducing DNA into animal cells. Statement II: Microinjection introduces DNA directly into the nucleus of an animal cell. Choose the correct answer.

    1. Both Statement I and Statement II are correct
    2. Both Statement I and Statement II are incorrect
    3. Statement I is correct but Statement II is incorrect
    4. Statement I is incorrect but Statement II is correct
    Answer

    D. Statement I is incorrect but Statement II is correct

    The gene gun is used mainly for plant cells, so Statement I is incorrect. Statement II is correct.

20. Given below are two statements: Statement I: Lysozyme is used to break the cell wall of fungi. Statement II: Chitinase is used to break the cell wall of bacteria. Choose the correct answer.

    1. Both Statement I and Statement II are correct
    2. Both Statement I and Statement II are incorrect
    3. Statement I is correct but Statement II is incorrect
    4. Statement I is incorrect but Statement II is correct
    Answer

    B. Both Statement I and Statement II are incorrect

    Lysozyme acts on bacteria and chitinase acts on fungi, so both statements are incorrect.

21. Which of the following statements are correct? 1. Taq polymerase is stable at the high temperature used for denaturation. 2. Primers used in PCR are long double-stranded DNA molecules. 3. In the annealing step, primers pair with complementary sequences on the template.

    1. 1 and 2 only
    2. 1 and 3 only
    3. 2 and 3 only
    4. 1, 2 and 3
    Answer

    B. 1 and 3 only

    Primers are short single-stranded oligonucleotides, so statement 2 is wrong. Statements 1 and 3 are correct.

22. Which of the following are essential features of a cloning vector? 1. An origin of replication 2. A selectable marker 3. Several recognition sites for the same restriction enzyme to give many cuts

    1. 2 and 3 only
    2. 1 and 3 only
    3. 1, 2 and 3
    4. 1 and 2 only
    Answer

    D. 1 and 2 only

    A vector needs ori and a selectable marker. It should have only one recognition site for the chosen enzyme, so statement 3 is wrong.

23. Given below are two statements: Statement I: Insertion of foreign DNA at the BamHI site of tetR in pBR322 inactivates the tetracycline resistance gene. Statement II: Such recombinant cells can grow on ampicillin but cannot grow on tetracycline. Choose the correct answer.

    1. Both Statement I and Statement II are correct
    2. Both Statement I and Statement II are incorrect
    3. Statement I is correct but Statement II is incorrect
    4. Statement I is incorrect but Statement II is correct
    Answer

    A. Both Statement I and Statement II are correct

    The insert interrupts tetR and ampR stays intact, so both statements are correct.

24. Given below are two statements: Statement I: White colonies in blue-white selection contain non-recombinant plasmids. Statement II: Blue colonies contain recombinant plasmids. Choose the correct answer.

    1. Both Statement I and Statement II are correct
    2. Both Statement I and Statement II are incorrect
    3. Statement I is correct but Statement II is incorrect
    4. Statement I is incorrect but Statement II is correct
    Answer

    B. Both Statement I and Statement II are incorrect

    Insertion inactivates β-galactosidase, so recombinants are white and non-recombinants are blue. Both statements are incorrect.

25. Given below are two statements: Statement I: Chilled ethanol precipitates DNA as fine threads. Statement II: Proteins in the cell lysate are removed by ribonuclease. Choose the correct answer.

    1. Both Statement I and Statement II are correct
    2. Both Statement I and Statement II are incorrect
    3. Statement I is correct but Statement II is incorrect
    4. Statement I is incorrect but Statement II is correct
    Answer

    C. Statement I is correct but Statement II is incorrect

    Proteins are removed by protease. Ribonuclease removes RNA, so Statement II is incorrect.

26. Given below are two statements: Statement I: Sticky ends form hydrogen bonds with their complementary cut counterparts. Statement II: A palindromic sequence reads the same on the two strands when read in the same 5′ → 3′ direction. Choose the correct answer.

    1. Both Statement I and Statement II are correct
    2. Both Statement I and Statement II are incorrect
    3. Statement I is correct but Statement II is incorrect
    4. Statement I is incorrect but Statement II is correct
    Answer

    A. Both Statement I and Statement II are correct

    Both statements are correct and describe sticky ends and palindromes.

27. Assertion (A): Taq polymerase is used in the polymerase chain reaction. Reason (R): Taq polymerase is not denatured at the high temperature of the denaturation step.

    1. Both A and R are true and R is the correct explanation of A
    2. Both A and R are true but R is not the correct explanation of A
    3. A is true but R is false
    4. A is false but R is true
    Answer

    A. Both A and R are true and R is the correct explanation of A

    Taq polymerase is thermostable, which is why it can be used through repeated cycles.

