See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/331558635

Minibook Series - Detailed Analysis of TECHNIQUES QUESTIONS CSIR-NET

2011-2018

Book · March 2019

CITATIONS READS

0 3,245

2 authors:

Aditya Arya Subhojit Paul

Pathfinder Research and Training Foundation Defence Research and Development Organisation
84 PUBLICATIONS   260 CITATIONS    24 PUBLICATIONS   41 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Drawing Pin Publishing View project

Diagnostic Biomarkers and therapeutic intervention for Hypobaric Hypoxia related Illness View project

All content following this page was uploaded by Aditya Arya on 06 March 2019.

The user has requested enhancement of the downloaded file.

 Minibook
Series CSIR-NET
2011-2018

Detailed Analysis of
TECHNIQUES QUESTIONS

Aditya Arya, Ph.D. | Subhojit Paul , Ph.D.

TM

First Edition
Revised
 CSIR-NET EXAM

Detailed Analysis
of Techniques
Questions

PY
O
C
R
O
TH
AU
All pages of this books are not available as per copyright agreement
with publisher.

PY
O
C
R
O
TH
AU
 CSIR-NET EXAM

Detailed Analysis
of Techniques
Questions

PY
O
C
2011- 2018
R
O

Aditya Arya, Ph.D.

TH

Subhojit Paul, Ph.D.

AU

TM
 TM

Drawing Pin Publishing

New Delhi, India.

Detailed analysis of Techniques Questions, 1st Edition.

Preprint version – 21 st April 2018 , Revised Reprint - 2 nd Feb 2019

ISBN: 978-81-936740-3-1

Copyright © 2017-19. Drawing Pin Publishing.

PY
All rights reserved (Except for questions, which have been
adapted from questions papers). No part of this publication may
be reproduced or transmitted in any form or by any means,
electronic or mechanical, including photocopying, recording, or

O
any other storage and retrieval system, without prior written
permission of the publisher. All the copyright related queries may
be directly sent to the publisher at drawingpinpublishing@gmail.com
C
Disclaimer
Although, utmost care has been taken to avoid any errors in the
preparation of answers keys, and most of the answers have been
R
matched with the key provided by CSIR, however in case of any
discrepancies or loss of any kind due to incorrect answers or
solutions, authors and (or) publishers shall not be responsible.
O

Value creativity and hard work

At drawing pin publishing, we value the efforts of all team
TH

members and therefore provide you with a glimpse of efforts that

have been put in to achieve this book in the form of human hours
involved and plagiarism declaration. We, therefore, request you to
value hard work and insist not to copy or discredit the hardwork.
Discrediting, not only causes a financial loss to publisher but also
AU

a moral loss to authors and community. Value originality and

creativity by respecting copyright. Drawing pin publishing strictly
follows the anti-plagiarism policy and acknowledges the hard
work, originality and creativity.

Plagiarism declaration
It is certified by the authors that anti-plagiarism check was done
before publishing this book and the content was found to be > 75 % unique.

 Human hours involved ~ 1400 Hh

Cover Design: Dr. Aditya Arya (Author)
Typesetting Editor: Dr.Amit Kumar
Copy Editor: Ms. Anamika Gangwar
Field Biology/Ecology Suggestions: Mr. Shashank Arya

Printed in India
iv
iv
 Preface

Methods in molecular biology are one of the key areas in the nationwide
examinations for PhD entrance and more importantly CSIR-NET.
Although, the unitwise division of CSIR question paper is almost
uniform and contains nearly equal number of questions from each unit
prescribed in the syllabus, a significant portion of questions in C part
from other units also involves the knowledge and understanding of
various methods in molecular biology. A number of questions from
biochemistry and cell biology may involve methods like circular
dichroism, spectrophotometry, flow cytometry etc. which are

PY
otheriwise component of unit 13. This leads to relatively higher number
of questions from this unit on yearly basis. In addition to this, the
standard of question is praiseworthy and understanding each and
every previous year question can certainly enhance the knowldge and

O
help in various PhD interviews and infact experimental data analysis
in future. This book has been particularly developed with an objective
C
to fulfill the demands of a number of students as well as researchers
and academicians, who spend a lot of time in exploring the solutions
for previous year CSIR-questions particularly related to techniques.
R
This book contains an exhaustive and detailed solutions to most of the
questions, so that the book in itself becomes complete preparatory
O

guide for enhacning the understanding of techniques and help students

to develop prolem-solving approaches. There are a number of books
TH

in the market with key and answers of previous year questions, but
most of them leave the students intrigued and puzzled about how to
reach that answer. This books has been prepared in consulatation with
experts who actually have vast experience on various technieus and
AU

therefore undertand the problems much better. In case of ambiguity,

with CSIR key, advice and expert commets have been obtained from
subject experts globally and justification has been provided. We, deeply
acknowledge and recognise the efforts of annonymous persons
involved in preparing those questions and CSIR, for using their
questions in this book. We wish that the book will fulfill the
requirement. We also welcome the suggestions and comments or
rectification of errors if any.
We wish you a good luck in CSIR-NETand sucess in Career !

