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#	Question	Answer
1	Who was Rene Descartes?	Rene Descartes was a French philosopher, mathematician, and biologist of the seventeenth century.
2	What was the main direction of natural sciences after the time of Rene Descartes?	Natural sciences were directed to develop technologies that add to creature comforts and value to human life.
3	Which sciences gave rise to engineering, technologies, and industries?	Physics and chemistry.
4	What is described as the major utility of the biological world?	The biological world is described as a source of food.
5	What is biotechnology described as in the text?	Biotechnology is described as the twentieth century off-shoot of modern biology.
6	What is the importance of sticky ends?	Sticky ends made pasting together pieces of DNA a precise exercise.
7	Which Stanford scientist collaborated conceptually with Herbert Boyer?	Stanley Cohen.
8	What are plasmids?	Small ringlets of DNA that float freely in the cytoplasm of certain bacterial cells and replicate independently from chromosomal DNA.
9	What method had Stanley Cohen developed?	A method of removing plasmids from a cell and reinserting them into other cells.
10	What did Boyer and Cohen achieve together?	They recombined DNA segments in desired configurations and inserted them into bacterial cells.
11	What does biotechnology deal with?	Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.
12	Give examples of traditional biotechnology mentioned in the text.	Making curd, bread, and wine.
13	How is biotechnology used in a restricted modern sense?	It refers to processes using genetically modified organisms to achieve products on a larger scale.
14	Name some techniques included under biotechnology.	In vitro fertilisation, synthesising a gene, developing a DNA vaccine, and correcting a defective gene.
15	What are the two core techniques that enabled modern biotechnology?	1. Genetic engineering, 2. Bioprocess engineering
16	What is genetic engineering?	Techniques to alter the chemistry of genetic material (DNA and RNA), introduce these into host organisms, and change the phenotype of the host organism.
17	What is bioprocess engineering?	Maintenance of sterile, microbial contamination-free conditions to grow desired microbes/eukaryotic cells for manufacturing products like antibiotics, vaccines, and enzymes.
18	What advantage does sexual reproduction provide over asexual reproduction?	Sexual reproduction provides opportunities for variations and unique combinations of genetic setup.
19	What is preserved in asexual reproduction?	Genetic information is preserved in asexual reproduction.
20	What is a limitation of traditional hybridisation procedures?	They often lead to inclusion and multiplication of undesirable genes along with desirable genes.
21	How does genetic engineering overcome the limitation of traditional hybridisation?	It allows isolation and introduction of only desirable genes without introducing undesirable genes.
22	What happens if alien DNA integrates into the genome of a recipient organism?	It may multiply and be inherited along with the host DNA.
23	What is the role of the origin of replication?	It is a specific DNA sequence responsible for initiating replication.
24	Why must alien DNA be linked to the origin of replication?	So that the alien DNA can replicate and multiply itself in the host organism.
25	What is cloning in biotechnology?	Making multiple identical copies of any template DNA.
26	What led to the construction of the first recombinant DNA molecule?	Linking a gene encoding antibiotic resistance with a native plasmid of *Salmonella typhimurium*.
27	What is a plasmid?	An autonomously replicating circular extra-chromosomal DNA.
28	Who accomplished the first recombinant DNA construction in 1972?	Stanley Cohen and Herbert Boyer.
29	What acts as a vector to transfer a piece of DNA?	Plasmid DNA acts as a vector to transfer a piece of DNA.
30	Which enzyme joins cut DNA molecules and their ends?	DNA ligase.
31	What is recombinant DNA?	A new combination of circular autonomously replicating DNA created in vitro.
32	Which bacterium was used for cloning the antibiotic resistance gene?	*Escherichia coli* (*E. coli*).
33	What is cloning of an antibiotic resistance gene?	The ability to multiply copies of the antibiotic resistance gene in *E. coli*.
34	What are the three basic steps in genetically modifying an organism?	1. Identification of DNA with desirable genes, 2. Introduction of identified DNA into the host, 3. Maintenance and transfer of DNA to progeny
35	What are the key tools required for recombinant DNA technology?	Restriction enzymes, polymerase enzymes, ligases, vectors, and host organism.
