NAME: ______________________________
FREEDOM CONCEPT SCHOOL, POIGAI, VELLORE-632114
(A.Y.2024-25) Revision exam -4
FREE KIDS: XII-A BIOLOGY (044) THEORY [Link]: 70
Reading Time: 15 min. CH:9 Writing Time: 3 hrs.
Teacher’s Name: R PADMAVATHI DATE: 19.10.2024
Answer the following questions:
1 A 1
2 C 1
3 C 1
4 B 1
5 B 1
6 a 1
7 C 1
8 C 1
9 A
10 d
11 a
12 B 1
13 a- vector DNA, b- Foreign DNA ii) Restriction 1
endonuclease Bam H1, iii) DNA ligase
14 Sticky ends on DNA are formed by action of enzymes restriction 3
endonucleases. These enzymes cut the strand of DNA a little away
from the centre of the palindrome sequence between the same two
bases on both the strands. This results in single stranded stretches
on both the complementary strands at their ends. These
overhanging stretches are called sticky ends as they form hydrogen
bonds with the complementary base pair sequences.
15 (i) Denaturation of ds-DNA 3
(e) Genomic DNA template
(g) Primers [1]Mark
(b) Chemically synthesized oligonucleotides
(c) Enzyme DNA-polymerase
(h) Thermostable DNA-polymerase (from Thermus
aquaticus) [1]Mark
(f) Nucleotides provided
(d) Complementary region of DNA
(a) In vitro synthesis of copies of DNA of interest
1
[1]Mark
16 Since, DNA molecules are hydrophillic, they cannot pass through cell 3
membranes. For recombinant DNA to be integrated into vector or
host genome it is necessary for the DNA to be inserted in the cell.
Therefore, making the host cells competent is necessary in
biotechnology experiments. The two ways by which cells can be
made competent to take up DNA are: (i) Chemical action By
increasing concentration of divalent cation, calcium, thereby
increasing the efficiency of DNA entering through pores in cell, wall.
(ii) Heat shock treatment Incubating the cells with recombinant DNA
on ice, followed by brief treatment of heat at 42 °C and again putting
them back on ice.
17 3
18 (i) Bioreactors are large vessels in which raw materials are 5
biologically converted into specific products by microbes, plant and
animal cells or human cells. The bioreactors work by providing
optimal conditions to process the culture as well as the production
of desired product by maintaining optimum pH, temperature,
oxygen and other growth conditions required. (ii) The two commonly
used bioreactors are: (a) Simple stirred-tank bioreactors (ii) Sparged
stirred-tank bioreactors
19 . i) Identification of DNA with desirable Genes:- Other molecules in 5
the target cell can beremovedby appropriate treatment & purified
DNA ultimately precipitates out after addition ofchilled ethanol.
ii) Cutting the DNA at specific location :- After having cut the source
DNA as well as vector DNA with Specific restriction enzyme, the cut
out “gene of interest” from the source DNA & the cut vector with
2
space are mixed & ligase is added.
iii)Insertion of Recombinant DNA into host cell :- Recipient cells after
making them competent to receive takes up DNA in its surrounding.
Recombinant DNA is introduced into suitable host cell by vector –
based or vector – less method
v)Selection & Screening :- If a recombinant DNA bearing gene for
resistance to an antibiotic is [Link] into E-coli the host – cell
become [Link] into ampicillin – resistant cells. Due to this
amp gene one is able to select a [Link] cell in the presence of
ampicillin. This amp r gene is called selectable marker.
v)Obtaining the foreign Gene product :- After having cloned the
gene of interest & having optimized the conditions to induce
expression of the target protein, one has to consider producing it on
large scale
20 Gel electrophoresis helps in separating DNA fragments. DNA 3
fragments are negatively charged then they are forced to move
towards anode under an electric field through agarose gel matrix.
The fragments separate according to their size through sieving
effect. Hence the smaller fragments move faster and further than
the larger ones. Separation and Isolation of DNA Fragments (Gel
Electrophoresis) ▪ Gel electrophoresis is a technique for separating
DNA fragments based on their size. ▪ Firstly, the sample DNA is cut
into fragments by restriction endonucleases. ▪ The DNA fragments
3
being negatively charged can be separated by forcing them to move
towards the anode under an electric field through a medium.