Authors

v
vi
AU
TH
O
R
C
O
PY

vi
Table of Contents

01. Review of Trend

02. Questions and Solutions June- 2011

PY
03. Questions and Solutions December- 2011
04. Questions and Solutions June- 2012

O
05. Questions and Solutions December - 2012
06. Questions and Solutions June- 2013
C
07. Questions and Solutions December - 2013
R
08. Questions and Solutions June - 2014
O

09. Questions and Solutions December - 2014

10. Questions and Solutions June- 2015
TH

11. Questions and Solutions December - 2015

12. Questions and Solutions June - 2016
AU

13. Questions and Solutions December- 2016

14. Questions and Solutions June - 2017
15. Questions and Solutions December - 2017
16. Questions and Solutions June - 2018
17. Questions and Solutions December - 2018
18. References

vii
AU
TH
O
R
C
O
PY
 Review of
Trend 01
Introduction

PY
CSIR NET JRF-LS is one of the most important exam for those who seek
research and college teaching as their career. After qualifying NET or
national eligibility test, one is eligible for applying to the post of assistant

O
professor or above in colleges and by qualifying JRF (junior research
fellowship), one get the research fellowship for the tenure of five years.
The pattern of this exam is set to enable students from various streams of
C
science to attempt with equal ease, therefore as per the prescribed syllabus
which has 13 units, a diverse variety of topics and subjects has been
included. Nevertheless, in exam ample choice is provided to facilitate a
R

fair competition. In section A, 15 out of 20 questions, in section B, 35 out

of 50 questions and in section C, 25 out of 75 questions have to be
O

attempted. In total only 75 out of 145 questions are required to be

attempted.
TH

Question paper has a very defined and set pattern of questions and
weightage of each unit is well maintained in the paper. Part A has most of
the aptitude questions involving basic Physics, Chemistry, Biology and
AU

Mathematics. Section B and C has questions from the defined syllabus. It

has been observed in the past years the questions are often arranged in
the order of units prescribed in the syllabus.

Methods in Biology is a very important domain of life sciences that enables

researchers to prove and justify their hypothesis. There is an ever-
increasing list of methods in biology and rapid advancements in methods
is also taking place. CSIR-NET, a national level exam for research
fellowship and lectureship in India, also considers methods in biology as
critical for evaluation of students for their research and teaching
temperament and hence, included in their syllabus as one full-fledged
unit. The unit is divided in eight sub-sections based on the major clad of
the technique or method. Although first sub-part is much cumbersome

1
and encompass a large number of recombinant DNA methods and
biomolecule analysis, however the weightage of questions has been
uniform across the period of past 7 years. Sometimes, one of two topics
were repeatedly asked and given more emphasis such as microscopy and
spectroscopy.

Analysis of Techniques’ section

If we, look at the overall trend of questions over a period of 7 years, we

will find that number of techniques questions fluctuated between 9 -16,
with lowest in June 2013 (9 questions) and highest in June 2014 and Dec
2015 (16 questions). This fluctuation was probably due to the involvement

PY
of methods in questions of various other units (which we considered in
this analysis), otherwise the distribution was almost uniform across the
years.

O
Year wise distribution of Questions from part A and B in techniques is
illustrated below.
C
Part B Part C Total
100% 20
16 16
13 13 15 14 15
14 11 7 12 12
10 12 11 10 8 10 9
8 10 8 6 7
8 9
50% 10
R
5 6

3 4 5 3 4 3 8 6 5 5 7 6 5 6
0% 0
1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2
O

2011 2012 2013 2014 2015 2016 2017 2011 2012 2013 2014 2015 2016 2017
TH

Fig 1. Graphical presentation of biochemistry questions in CSIR exams 2011- 2017. a. total
vs techniques questions, b. Techniques questions only.

As mentioned, the number of techniques questions were not variable much

and reached a highest of 16 in some exams. The relative numbers between
AU

part B and part C also varied and did not show an uniform trend.

However, this information just hints that just like all other subjects (not
analysed here) technique was also persistent in all the papers throughout.
A deeper analysis into the sub topics of techniques provides a better insight
to develop strategy for preparation of this exam. Prescribed syllabus of
CSIR-NET contains methods in biology in unit 13 and has been categorised
into eight sections. Based on the importance and distribution of topics,
we divided the eight sections into four main groups. First, rDT and
biomolecular analysis; second, microscopic methods; third, immunological
and spectrometric techniques and fourth, included radioactive methods,
statistical methods, methods in field biology etc.
2
2
We also noticed a unique pattern of questions based on similar logic and
mathematical relationshipes were asked persistently (but this information
should not be extrapolated to predict next trend). topics may help in the
determining the focus areas. Based on trend we have demarkated some
high value areas and those must not be missed by studnets while preparing
for future exams, these topics include, microscopy (in particular the
resolution), spectrometry (beer lambert law), cricular dichroism, flow
cytometry data analysis, ELISA and western blot and allied methods. A
more detailed discussion on section-wise trend is provided below:

Section-wise trend

In the first section, we considered to include recombinant DNA technology

PY
(rDT) and biomolecular analysis. Although the syllabus is very extensive
and begins with the isolation and purification of biomolecules , passing
through extensive vector design for cloning and expression, genome
seqeuncing strategies etc., yet the questions were limited to fewer

O
categories such as problem solving approaches in restriction digestion
(about 4-5 question in past 7 years), next set of questions were based on
C
cloning vectors and selection strategies, more focus was on yeast artificial
chromosomes (YACs). Besides this some of the biomolecular analysis and
purification methods such as gel electrophoresis, electrophoretic mobility
R
shift assay (EMSA) and chromatograhy were also focus of some questions.
O

The second section, microscopy, seems to be more important and

questions were observed across all the years on regular basis. there were
close to 16 questions across 2011-2017,which were based on principles of
TH

microscopy. The common feature of most of the questions was Abbe’s

formula of microscopic resolution which relates limit of resolution with
wavelength, angular aperture and refractive index, a distincition betrween
which microscopy can be used for the analysis of live cells and dead cells
AU

was also cosidered meaningful. besides the basic questions on resolution,

some questions were based on the properties and features of confocal
microscopy such as their ability to provide three dimentional images and
provision of optical sectioning.