36	Which two enzymes restricting bacteriophage growth in *E. coli* were isolated in 1963?	One enzyme added methyl groups to DNA, while the other cut DNA (restriction endonuclease).
37	What was the first restriction endonuclease discovered?	Hind II.
38	What is a recognition sequence?	A specific base sequence recognised by a restriction enzyme.
39	Approximately how many restriction enzymes are known today?	More than 900 restriction enzymes.
40	From how many bacterial strains have restriction enzymes been isolated?	Over 230 strains of bacteria.
41	How are restriction enzymes named?	The first letter comes from the genus and the next two letters from the species of the prokaryotic cell.
42	From which organism is EcoRI derived?	*Escherichia coli* RY13.
43	What do Roman numerals in restriction enzyme names indicate?	The order in which the enzymes were isolated from that bacterial strain.
44	To which larger class do restriction enzymes belong?	Nucleases.
45	What are the two kinds of nucleases?	Exonucleases and endonucleases.
46	What is the function of exonucleases?	They remove nucleotides from the ends of DNA.
47	What is the function of endonucleases?	They cut DNA at specific positions within the DNA.
48	What does a restriction endonuclease do after recognising its sequence?	It binds to DNA and cuts both strands of the double helix at specific points.
49	What type of nucleotide sequence is recognised by restriction endonucleases?	Palindromic nucleotide sequences.
50	How do restriction enzymes cut DNA strands?	They cut DNA strands a little away from the centre of palindrome sites but between the same two bases on opposite strands.
51	What are sticky ends?	Single-stranded overhanging stretches formed at the ends of DNA after restriction enzyme cutting.
52	How do sticky ends help recombinant DNA formation?	Their stickiness facilitates the action of DNA ligase.
53	What are recombinant DNA molecules?	DNA molecules composed of DNA from different sources/genomes.
54	What happens when DNA fragments are cut by the same restriction enzyme?	They have the same kind of sticky ends and can be joined together using DNA ligase.
55	Why must vector DNA and source DNA be cut with the same restriction enzyme?	Otherwise, recombinant vector molecules cannot be created.
56	Which technique is used for separation of DNA fragments?	Gel electrophoresis.
57	Why can DNA fragments move in an electric field during gel electrophoresis?	Because DNA fragments are negatively charged molecules.
58	Towards which electrode do DNA fragments move during electrophoresis?	Towards the anode (positive electrode).
59	What is the commonly used matrix in gel electrophoresis?	Agarose.
60	From where is agarose extracted?	Sea weeds.
61	On what basis are DNA fragments separated in agarose gel electrophoresis?	According to their size.
62	How does fragment size affect movement in agarose gel?	Smaller fragments move farther.
63	Which compound is used to stain DNA fragments in gel electrophoresis?	Ethidium bromide.
64	Why is UV radiation used after staining DNA with ethidium bromide?	To visualise DNA fragments as bright orange coloured bands.
65	What is elution?	The process of cutting out separated DNA bands from agarose gel and extracting them.
66	What are cloning vectors?	DNA molecules like plasmids and bacteriophages used to multiply alien DNA inside bacterial cells.
67	Why do bacteriophages have high copy numbers inside bacterial cells?	Because they replicate independently and occur in high numbers per cell.
68	How many copies per cell may some plasmids have?	Some plasmids may have 1–2 copies, while others may have 15–100 copies per cell.
69	What is the advantage of linking alien DNA with plasmid or bacteriophage DNA?	The alien DNA can multiply equal to the copy number of the plasmid or bacteriophage.
70	How are modern vectors engineered?	They are engineered to allow easy linking of foreign DNA and selection of recombinants from non-recombinants.
71	What is the origin of replication (ori)?	It is a sequence from where replication starts.
72	What happens when a piece of DNA is linked to the origin of replication?	It can replicate within the host cells.
73	What controls the copy number of linked DNA in a vector?	The origin of replication (ori).
74	Which type of vector is preferred to obtain many copies of target DNA?	A vector whose origin supports high copy number.
75	What is a selectable marker?	A feature in a vector that helps identify and eliminate non-transformants while selectively permitting growth of transformants.