Commonly used matrix is agarose, which is a natural linear polymer
of D-galactose and 3, 6-anhydro-L-galactose which is extracted from
sea weeds. ▪ The DNA fragments separate-out (resolve) according to
their size because of the sieving property of agarose gel. Hence,
smaller the fragment size, the farther it will move. A typical agarose
gel electrophoresis showing migration of undigested (lane 1) and
digested set of DNA fragments (lane 2 to 4) ▪ The separated DNA
fragments are visualised after staining the DNA with ethidium
bromide followed by exposure to UV radiation. ▪ The DNA fragments
are seen as orange coloured bands. ▪ The separated bands of DNA
are cut out and extracted from the gel piece. This step is called
elution. ▪ The purified DNA fragments are used to form recombinant
DNA which can be joined with cloning vectors.
21 Stanley Cohen and Herbert Boyer constructed first artificial 2
recombinant DNA. They did this by isolating the antibiotic resistant
gene by cutting out a piece of DNA from a plasmid which was
responsible for giving antibiotic resistance.
22 3
(a) EcoRI (b) (c) These are
named sticky ends, because they form hydrogen bonds with their
complementary cut parts
23 Agrobacterium tumefaciens is a pathogen in many dicot plants. It is 3
able to deliver a piece of DNA (T–DNA) to transform normal plant
cell into a tumor and directs these tumor cells to produce the
chemicals required by pathogen.
24 ❑ Separation and Purification ❑ This process is essential because 2
before reaching into market, the product has to be subjected for
clinical trial and quality control.
25 Number of fragments of linear DNA = 4 Number of fragments of 2
plasmid = 3.
26 PCR stands for Polymerase Chain Reaction. In this reaction, multiple 5
copies of the gene (or DNA) of interest is synthesised in vitro using
two Figure 9.6 Polymerase chain reaction (PCR) : Each cycle has
three steps: (i) Denaturation; (ii) Primer annealing; and (iii) Extension
4
of primers 172 2024-25 BIOTECHNOLOGY : PRINCIPLES AND
PROCESSES 173 sets of primers (small chemically synthesised
oligonucleotides that are complementary to the regions of DNA) and
the enzyme DNA polymerase. The enzyme extends the primers using
the nucleotides provided in the reaction and the genomic DNA as
template. If the process of replication of DNA is repeated many
times, the segment of DNA can be amplified to approximately billion
times, i.e., 1 billion copies are made. Such repeated amplification is
achieved by the use of a thermostable DNA polymerase (isolated
from a bacterium, Thermus aquaticus), which remain active during
the high temperature induced denaturation of double stranded DNA.
The amplified fragment if desired can now be used to ligate with a
vector for further cloning
27 A stirred-tank reactor is usually cylindrical or with a curved base to 5
facilitate the mixing of the reactor contents. The stirrer facilitates
even mixing and oxygen availability throughout the bioreactor.
Alternatively air can be bubbled through the reactor. If you look at
the figure closely you will see that the bioreactor has an agitator
system, an oxygen delivery system and a foam control system, a
temperature control system, pH control system and sampling ports
so that small volumes of the culture can be withdrawn periodically
28 Plasmid and bacteriophage 2
5
29
30 Since DNA is a hydrophilic molecule, it cannot pass through cell 5
membranes. Why? In order to force bacteria to take up the plasmid,
the bacterial cells must first be made ‘competent’ to take up DNA.
This is done by treating them with a specific concentration of a
divalent cation, such as calcium, which increases the efficiency with
which DNA enters the bacterium through pores in its cell wall.
Recombinant DNA can then be forced into such cells by incubating
the cells with recombinant DNA on ice, followed by placing them
briefly at 420C (heat shock), and then putting them back on ice. This
enables the bacteria to take up the recombinant DNA. This is not the
only way to introduce alien DNA into host cells. In a method known
as micro-injection, recombinant DNA is directly injected into the
nucleus of an animal cell. In another method, suitable for plants,
cells are bombarded with high velocity micro-particles of gold or
tungsten coated with DNA in a method known as biolistics or gene
gun. And the last method uses ‘disarmed pathogen’ vectors, which
when allowed to infect the cell, transfer the recombinant DNA into
the host. Now that we have learnt about the tools for constructing
recombinant DNA, let us discuss the processes facilitating
recombinant DNA technology.