The third section, which we cosidered to consist of immunological

methods and spectrometric methods was another critical topic which must
not be avoided during preparation, and must be helpful to a great extent
based on the previous trend. Some of the features of the trend from this
section included, numericals based on Beer-Lambert’s law (involvement
of mole concept was also seen), some fundamental questions based on
principles and numericals on circular dichroism were noted from
biophysical methods (about 10 questions). Aditionally, within
3
immunological methods, flow cytometry, immupreciptation and ELISA
wwere some of the critical topics that were repeatedly asked. The
questions on flow cytometry were mainly based on data analysis that
included cell cycle analysis, cell death analysis and sometimes coupled
to the concept of immunology such as information based on missing RAG
(recombinase) function. A single question on ChIP was based on the
experimental observation and knowledge of controls, while questions on
ELISA, were based on the fact that competitive ELSIA is more sensitve,
and prefered to detect small amounts of antigen. Seldom, sequence of
procedures was also asked for some immunological methods such as
chromatin immunoprecipitation and ELISA.

We considered statistical methods, radiolabelling methods and methods

PY
in field biology as section four, the questions from this section were less
frequent and limited to similar concepts (1-2 questions in each paper).
Most questions on statistics were based on the knowledge of measures of
central tendency (mean, median and mode) and central limit theorem.

O
Sometimes a knowdge of statistical tests and hypothesis testing was also
evaluated. In radiolabelling methods questions were based on use of
C
radiolabels and half life based calculations. In field biology, the question
were rare (0-1 question per paper), some of them were based on mark-
racpture method of population estimation, and recently a question on
R
remote sensing was also asked. However, no detailed questions on
algorithms of field biology methods were asked.
O

Difficulty level and preparation guide

TH

For a novice, who would not have studied methods in biology at any
level, these question might have been challenging. the quick ability to
solve questions. A throretical knowlegde and mere understanding of
pricniples was not only factor that can help you answer section C question
AU

on techniques, but teh trend suggests that a good understanding and

ability of data compresion should also be developed by the aspirants.
Questions on techniques such as flow cytometry, immunoprecipitation,
mass spectrometry, western blot were directly from the scietific research
observations and data comprehension was espected. Difficulty level of
these questions is higher than any other subject as most aspirants are not
exposed to these methods in real (dont have hands on experience) and
hence, it becomes challenging for them to comprehend data. A more recent
trend includes match the following type and choosing the correct or
incorrect statements has been observed that adds to the complexity and
needs a deeper understanding of application, pros and cons of various
techniques. Aspirants must also remain cautious about the key words
4
4
‘correct’ and ‘incorrect’ in the question and must keep a watch on the
time.

The question that how to prerpare for this section is more diverse and
difficult to answer. Students have to explore a lot for complete preparation
of this topic. But, if aspirants could develop enough reasoning and
analytical approach by building the understading of concepts and data
compresion, this section is more straightforward and rewarding than any
other section in CSIR-NET paper. A book by Dr. Arya and Dr. Amit
(PrepNotes, Module 13 - Methods in Biology) is also highly recomeded in
order to gain a detailed access to all the methods in biology prescribed in
CSIR syllabus in point to point manner with greater depth and conceptual
understanding. Publisher also provides a free weekly facebook series

PY
called Interpret, where a data analysis graphic is provided from a research
articles, to augment this part. Besides the above recomeded books and
resources, some of the well-known reference books for techniques include
gene cloning and DNA analysis by TA brown, for genetic engineering;

O
principles and techniques of biochemistry and molecular biology by
Wilson and Walker,; Biostatistics For Dummies by by John C. Pezzullo
C
which are expected to cover most of the topics of the syllabus. Sometimes
research articles containing data pertaining to a specific technique may
also be helpful in preparation.
R

This minibook would be reviewed after 2 years (in 2019) and updated for
a better preparation. Wishing good luck and successful preparation of
O

biochemistry for CSIR NET.

TH

Authors
AU

5
 June
2011 02
Total Questions: 145 (75), Techniques: 13 [Part B: 3, Part C: 10]

Part B

PY
Q1. Yeast artificial chromosomes (YAC) vectors contain selectable markers.
Loss of which marker at the cloning site distinguishes the re-ligated YACs
from the original vector marker.

O
1. TRP1 2. SUP4
3. URA3 4. CEN
C
Solution: (Correct answer – 2). Yeast artificial chromosomes are the highest
capacity vectors that contain a telomeric region of yeast chromosome to stabilize
R
the vector. Just like other class of vector YACs also have selectable markers and
some markers which can predict the orientation of gene as well. Based on the map
O

of YAC, it can be predicted a gene which is usually disrupted due to insertional

inactivation can be used to distinguish the self-ligated vectors from correct insert.
TH

Although in case of many other vectors of yeast Trp1 and URA3 are also used as
markers. But in case of YAC, cloning site is present within the SUP4 gene,
which is likely to lose its function after insertion of gene, hence it can be used to
differentiate the re-ligated YACs from original. Refer the map of YAC on next
AU

page (Fig 1).