76	What is transformation?	A procedure through which a piece of DNA is introduced into a host bacterium.
77	Which antibiotic resistance genes are commonly used as selectable markers in *E. coli*?	Resistance genes for ampicillin, chloramphenicol, tetracycline, and kanamycin.
78	Why are antibiotic resistance genes useful as selectable markers?	Normal *E. coli* cells do not carry resistance against these antibiotics.
79	What are cloning sites in vectors?	Specific recognition sites for restriction enzymes where alien DNA can be linked.
80	Why should vectors preferably have single recognition sites?	Multiple recognition sites generate several fragments and complicate gene cloning.
81	At which site can foreign DNA be ligated in vector pBR322?	BamHI site of the tetracycline resistance gene.
82	What happens to tetracycline resistance after insertion of foreign DNA into pBR322?	Recombinant plasmids lose tetracycline resistance.
83	How are recombinants selected in pBR322?	Recombinants grow on ampicillin medium but not on tetracycline medium.
84	How do non-recombinants behave on antibiotic media in pBR322 selection?	They grow on media containing both antibiotics.
85	What is insertional inactivation?	Inactivation of a gene due to insertion of recombinant DNA within its coding sequence.
86	Which enzyme gene is commonly inactivated for colour-based selection?	β-galactosidase gene.
87	What colour do non-recombinant colonies produce in the presence of chromogenic substrate?	Blue coloured colonies.
88	What colour do recombinant colonies produce after insertional inactivation?	They do not produce any colour.
89	Which bacterium naturally transfers T-DNA into plant cells?	*Agrobacterium tumifaciens*.
90	What is T-DNA?	A piece of DNA delivered by *Agrobacterium tumifaciens* into plant cells.
91	What is the role of Ti plasmid in biotechnology?	Modified Ti plasmid acts as a cloning vector for delivering genes into plants.
92	How are retroviruses useful in biotechnology?	Disarmed retroviruses are used to deliver desirable genes into animal cells.
93	Why must bacterial cells be made competent?	Because DNA is hydrophilic and cannot pass through cell membranes naturally.
94	Which divalent cation is commonly used to make bacterial cells competent?	Calcium ions.
95	How does calcium treatment help bacterial cells take up DNA?	It increases the efficiency with which DNA enters the bacterium through pores in the cell wall.
96	What is micro-injection?	A method in which recombinant DNA is directly injected into the nucleus of an animal cell.
97	What is biolistics or gene gun?	A method where plant cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA.
98	What role do disarmed pathogen vectors play in biotechnology?	They transfer recombinant DNA into the host cell.
99	What are the major steps involved in recombinant DNA technology?	Isolation of DNA, fragmentation by restriction endonucleases, isolation of desired DNA fragment, ligation into vector, transfer into host, culturing host cells, and extraction of desired product.
100	Why must DNA be in pure form before cutting with restriction enzymes?	Because restriction enzymes act effectively only on pure DNA free from other macromolecules.
101	Which macromolecules are released along with DNA when a cell is broken open?	RNA, proteins, polysaccharides, and lipids.
102	Which enzyme is used to break bacterial cell walls during DNA isolation?	Lysozyme.
103	Which enzyme is used to break plant cell walls during DNA isolation?	Cellulase.
104	Which enzyme is used to break fungal cell walls during DNA isolation?	Chitinase.
105	Which proteins are associated with DNA in chromosomes?	Histones.
106	Which enzyme removes RNA during DNA purification?	Ribonuclease.
107	Which enzyme removes proteins during DNA purification?	Protease.
108	How is purified DNA precipitated out?	By addition of chilled ethanol.
109	How does precipitated DNA appear in suspension?	As fine threads.
110	How are restriction enzyme digestions performed?	By incubating purified DNA molecules with restriction enzymes under optimal conditions.
111	Why is agarose gel electrophoresis used during restriction digestion?	To check the progression of restriction enzyme digestion.