28. Assertion (A): Bacterial cells can be made competent by treatment with calcium chloride. Reason (R): A heat shock at about 94°C is given to make bacteria take up DNA.

    1. Both A and R are true and R is the correct explanation of A
    2. Both A and R are true but R is not the correct explanation of A
    3. A is true but R is false
    4. A is false but R is true
    Answer

    C. A is true but R is false

    Assertion is true. The heat shock is at about 42°C, so the reason is false.

29. Assertion (A): Exonucleases cut DNA at specific recognition sequences inside the DNA. Reason (R): Endonucleases cut DNA within the molecule.

    1. Both A and R are true and R is the correct explanation of A
    2. Both A and R are true but R is not the correct explanation of A
    3. A is true but R is false
    4. A is false but R is true
    Answer

    D. A is false but R is true

    Exonucleases remove nucleotides from the ends, so the assertion is false. The reason is true.

30. Assertion (A): Bioprocess engineering is needed for large-scale production of antibiotics and vaccines. Reason (R): It maintains a sterile environment so that only the desired cells grow in large quantity.

    1. Both A and R are true and R is the correct explanation of A
    2. Both A and R are true but R is not the correct explanation of A
    3. A is true but R is false
    4. A is false but R is true
    Answer

    A. Both A and R are true and R is the correct explanation of A

    Sterility prevents contamination, which explains the need for bioprocess engineering.

31. Assertion (A): The Ti plasmid of Agrobacterium tumefaciens is used as a vector for plants after modification. Reason (R): The modified Ti plasmid still causes tumours in plant cells.

    1. Both A and R are true and R is the correct explanation of A
    2. Both A and R are true but R is not the correct explanation of A
    3. A is true but R is false
    4. A is false but R is true
    Answer

    C. A is true but R is false

    The plasmid is disarmed so that it is no longer pathogenic. The assertion is true and the reason is false.

32. Match List I with List II. List I: a. EcoRI b. Taq polymerase c. Lysozyme d. DNA ligase List II: i. Thermus aquaticus ii. Breaks the bacterial cell wall iii. Joins DNA fragments iv. Restriction endonuclease from Escherichia coli

    1. a-i, b-iv, c-ii, d-iii
    2. a-iv, b-i, c-ii, d-iii
    3. a-ii, b-i, c-iv, d-iii
    4. a-iv, b-ii, c-i, d-iii
    Answer

    B. a-iv, b-i, c-ii, d-iii

    EcoRI is from Escherichia coli (iv); Taq polymerase from Thermus aquaticus (i); lysozyme breaks bacterial walls (ii); ligase joins DNA (iii).

33. Match List I with List II. List I: a. Fungal cell b. Plant cell c. Bacterial cell d. RNA in the lysate List II: i. Cellulase ii. Chitinase iii. Lysozyme iv. Ribonuclease

    1. a-i, b-ii, c-iii, d-iv
    2. a-ii, b-i, c-iv, d-iii
    3. a-iii, b-i, c-ii, d-iv
    4. a-ii, b-i, c-iii, d-iv
    Answer

    D. a-ii, b-i, c-iii, d-iv

    Fungal wall - chitinase; plant wall - cellulase; bacterial wall - lysozyme; RNA - ribonuclease.

34. Match List I with List II. List I: a. Gene gun b. Microinjection c. Heat shock d. Disarmed pathogen vector List II: i. DNA injected into the nucleus of an animal cell ii. Competent bacterial cells at 42°C iii. Gold or tungsten particles coated with DNA iv. Ti plasmid of Agrobacterium

    1. a-iii, b-ii, c-i, d-iv
    2. a-iii, b-i, c-iv, d-ii
    3. a-i, b-iii, c-ii, d-iv
    4. a-iii, b-i, c-ii, d-iv
    Answer

    D. a-iii, b-i, c-ii, d-iv

    Gene gun - coated particles (iii); microinjection - nucleus of animal cell (i); heat shock - competent bacteria (ii); disarmed vector - Ti plasmid (iv).

35. Match List I with List II. List I: a. ori b. ampR c. tetR d. rop List II: i. Resistance to tetracycline ii. Codes for proteins for replication of the plasmid iii. Start of replication iv. Resistance to ampicillin

    1. a-iv, b-iii, c-i, d-ii
    2. a-i, b-iv, c-iii, d-ii
    3. a-iii, b-iv, c-i, d-ii
    4. a-iii, b-i, c-iv, d-ii
    Answer

    C. a-iii, b-iv, c-i, d-ii

    ori - start of replication (iii); ampR - ampicillin resistance (iv); tetR - tetracycline resistance (i); rop - replication proteins (ii).