Q2. The μ and σ of wing length (normally distributed parameter) in a

population of fruit flies are 4 and 0.2 mm, respectively. In a random
sample of 400 fruit flies, how many individuals are expected to have wing
length greater than 4.4 mm?
1. 20 2. 64
3. 10 4. 336

Solution: (Correct answer – 3). Normal distribution means, a probability

distribution in which the values are present in a symmetrical fashion, and most
of the results are situated around the probability mean. Values are equally likely
6
6
to plot either above or below the mean. As the values of mean and standard
deviation are already provided, based on the central limit theorem and three sigma
rule (or 68-95-99 rule). As per this rule, the values of a data represented by a
range, mean ± standard deviation (μ ± σ), shall cover only 68% of the
population of data, while μ ± 2σ shall cover only 95% of the population of
data and μ ± 3σ shall cover only 99.7% of the population of data. Hence, for
the given question wing length of 68% of the total butterflies (272) shall be
in range 3.8 to 4.2 cm (4.0±0.2), while 95% of the total butterflies, i.e (380)
shall have a wing span in the range 3.4 to 4.4 cm (4.0±0.4). So only 20
butterflies shall be remaining that will have wings either larger than 4.4 cm
or smaller than 3.8 cm. As the data is normally distributed so set of 20
butterflies will be distributed equally on both sides, i.e. 10 butterflies will

PY
have wings smaller than 3.8 cm and 10 butterflies will have larger than 4.4
cm.

O
Cleave with
BamHI and SnaBI
CEN4
C
ori SnaBI SnaB1
SnaB1
SUP4
TRP1 Left arm Right arm
R
pYAC3
Insert
URA3 DNA
O

(> 100 kb)

TEL TEL
BamHI BamHI TEL TRP1 CEN4 Insert URA3 TEL
TH

DNA

Fig 1. Schmatic of typical YAC vector

AU

Q3. ELISA assays uses

1. An enzyme which can react with secondary antibody
2. An enzyme which can react with antigen
3. A substrate which gets converted into a coloured product
4. A radiolabelled secondary antibody

Solution: (Correct answer – 3). ELISA or enzyme linked immunosorbent assay

is based on the detection of specific antigen using specific antibodies, which then
interact with secondary antibodies coupled to an enzyme. The presence of this
enzyme and therefore binding of antibody to antigen is detected using the enzyme
catalysed reaction where a substrate is converted into coloured compound, such
as p-dinitrobenzene. Enzyme does not react with antigen or antibody. Radiolabeled
antibody is used in case of radioimmunoassay or RIA.
7
Part C

Q4. You are studying the binding of protein to the cytoplasmic face of
cultured liver cells and have found a method that gives good yield of
inside out vesicles from plasma membrane. Unfortunately, your
preparations are contaminated with variable amount of right-side-out
vesicles. Nothing you have tried to avoid this contamination. Somebody
suggests that you pass the vesicles over an affinity column made of lectin
coupled to sepharose beads. What is the rationale of this suggestions?
1. Right-side-out vesicles will be lysed by Lectin coupled to Sepharose
beads.

PY
2. Right-side-out vesicles will simply bind to the lectin coupled
sepharose beads.
3. Lectin will bind to the carbohydrates residues present only on the
inside-out- vesicles.

O
4. Lectin will bind only to glycoprotein and glycolipids present on the
inside-out-vesicle.
C
Solution: (Correct answer – 2). Following figure depicts the type of vesicles
that may be formed after resealing.
R
O

leaky ghost
TH

sh
Wa
sealed ghost
Hypotonic
Lysis Wash and reseal
AU

right- side out

D
an isrup vesicles
dr
ese t
al
inside-out
vesicles

Right side out (means correct side out, don’t confuse with right and left), and
inside out means the cytosolic leaflet becomes extra cytosolic and vice versa. As
a matter of fact, the external leaflet of plasma membrane contains a number of
sugar moities (due to glycosylation) whereas inner leaflet (cytosolic face does not
contain any sugars. Sugars can interact with lectin (due to affinity). Therefore,
8
8
3. Change both r and m
4. Not change r or m

Solution: (Correct answer - 1). Correlation coefficient establishes the relationship

between or inter-dependence between two variables, which is independent of axis.
However, the value of slope of the regression line is calculated from equation of
line y= mx +c , on changing the axis the equation will be equivalent to x = my +
c. hence, the slope will change. Unless, the line is emerging from origin (c = 0)
and the slope is 45 degrees.

Q3. The use of biotinylated secondary antibody in ELISA

1. Increases the sensitivity of the assay but compromises the specificity

PY
2. Increases the sensitivity of the assay without compromising the
specificity
3. Does not alter either sensitivity of specificity
4. Decreases both sensitivity and specificity

O
Solution: (Correct answer - 2). Biotinylation of secondary antibodies allows one
C
to detect the presence by using avidin conjugated enzyme or fluorophore. As
more than one secondary antibody binds to a primary antibody and many avidin
can bind to one biotin, its presence significantly increase the signal that means
R

increases the sensitivity. However, it is an observed experimental fact that binding

of biotin to antibody does not affect its specificity (as it is conjugated to some
O

other site not involved in direct antigen interaction).

