Class Assignment

Q1. (A) Define the following (Any five)​ ​ ​ ​ ​ ​ (5x1=5)

(1) Blotting
Blots are techniques for transferring DNA , RNA and proteins onto a carrier so they can be
separated, and often follows the use of a gel electrophoresis. The Southern blot is used for
transferring DNA, the Northern blot for RNA and the western blot for proteins.
(2) Homopolymer tailing
Its a general method for joining dna molecules by making use of the complementary
homopolymer sequences. The enzyme terminal deoxynucleotidyl transferase adds oligo(dA)
sequences to the 3′ ends of one population of DNA molecules and oligo(dT) blocks to the 3′
ends of another population, the two types of molecule then anneal to form mixed dimeric circles

(3) DNA fingerprinting

DNA fingerprinting or DNA profiling is a process used to determine the nucleotide sequence at a
certain part of the DNA that is unique in all human beings.

The process of DNA fingerprinting was invented by Sir Alec Jeffrey

(5) Bt Toxin
Bt toxin is produced by a bacterium called Bacillus thuringiensis. The Bt toxin gene is
cloned from the bacteria and expressed in plants to provide resistance from insects
without the need for insecticides.
(6) Gene therapy
Gene therapy is a medical approach that allows doctors to treat a disease by altering the
patient’s genetic make-up instead of using surgery or medicines. The effects of gene therapy
have a longer effect than other treatment methods. It can be of two types- Somatic gene therapy
germline gene therapy.

(B) Differentiate between the following (Any Three)​ ​ ​ (3x2=6)

(1) SDS PAGE and Native PAGE

SDS Native

SDS Page or Sodium-dodecyl sulfate Page Native Page uses non-denaturing gels and
separates proteins based on their separates proteins based on their size,
molecular weight, charge and the shape

SDS is present as a detergent to impart a SDS is not present in the native page.
negative charge on the sample in SDS
page.
 Stability of the protein is low in SDS page Stability of protein is high in the native
page.

(3) cDNA library and genomic library

cDNA library Genomic library

▪​A cDNA library is defined as a collection of

Genomic library is a collection of the clones
cDNA fragments, each of which has been bearing the total genomic DNA of an
organism.
cloned into a separate vector molecule.

Starting material is mRNA The starting material is DNA.

cDNA library is small. Genomic library is large.

(4) Ex-vivo gene therapy and in-vivo gene therapy

Ex-vivo gene therapy In-vivo gene therapy

Gene modification is done outside the body. Genes are changed when they are still
inside the body.

Cells are incubated with vector then returned Vector is directly injected into the body.
to the body.

This method does not introduce adverse This method introduces adverse
immunological responses in the patient’s immunological responses in the patient’s
body. body.

Q2. (a) Describe the principle and methodology of enzymatic method of DNA sequencing.
Enzymatic method of DNA sequencing, also known as chain termination method was given by
frederick sanger in 1977.
In a normal nucleotide in dna or dNTP, the third carbon of deoxyribose sugar has a free OH
group which is needed for ester bond formation during template strand synthesis. In case of
ddNTP, it lacks the free 3’ OH group. When it’s incorporated into a growing chain, the 3’ H
cannot form an ester bond with the next nucleotide and chain termination occurs.
The steps include:
1.​ Preparation of reaction mixtures: In four different test tubes, four reaction mixtures are
prepared containing an unknown DNA sample. Radio-labelled primer, DNA polymerase,
4 dNTPs (dATP, dCTP,dGTP and dTTP) and different ddNTPs in each test tube
(ddATP…)
 2.​ Chain termination by ddNTP: The added ddNTPs are randomly incorporated into some
of the newly synthesized strands in the place of dNTPs. As a result, differently sized
DNA fragments are formed in all the tubes, ending in their respective ddNTPs
3.​ Gel electrophoresis: The products obtained are then loaded into four different wells of a
polyacrylamide gel and the fragments are separated.
4.​ The radiolabelled DNA bands are then visualised using autoradiography.

Q3. What are primers? Describe the various factors being considered while designing primers.
A primer is a short synthetic oligonucleotide which is used in many molecular techniques. These
primers are designed to have a sequence which is the reverse compliment of a region of
template dna.
Factors that are considered while designing primers are (slide 119 to 124)

Q4. Describe the process of recombinant screening in E.coli using Blue white screening method
This is an effective method to identify recombinant bacteria from the non-recombinants. For
selecting recombinants from non-recombinants, , a chromogenic substrate X-gal is added to the
agar plate. If beta-gal is present then this produces a bright blue colour and thus the
non-recombinants can be identified. The recombinant colonies, which don’t contain beta-gal,
appear as white.

Q7. What is transgenic? Describe it with example in which Bt toxin gene has been used?
Transgenic is used to define any organism whose genome has been altered by introduction of
foreign DNA by artificial means. Transgenic animals are used to produce hormones and other
products for human benefit. Some transgenic animals are also used as models for testing cures
for many human diseases such as alzheimer’s. Bt toxin, derived from Bacillus thuringiensis,
has been used to produce many varieties of pest resistant varieties of transgenic plants. Plants
such as Bt cotton, Bt brinjal etc. are resistant to attack by pests. When the insects consume the
plant, the spore of Bacillus thuringinesis are releases “cry proteins” which are toxic. These
cause the lysis of the stomach wall of the insect which leads to its death, thus protecting the
crop from any further damage by the pests.

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