Molecular biotechnology vol 2 issue 2

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 Cell transformation
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 Molecular and cell biology automation
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 Cells genetic and metabolic capacity
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Department of Biotechnology, Graphic Era
University, Uttarakhand, India
Dr. Brijesh Pandey
Amity University, Lucknow (U.P.),
India
Dr. P Mary Anupama
Department of Biotechnology, ANITS,
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Calcutta University Institute of Post Graduate
Medical Education and Research [IPGMER]
/University College of Medicine [UCM],
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Central Insititute for Cotton Research,
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Basavatarakam Indo-American Cancer Hospital
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St. Xavier's College (Autonomous),
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From the Editor's Desk
Dear Readers,
We would like to present, with great pleasure, the inaugural volume of a new scholarly
journal, International Journal of Molecular Biotechnology. This journal is part of the
Applied Sciences, and is devoted to the scope of present Molecular Biotechnology issues,
from theoreticalaspectstoapplication-dependentstudiesand thevalidationofemergingtechnologies.
This new journal was planned and established to represent the growing needs of Molecular Biotechnology
as an emerging and increasingly vital field, now widely recognized as an integral part of scientific and
technical investigations. Its mission is to become a voice of Molecular Biotechnology, addressing
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The core vision of International Journal of Molecular Biotechnology in JournalsPub is to propagate novel
awareness and know-how for the profit of mankind ranging from the academic and professional research
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acts as a pathfinder for the scientific community to publish their papers at excellently, well-timed &
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in the realm of Cell transformation, Gene cloning systems, Next generation sequencing, Molecular and cell
biology automation, Protein purification and analysis, Transgenic species, Therapeutic proteins, Cells
geneticandmetaboliccapacity,Molecularmarkers etc.
The Journal is intended as a forum for practitioners and researchers to share the techniques of Molecular
Biotechnology and solutions in the area. Many scientists and researchers have contributed to the creation
and the success of Molecular Biotechnology. We are very thankful to everybody within that community who
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It is our hope that this fine collection of articles will be a valuable resource for Molecular Biotechnology
readers andwillstimulatefurtherresearchintothevibrantareaofMolecularBiotechnology.
PuneetMehrotra
ManagingDirector

1. Diagnosis of Forest Pathogens: A Molecular Approach – A Review
S. Rawat, A. Bagwari, Y.P. Singh 1
2. Breeding for Disease Resistance in Crops by Molecular Method
Archana Bagwari 8
3. Molecular Markers in Livestock Improvement
Sanjay Kumar 14
4. Production of Therapeutic Proteins: A Review
Amandeep Kushan 20
5. Rapid DNA Isolation Protocol for ISSR and SSR Markers Analysis in Trans-Himalayan Cabbage Species
S. Allen, S. Kapoor, M.K. Patel, V. Kumar, O.P. Chaurasia, B. Kumar 26
Contents

IJMB (2016) 1–7 © JournalsPub 2016. All Rights Reserved Page 1
International Journal of Molecular Biotechnology
Vol. 2: Issue 2
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Diagnosis of Forest Pathogens: A Molecular Approach – A
Review
S. Rawat*, A. Bagwari, Y.P. Singh
Forest Pathology Division, FRI, Dehradun, India
ABSTRACT
In India forest is the main source of timber and livelihood for many farmers. But the
condition of forestry is not good in India as many factors affect their production. One of the
main factor is pathogens especially fungi. Many wood rotting fungi are reported to cause
mortality in important forestry trees in India. Accurate identification of pathogen is an
important strategy to initiate curative measures. Diagnosis of any disease is the first stage of
its management. In recent time, researcher use many new methods, as manual methods for
the detection of pathogens is time consuming and needs expert supervision. These limitations
led to the development of molecular approaches due to high accuracy and specificity.
Molecular technique will be effective for the detection of pathogens. By using molecular
approaches forest pathogens can be managed easily. So, aim of this review is to study the
role of molecular methods in the management of forest pathogens.
Keywords: diagnosis, fungi, molecular markers
INTRODUCTION
Forest is affected by various biotic agents
or there are several agents which cause
disease on tree. For example, bacteria,
virus, protozoans, fungi, etc. Fungi cause
the majority of forest diseases (Edmonads,
et al., 2010).
Some examples of fungal disease which
causes death of the plants are, wilt disease
of Dalbergia sissoo Roxb. Caused by
Fusarium solani (Bagchee, 1945; Bakshi,
1954), Ganoderma root rot caused by
Ganoderma lucidum on various tree,
damping off by Rhizoctonia solani, leaf
spot and leaf blight by various fungal
pathogens. Further, wood decaying fungi
are the important elements of forest
ecosystems, as they take Care of the
decomposition of coarse woody debris
(fallen trunks, branches, stumps, etc.).