TH

Avidin/Strepavidin
AU

Biotin

Secondary antibody

Primary antibody

Antigen

Q4. Among existing technologies, which of the following vector systems

would you prefer to use for generating a library for 140 kb eukaryotic

27
experiment, hence incorrect (or right answer). In order to make such conclusion,
purified protein has to be compared on native PAGE and SDS-PAGE. In case
native PAGE gives one band and SDS PAGE shows three bands, it will represent
three subunits and if both represent three bands they represent three different
proteins.

Q10. The diagram below represents a 2kb insert successfully introduced

in between two BamHI sites of a 3.8 kb vector in desired orientation. The
Hind III site on the insert and EcoRI site on the vector is also indicated. If
the insert was introduced in opposite direction, which one of the following
statements is incorrect?

PY
HindIII1
.2 kb
8 kb BamHI
BamHI 0.
kb

2 kb
0.9

EcoRI

O
Vector
3.8 kb
C
2.9 kb
R

1. Digestion with EcoRI will linearize the 5.8 kb plasmid

O

2. Digestion with BamHI will yield one 3.8 kb and one 2.0 kb fragment
3. Digestion with EcoRI and HindIII will yield one 1.7 kb and one 4.1 kb
fragment
TH

4. Digestion with EcoRI and HindIII will yield one 2.1 kb and one 3.7 kb
fragment

Solution: (Correct answer – 3) Statement 1 is true that EcoRI digestion will

AU

linearize the plasmid into a 5.8 kb DNA, but this cannot predict the correct
orientation of insert. Also, statement 2 is also correct, that digestion with BamHI
will produce two fragments of 3.8 kb and 2.0 kb by separating vector and insert,
but this can also not predict the orientation. The orientation can only be predicted
by double digestion with EcoRI and HindII, which will produce 1.7 kb and 4.1 kb
fragments respectively in correct orientation and 2.1, 3.7 kb fragments in opposite
orientation. (see figure below)

58
58
 Hind III Hind III
kb
kb 1.2 kb 2.1 0.8
0.8 kb kb
1.2
b

b
k

kb

k
0.9

0.9
EcoRI 1.7
EcoRI
Correct Opposite
orientation orientation
Site of homology

neor tk
1.

neo
r
tk
2.

neo
r
tk

3.
neo tk
r

4.

4.1 kb 3.7 kb

PY
Refer an article (Arya and Kumar, 2017). Problem solving in restriction digestion based questions”
high value note. Drawing pin publishing for details on such problems.

O
Q11. Given below are some of the method used to assess evolutionary
phylogenetic relationship among plant taxa.
C
P. 18S rRNA sequence
Q. Mitochondrial microsatellite
R. Biochemical characterization
R
S. Morphology
O

Which of the two can best reveal the evolutionary phylogenetic

relationship?
TH

1. P and Q 2. Q and R
3. R and S 4. P and S

Solution: (Correct answer – 1). Although, morphology was used for classification
AU

of plants several decades ago, but confusing results and ambiguity makes this as
less reliable feature. Similarly, due to huge similarities in basic biochemical
pathways and temporal or environmental effect on metabolites levels, biochemical
characterization can also lead to erroneous results. However, due to highly
conserved nature of 18S rRNA and mitochondrial satellite DNA are most suitable
and infact most widely used approaches in plant phylogenetic relationship.

Q12. The hydrogen atoms in the δ (delta) methylene group of lysine will
give the following splitting pattern in the 1H NMR spectra of lysine.
1. Triplet of triplets
2. Quintet
3. Doublet of triplets
4. Triplet of Doublets
59
Q12. As cancer progresses, several genome rearrangement including
translocation, deletion, duplications, etc. occur. If these rearrangements
are to be identified, which of the following techniques would be most
suitable?
1. RAPD 2. Microarray
3. Multi-colour FISH 4. Flow cytometry

Solution: (Correct answer - 3). Florescence in situ hybridization involving various

colours can identify the location of a particular DNA fragment in its original
location in cell or tissue. This can help in detection of deletion, duplication and
other chromosomal abreactions. Infact, this is also a clinical technique for pre-
natal diagnosis of genetic defects. Other techniques cannot be used to detect

PY
these changes. RAPD is used for phylogenetic analysis based on random primers,
Flow cytometry is used to analyse various properties of cells and microarray is
used for the large scale gene expression analysis.

O
Q13. A mixture of two proteins was subjected to following three
chromatographic columns:,a) Cation exchange, b) Size exclusion
C
(Sephadex 100) and (c ) Reverse phase. Following elution profiles were
obtained:
R
Cation exchange Size Exclusion Reverse phase
A B B A B A
O
Absorbance

Absorbance

Absorbance

NaCl Aceto-
TH

nitrile

Time Time Time

AU

Which of the following statements is correct?

1. A is larger and more hydrophobic than B
2. B is more anionic and more hydrophobic than A
3. A is more hydrophobic and smaller than B
4. A is more cationic and smaller than B

Solution: (Correct answer - 3). In order to solve this question we must be well
versed with the principle of these three type of chromatographies, and understand
the meaning of graph (Readers are advised to refer to biannual reviews of technique
by drawing pin publishing and Tips and TricksTM Series). In chromatographic
techniques the detection of protein is done using spectrophotometry and when
protein is eluted at particular time, a peak is observed, so in this case if we see

89
4. One electrode is applied to the plasma membrane containing one or
few ion channels and one electrode inserted into the cytosol.
Solution: (Correct answer – 3). Patch clamp technique involves the study of
small patch of a cell by making a contact using a fine pipette. The difference in
voltage across the patch is used to asses the pysico-chemical environment. There
are different variants of patch clamp such as whole cell (WC), Cell attached patch
(CAP), outside out patch (OOP) and inside out patch (IOP) depending upon the
format and objective of analysis (details in Prepnote module 13, chapter G). In
case of intact (or cell attached patch), two electrodes are applied to the plasma
membrane, one in a region containing only lipids and other in a region containing
one or few ion channels.