112	Why does DNA move towards the positive electrode during electrophoresis?	Because DNA is negatively charged.
113	What is added after mixing cut vector DNA and gene of interest?	DNA ligase.
114	What is the result of ligating cut vector DNA and gene of interest?	Formation of recombinant DNA.
115	What does PCR stand for?	Polymerase Chain Reaction.
116	What is the purpose of PCR?	To synthesise multiple copies of the gene or DNA of interest in vitro.
117	What are primers in PCR?	Small chemically synthesised oligonucleotides complementary to regions of DNA.
118	Which enzyme extends primers during PCR?	DNA polymerase.
119	What acts as the template during PCR?	Genomic DNA.
120	What happens when DNA replication is repeated many times in PCR?	The DNA segment is amplified to approximately one billion copies.
121	Which type of DNA polymerase is used in PCR?	Thermostable DNA polymerase.
122	From which bacterium is thermostable DNA polymerase isolated?	*Thermus aquaticus*.
123	Why is thermostable DNA polymerase important in PCR?	It remains active during high-temperature induced denaturation of double-stranded DNA.
124	What can be done with the amplified DNA fragment after PCR?	It can be ligated with a vector for further cloning.
125	What are competent recipient cells capable of doing?	Taking up DNA present in their surroundings.
126	What happens when recombinant DNA carrying ampicillin resistance is transferred into *E. coli*?	The host cells become transformed into ampicillin-resistant cells.
127	What happens when transformed cells are spread on agar plates containing ampicillin?	Only transformants grow, while untransformed cells die.
128	Why is the ampicillin resistance gene called a selectable marker?	Because it helps select transformed cells in the presence of ampicillin.
129	What happens when alien DNA is inserted into a cloning vector and transferred into host cells?	The alien DNA gets multiplied.
130	What is the ultimate aim of most recombinant technologies?	To produce a desirable protein.
131	What is a recombinant protein?	A protein produced when a protein-encoding gene is expressed in a heterologous host.
132	Why are continuous culture systems used in biotechnology?	To maintain cells in their physiologically most active log/exponential phase.
133	What is the advantage of continuous culture systems?	They produce larger biomass leading to higher yields of desired protein.
134	Why are bioreactors needed?	Small volume cultures cannot yield appreciable quantities of products.
135	What are bioreactors?	Vessels in which raw materials are biologically converted into specific products using microbial, plant, animal, or human cells.
136	What conditions does a bioreactor provide?	Optimum growth conditions such as temperature, pH, substrate, salts, vitamins, and oxygen.
137	Which type of bioreactors are most commonly used?	Stirring type bioreactors.
138	What is the shape of a stirred-tank bioreactor?	Usually cylindrical or with a curved base.
139	What is the function of the stirrer in a bioreactor?	It facilitates even mixing and oxygen availability throughout the bioreactor.
140	Which systems are present in a bioreactor?	Agitator system, oxygen delivery system, foam control system, temperature control system, pH control system, and sampling ports.
141	What is downstream processing?	The series of processes a product undergoes after the biosynthetic stage before marketing as a finished product.
142	Which processes are included in downstream processing?	Separation and purification processes.
143	Why are preservatives added during downstream processing?	To formulate the product suitably before marketing.
144	What must biotechnology products undergo before marketing?	Thorough clinical trials and strict quality control testing.
145	On what does downstream processing and quality control testing depend?	They vary from product to product.
146	What does biotechnology deal with according to the summary?	Large-scale production and marketing of products and processes using live organisms, cells, or enzymes.
147	What made modern biotechnology using genetically modified organisms possible?	Learning to alter the chemistry of DNA and construct recombinant DNA.
148	What is recombinant DNA technology also called?	Genetic engineering.
149	Which enzymes are involved in recombinant DNA technology?	Restriction endonucleases and DNA ligase.
150	What is the role of plasmid or viral vectors in recombinant DNA technology?	They isolate and ferry foreign DNA into host organisms.
151	What happens after foreign DNA enters the host organism?	The foreign gene is expressed and the functional protein is purified.
152	What is required for large-scale production in biotechnology?	Use of bioreactors.
153	What is the purpose of restriction enzymes?	They cut DNA at specific sites.
154	What is the function of DNA ligase in recombinant DNA technology?	It joins DNA fragments together.
155	What is the ultimate product purified in recombinant DNA technology?	Functional protein.
156	Why are bioreactors important in biotechnology?	They are used for large-scale production.