36. Match List I with List II. List I: a. Denaturation b. Annealing c. Extension d. Result of about 30 cycles List II: i. Primers pair with the template ii. The two strands of DNA separate iii. Taq polymerase adds nucleotides to primers iv. About a billion copies of the target

    1. a-ii, b-iii, c-i, d-iv
    2. a-ii, b-i, c-iii, d-iv
    3. a-i, b-ii, c-iii, d-iv
    4. a-ii, b-i, c-iv, d-iii
    Answer

    B. a-ii, b-i, c-iii, d-iv

    Denaturation separates strands (ii); annealing pairs primers (i); extension by Taq (iii); about a billion copies after about 30 cycles (iv).

37. Match List I with List II. List I: a. Agarose b. Ethidium bromide c. Anode d. Elution List II: i. Gives orange bands under UV ii. Polymer from sea weeds iii. Electrode towards which DNA moves iv. Extraction of DNA from a cut gel band

    1. a-iii, b-i, c-ii, d-iv
    2. a-i, b-ii, c-iii, d-iv
    3. a-ii, b-i, c-iv, d-iii
    4. a-ii, b-i, c-iii, d-iv
    Answer

    D. a-ii, b-i, c-iii, d-iv

    Agarose - sea weed polymer (ii); ethidium bromide - orange bands (i); anode - DNA moves towards it (iii); elution - extraction from gel (iv).

38. A gene is ligated at the BamHI site of tetR in pBR322. E. coli cells transformed with the ligation mixture form colonies on ampicillin but fail to grow on tetracycline. These colonies most likely carry

    1. plasmids with the foreign gene inserted in tetR
    2. plasmids that have lost ampR
    3. no plasmid at all
    4. plasmids without any insert
    Answer

    A. plasmids with the foreign gene inserted in tetR

    Loss of tetracycline resistance with retained ampicillin resistance shows insertional inactivation of tetR by the insert.

39. A mixture of DNA fragments of 2 kb, 5 kb and 8 kb is run on an agarose gel. The band nearest the wells after the run is the

    1. fragment of all three sizes equally
    2. 8 kb fragment
    3. 5 kb fragment
    4. 2 kb fragment
    Answer

    B. 8 kb fragment

    Larger fragments move more slowly through the gel, so the 8 kb fragment stays nearest the wells.

40. A student wants to isolate DNA from yeast cells and remove the RNA from the lysate. The correct pair of enzymes is

    1. lysozyme and protease
    2. cellulase and ribonuclease
    3. chitinase and ribonuclease
    4. chitinase and protease
    Answer

    C. chitinase and ribonuclease

    Yeast is a fungus, so chitinase breaks the wall. Ribonuclease removes RNA.

41. In a PCR, the enzyme was replaced by a DNA polymerase that is destroyed at high temperature. The most likely result is that

    1. amplification becomes faster
    2. no denaturation of DNA can take place
    3. primers fail to anneal at all
    4. amplification fails because the enzyme is inactivated during denaturation
    Answer

    D. amplification fails because the enzyme is inactivated during denaturation

    Without a thermostable enzyme like Taq polymerase, the enzyme is lost in the first denaturation step.

42. A plant biologist wants to introduce a gene into plant cells but Agrobacterium is not available. The suitable method is

    1. treating cells with calcium chloride and heat shock at 42°C
    2. injecting DNA into the nucleus of the cell
    3. bombarding the cells with DNA-coated gold particles
    4. using a disarmed retrovirus
    Answer

    C. bombarding the cells with DNA-coated gold particles

    Biolistics (gene gun) is used for plant cells. Microinjection is for animal cells and retroviruses are for animal cells.

43. Bacterial cells are kept with recombinant DNA on ice, moved briefly to 42°C and returned to ice. The purpose of the 42°C step is to

    1. denature the recombinant DNA
    2. help the cells take up the recombinant DNA
    3. activate the restriction enzyme
    4. kill non-transformed cells
    Answer

    B. help the cells take up the recombinant DNA

    The heat shock helps competent cells take up DNA through pores.

44. In a large culture vessel, cells at the bottom are starved of oxygen even though sterile air is supplied. The feature that corrects this is

    1. the stirrer that mixes the contents evenly
    2. the sampling port
    3. the foam control system
    4. the pH control system
    Answer

    A. the stirrer that mixes the contents evenly

    Stirring mixes the culture and makes oxygen available throughout the vessel.