PY
Part C
Q7. To assess the impact of newly identified drug when added to a culture
of sub-confluent HeLa cells a researcher analyses the fluorescence

O
activated cell sorting (FACS) profile of untreated (- drug) versus (+drug)
cells.
C
- drug + drug
No. of cells
No. of cells

R
O

2n 4n 2n 4n
TH

Relative amount of DNA Relative amount of DNA

Based on the FACS profile above, this drug inhibits

1. G1 phase of the cell cycle
AU

2. S phase of the cell cycle

3. G2/M phase of the cell cycle
4. G0 phase of the cell cycle

Solution: (Correct answer - 3). A typical cell cycle analysis histogram contain
two distinct peaks (first with lower DNA content represents G0/G1 phase and
the second with higher DNA content represents G2/M phase, while a region in
between those two peaks represents cells in S phase). The area of these peaks can
be an indicative of total cells present in a given phase. An increase in the area of
one peak (swelling) may indicate in number of cells in that phase and a decrease
in the area of any peak (shrinking) indicate decrease in the number of cells in that
given phase. The given example is typical results shown by G2 arrest in cell

121
cycle analysis using propidium iodide. The drug most likely caused the arrest of
cells in G2 phase, which prevented the return of those cells from G2 to G1 again
and therefore in such condition, the G2 fractions of cells will swell (increased)
while the area of G1, G0 and S phase cells will shrink. Following is the
representative image of cell cycle analysis under normal condition, G1/S arrest
and G 2 arrest.

Control G1/S arrest G2/M arrest

No. of cells
No. of cells
No. of cells

Replicating cells

PY
DNA content DNA content DNA content

Fig. Sample experiment using ab139418 on HeLa cells treated with Thymidine and Nocodazole. (A)

O
Untreated HeLa cells with the expected distribution of cells with 2N, 2N-4N and 4N DNA content.
(B) HeLa cells treated with Thymidine have predominantly 2N DNA content. Thymidine inhibits
DNA synthesis. (C) HeLa cells treated with Nocodazole have predominantly 4N DNA content.
C
Nocodazole is a microtubule inhibitor that causes mitotic arrest.

Q8. .A researcher attempted to clone a 630 bp coding sequence of a gene

R

downstream to a bacterial promoter (500 bp in length) for over expression

and purification of encoded protein. The gene sequence was isolated as
O

SmaI fragment (recognition sequence CCC/GGG) and cloned at the SmaI

site located downstream to the promoter. The gene sequence contained a
TH

single site for EcoR1 located 30 bp downstream of the start codon. A

schematic representation of the plasmid along with location(s) of some
restriction enzyme sites of the vector is given below:
AU

Gene

Promoter

Hind III SmaI EcoRI

The researcher screened the obtained colonies by a double digestion using

HindIII and EcoRI. Which one of the following restriction digestion
122
122
patterns would represent the restriction profile of the desired clone that
could be used for overexpression?
1. ~530 bp + ~600 bp + vector backbone
2. ~1100 bp + ~30 bp + vector backbone
3. ~500 bp + vector backbone
4. ~1130 bp + vector backbone

Solution: (Correct answer – 1). In this question we have to first prepare a complete
map with of the construct. Information about the position of the restriction sites
and gene length is given in question. It may be noted that Eco RI restriction sites
indicated in question has not been shown in the given figure, Infact that is an
additional site present in the vector. So follwing is the map based on the given

PY
information.

Eco R1
Gene
bp

O
30 530 bp
Hind III EcoR1
Promoter Eco R1
630 bp
Hind III 630 bp
EcoR1 EcoR1
500 bp 30 bp
C
Hind III, EcoR1
double digestion

Vector
R
backbone
O

hence, statement 1 is correctly indicate the outcome of double digestion

TH

Q9. To achieve the best resolution using a fluorescence microscope, what

combination of wavelength of emitted light (λ), refractive index and the
angle (2θ) by which light enters into the microscope would be the best
AU

choice for the user:

1. λ= 405; refractive index =1.33; 2θ=90o
2. λ= 420; refractive index =1.51; 2θ=180o
3. λ= 520; refractive index =1.51; 2θ=90o
4. λ= 405; refractive index =1.51; 2θ=90o

Solution: (Correct answer – 4). The limit of resolution of a microscope is given

by abbe’s formula d= 0.61 λ/μ sin θ, hence for a small value of d or better resolution,
wavelength (λ) should be small, refractive index (μ) and aperture angle (θ) should
be large.

123
 December
2018 17
Total Questions: 145 (75), Techniques: 15 [Part B: 6, Part C: 10 ]

Part B

PY
1. Which of the following statements regarding restriction/modifying
enzymes used in recombinant DNA technology is correct?
1. Endonucleases remove nucleotides, one by one at a time from
the ends of a sequence

O
2. Type II class of restriction enzymes do not recognise
C
palindromic sequence
3. Mung bean nuclease act on double stranded DNA or RNA
termini
R
4. Type II class of restriction enzymes can generate either “sticky”
(staggered) or “blunt” ends.
O

Solution: (Correct answer – 4). It is incorrect that endonucleases remove

nucleotides from ends, as the endo represents within, only exonucleases can
TH

perform such function. All type II restriction endonuclease have a characteristic

that they recognise only palindromic sequences (one that is read same from both
sides (e.g. G/AATTG is the restriction site recognised by Eco RI), so this statement
is also incorrect. Mung bean nuclease is capable of degrading single stranded
AU

DNA, single stranded overhands from sticky ends, or single stranded RNA, so
this is also incorrect. However, this is true that type II restriction endonuclease
can create sticky or blunt ends. ECo RI is an example of sticky end cutter, while
SmaI is an example of blunt end cuter.

2. Which of the following is used as a source of excitation in a confocal

microscope?
1. Laser
2. Electron beam
3. Mercury lamp
4. Masers

139
Solution: (Correct answer – 3). Chi square test is non-parametric test. A non-
parametric test is performed when the variable has non-numeric values such as
index of pain, degree of tiredness etc. A parametric test is always more powerful
than non-parametric one. Also, non-parametric test do not assume a normal
distribution (A normal distribution means the values of experimental tests/assays/
estimates lie equally on both sides of the mean). So statements 1, 2 and 4 are
incorrect. However, statement 3 is correct as presence of outliers (extremely large
or small values) in a dataset of experimental outcome can affect the outcome of a
parametric test.

Part C

7. Three proteins, Blm1, Blm2 and Blm 3 were shown to be involved in

PY
repair of DNA double strand breaks. A chromatin immunoprecipitation
experiment was performed for the three proteins. The pattern obtained is
shown below.

0 min
(before break)

O 30 min
(After break)

0 kb 1 kb
C 0 kb 1 kb
from from from from
INPUT break break
INPUT break break
R
Blm 1

Blm 2
O

Blm 3
TH

Based on the above figure choose the option that correctly interpret the
data.
1. Blm 1, Blm 2 and Blm 3 binds to DNA break sites
AU

2. Blm 1 binds to the break site; Blm 3 binds to the break site
and beyond
3. Blm 2 remains bound to DNA after the break is induced
4. Blm 3 binds to DNA irrespective of the break

Solution: (Correct answer – 2). Chromatin immunoprecipitation or ChiP is a

method in which a specific part of DNA pulled down by using antibodies specific
to the protein interacting with that part of DNA. Then, this pulled DNA is
amplified and results are viewed on gel. Higher the intensity of band, higher is
the protein bound to DNA, Input represents a positive control and contains total
chromatin that was used in the experiment. As we can observe in the figures
from two set of experiments on before and another after the DNA break, Blm 1
shows no binding before the break, but shows binding at the site of break alone,
30 min after the break. Blm 2 on the other hand, binds to DNA before the 142
break
142
 NOTES

PY
O
C
R
O
TH
AU

148
148
 References
16
2011
Markie D., Markers, selection, and media in yeast artificial chromosome
cloning. Methods Mol Biol. 2006;349:1-12. –Image of YAC used in this book was
based on this article.
Lindsay JG, Reid GP, D’Souza MP. Lectins as biochemical agents for the

PY
isolation of sealed membrane vesicles of defined polarity. Biochim Biophys
Acta. 1981 Feb 6;640(3):791-801.Methods for separation of Lectins
https://www.jax.org/news-and-insights/2006/may/the-cre-lox-and-flp-frt-
systems - Image on cre-lox system was adapted from Jackson Laboratory

O
website.

2012
C
Garner CM1, Hubbold LM, Chakraborti PR., Mycoplasma detection in cell
cultures: a comparison of four methods. Br J Biomed Sci. 2000;57(4):295-301. –
Methods for detection of mycoplasma.
R
Liu H, Bebu I, Li X. Microarray probes and probe sets. Front Biosci (Elite Ed).
2010;2:325-38. Published 2010 Jan 1. – Probes for Microarray
O

2013
Ronda L, Bruno S, Viappiani C, et al. Circular dichroism spectroscopy of
TH

tertiary and quaternary conformations of human hemoglobin entrapped in wet

silica gels. Protein Sci. 2006;15(8):1961-7. – For circular dichroism of human
haemoglobin.
A Huete, K Didan, T Miura, E.P Rodriguez, X Gao, L.G Ferreira, Overview of
AU

the radiometric and biophysical performance of the MODIS vegetation indices,

Remote Sensing of Environment, 2002 Volume 83, Issues 1–2, 195-213, - For
remote sensing.
Galas DJ, Schmitz A. DNAse footprinting: a simple method for the detection of
protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157-70. –
DNA Footprinting assay.

2014.
Small molecule fluorescent voltage indicators for studying membrane
potential. Curr Opin Chem Biol. 2016;33:74-80. – An article on sensing
membrane potential with spatial resolution.
https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html - Expasy
- protease cleavage rules.

149
2015
Simon-Lukasik KV, Persikov AV, Brodsky B, et al. Fluorescence determination
of tryptophan side-chain accessibility and dynamics in triple-helical collagen-
like peptides. Biophys J. 2003;84(1):501-8.
http://www.abcam.com/free-glycerol-assay-kit-ab65337.html -
Spectrophotometry based assay for glycerol
http://www.cyberlipid.org/acylglyt/acyl0002.htm - spectrophotometric/
fluorimetric methods for glycerol estimation
https://pdfs.semanticscholar.org/b464/
9415b0fc5da580b0038d4a40488064edeed6.pdf - viscosity based glycerol
http://mbu.iisc.ernet.in/~pbgrp/374.pdf

2016.
Ruoho AE, Kiefer H, Roeder PE, Singer SJ. The mechanism of photoaffinity

PY
labeling. Proc Natl Acad Sci U S A. 1973;70(9):2567-71. – For photo affinity
labelling.
Till BJ1, Zerr T, Comai L, Henikoff S. , A protocol for TILLING and Ecotilling in
plants and animals. Nat Protoc. 2006;1(5):2465-77. – For TILLING Assay.

O
Paun O, Schönswetter P. Amplified fragment length polymorphism: an
invaluable fingerprinting technique for genomic, transcriptomic, and epigenetic
studies. Methods Mol Biol. 2012;862:75-87.
C
Deranieh RM, Joshi AS, Greenberg ML. Thin-layer chromatography of
phospholipids. Methods Mol Biol. 2013;1033:21-7. – Thin Layer
chromatography for phospholipids.
R
https://www.nature.com/scitable - (A graphic of AFLP is avaible on the
website of cited source – accessed on12th July 2017)
O

https://www.nature.com/scitable/content/outline-of-the-aflp-procedure-
41047 - AFLP illustration
http://lipidlibrary.aocs.org/History/content.cfm?ItemNumber=40365 – HPLC
TH

for phospholipids
https://www.omicsonline.org/open-access/spectroscopic-chromatographic-
methods-for-quantitative-analysis-of-phospholipid-complexes-of-flavonoids-
2153-2435.1000322.php?aid=36729 - Various spectrometric methods for lipid
AU

analysis
http://www.abcam.com/secondary-antibodies/secondary-antibody-selection-
guide - Secondary antibodies - anti-isotypic
https://www.neb.com/protocols/1/01/01/fusion-protein-expression-e6901 -
factors affecting fusion protein expression.
http://www.pnas.org/content/pnas/94/10/4978.full.pdf - Trigger factor to
enhance bacterial survival
https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2958.2003.03785.x-
Temperature effect on inclusion bodies.

2017
Malzahn A, Lowder L, Qi Y. Plant genome editing with TALEN and
CRISPR. Cell Biosci. 2017;7:21. Published 2017 Apr 24. doi:10.1186/s13578-017-
0148-4 – TALEN and CRISPR.
150
Genome engineering with zinc-finger nucleases. Genetics. 2011;188(4):773-82.
150
Zhang YZ1, Paterson Y, Roder H. Rapid amide proton exchange rates in
peptides and proteins measured by solvent quenching and two-dimensional
NMR. Protein Sci. 1995 Apr;4(4):804-14.
http://www.pnas.org/content/pnas/93/3/1156.full.pdf - ZFN based genome
editing.
http://www.cryst.bbk.ac.uk/PPS2/course/section8/ss-960531_22.html
(accessed on 17th Aug 2017)
More details on NOE and structure determination using NOE :
Annual review on 2D NMR and protein structure - https://
spin.niddk.nih.gov/bax/lit/508/110.pdf
https://www.labome.com/method/Recombinant-Protein-Expression-Vector-
Host-Systems.html - key features of expression systems
http://www.abcam.com/propidium-iodide-flow-cytometry-kit-ab139418.html

PY
2018
https://onlinelibrary.wiley.com/doi/abs/10.1002/mas.10017 - Mass
spectrometry to study protein conformations
https://www.ncbi.nlm.nih.gov/pubmed/24061915- Fluorescence microscopy

O
to study protein conformations.
https://www.pnas.org/content/96/3/893.short - Fluorescence microscopy to
study protein conformations.
C
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409303/- Differential
calorimetry to study protein dynamics
https://www.pnas.org/content/95/13/7406.short- DSC for conformational
R
dynamics
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3001252/ - NMR to study
O

dynamics of protein
https://www.ncbi.nlm.nih.gov/pubmed/11050940 - DNA vaccines
https://www.inmunologia.org/Upload/Articles/6/2/621.pdf
TH
AU

151
 About The Book

T his booklet has been prepared as a complementary book to

PrepnotesTM Module 13 from the publisher as a comprehen-
sive preparatory package for CSIR-NET examination.
Considering CSIR-NET as one of most foremost examinations for
research and teaching eligibility, preparation is incomplete without
practicing previous year questions. As the exam is very diverse and
consist of various domains of life sciences, ample choice is provided
in the paper that enables students to choose the domain of their
interest and expertise. A comprehensive preparation of methods in
molecular biology can enable students to attempt nearly 30% of
the questions in part C. Lack of detailed and authentic solutions to
the questions leaves the preparation incomplete and most students
either remember the choice from key provided by the examiner or
several other publishers. This does not enhance knowledge. This
books aims not only to provide solutions but expert comments and
advice on different questions with elaborate discussion on various
techniques in consultation with active researchers in the area.
Besides CSIR-NET this booklet may also be used by students
preparing for GATE or M.Sc. entrance examinations for improving
their analytical skills in common techniques in life sciences. This
booklet will be updated on biannual basis with new questions and
solutions.

MRP. INR 17500

TM

DPP Minibooks

View publication stats