Vol. 2: Issue 2
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Breeding for Disease Resistance in Crops by Molecular
Method
Archana Bagwari*
Forest Pathology Division, FRI, Dehradun, India
ABSTRACT
Tree/crops are the prime source of food in India. Crops also contribute to the national
exchequer. At the same time, plants are the reservoir of many organisms (microorganism,
nematodes, insect, etc.) as they have rich source of nutrient in it. These organism cause
disease on plants and sometimes caused death of the plants. So, it can create severe food
threat in our system. Plants lack immune system to fight against such disease but have a
defense mechanism. Plant pathologists discover various methods for the control of the
disease. Breeding for disease resistance is one of the best examples to overcome against
severe pathogen. It is very quick methods for disease resistance.
Keywords: disease, pathogens, resistance
INTRODUCTION
Plant disease is a serious problem for plant
pathologist. Although, many advance
technique have been developed to control
the disease, but our universal food supply
is still vulnerable by a several pathogens
and pests (Moffat, 2001; Garelik, 2002
and Many authors, 1999). Diseases caused
by the pathogens can greatly reduce crop
outturn and the effect of disease outburst
is mainly acute in developing nations like
in India. Oerke (2006) reported that an
average of 26% of the worldwide crop
yielding is lost each year due to pre-
harvest pests and pathogens. Chemicals
(Fungicides, pesticides, etc.) provide
effective protection but their suitableness
can be affected by adverse environmental
conditions and by the emergence of
resistant pathogen strains (McDowell and
Woffenden, 2003) [1–5].
Expanding rate of human population, loss
of farming area because of environmental
change, disintegration, for example, those
created by pathogens however much as
could be expected (Bebber et al., 2013).
The four major crop yielded universally,
are rice (which feeds more than half the
world population), wheat, maize and
banana are continuously exposed by
destroying emerging infectious diseases in
both developing and developed countries
(Fisher et al., 2012). Magnaporthe oryzae
caused Rice blast is one of the most
destructive diseases of the rice in several
part of the world (Skamnioti and Gurr,
2009; Helliwell and Yang, 2013; Helliwell
et al., 2013). Another serious threat to
food security is Basidiomycetes fungus
Puccinia graminis f. sp. tritici (the causal
agent of wheat stem rust). Another major
fungi, the oomycete pathogen
Phytophthora infestans is the causative
agent of late blight of potato, resulting in
the deaths of 1.25million people during
the Irish potato famine of 1845 (Haverkort
et al., 2008; Pennisi,2010) (Figure 1,
Table 1) [6–10].

Vol. 2: Issue 2
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Molecular Markers in Livestock Improvement
Sanjay Kumar
Central University of Rajasthan, Ajmer, India
BACKGROUND
Recent advancements in DNA
technologies have made it conceivable to
reveal an extensive number of genetic
polymorphisms at the DNA sequence
level, and to utilize them as markers for
assessment of the genetic basis for the
watched phenotypic variability. The
markers revealing varieties at DNA level
are alluded to as the molecular markers.
Based on techniques utilized for location,
these markers are arranged into two
noteworthy classifications:
 Hybridization-based markers
 PCR-based markers
The molecular markers have interesting
genetic properties and methodological
advantages that make them more helpful
and manageable for genetic examination
contrasted with other genetic markers. The
conceivable uses of molecular markers in
domesticated animals change have been
surveyed with reference to routine and
transgenic reproducing procedures.
In breeding strategies, molecular markers
have a several short-range or immediate
applications (viz. parentage determination,
genetic distance estimation, determination
of twin zygosity and free martinism, and
sexing of pre-implantation embryos and
identification of disease carrier) and long-
range applications (viz. gene mapping, and
marker-assisted selection) and long-go
applications (viz. quality mapping, and
marker-helped choice) [1, 2] In transgenic
breeding, molecular markers can be
utilized as reference focuses for
identification, isolation and manipulation
of the relevant genes, and for
identification of the animals carrying the
transgenes. The advance being developed
of molecular markers proposes their
potential use for genetic change in
domesticated animal’s species.
The advance in recombinant DNA
technology and gene cloning during the
most recent two decades has gotten
revolutionary changes in the field of
fundamental and additionally of connected
genetic by giving a few new ways to deal
with genome analysis with more
prominent genetic resolution. It is
currently conceivable to reveal countless
polymorphisms at the DNA sequence
level, and to utilize them as markers for
assessment of the genetic reason for the
watched phenotypic variability [3].
Despite the fact that hypothetically DNA
sequencing is the immediate way to deal
with uncovers such DNA polymorphism,
it has two reasonable impediments:
Sequencing needs initial cloning of(i)
the quality or DNA fragment at which
allelic variety of intrigue exists, and
(ii) It requires appropriate and savvy
technique for scoring DNA sequence
variety. In any case, the aberrant approach
for revealing of genetic variation at the
DNA level utilizing molecular or DNA
markers blocks the above restrictions.
Since the primary show of DNA-level
polymorphism, known as the limitation
fragment length polymorphism (RFLP), a
practically boundless number of molecular
markers have aggregated. Currently, more

Vol. 2: Issue 2
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Production of Therapeutic Proteins: A Review
Amandeep Kushan
Department of Biotechnology, Mewar University, Chittorgarh, Rajasthan, India
BACKGROUND
Therapeutic proteins will be proteins that
are engineered in the lab for
pharmaceutical utilize. Insulin was the
main remedial protein to be acquainted
with treat diabetes in the 1920s. Because
of recombinant DNA-technology proteins
can be produced in particular host cells (e.
g. bacteria, yeast or mammalian cells)
under characterized conditions. Contingent
upon the necessities of the protein the
production host must be picked precisely.
Mammalian cells are the primary host of
decision in light of the fact that their
posttranslational changes, for example,
glycosylation and sialylation has the
greatest effect on the protein's
pharmacokinetics and effectiveness.
Protein-based treatments are utilized for
instance as a part of the treatment of:
 Cancer
 Infectious diseases
 Hemophilia
 Anemia
 Multiple sclerosis
 Hepatitis B/C
Therapeutic proteins allow an
individualized treatment approach by
supporting a particularly focused on
Therapeutic process by remunerating the
lack of an essential protein.
The quickest developing class of
Therapeutic proteins is antibodies. From
one perspective they bolster the safe
reaction and then again they can prompt
the aggregation of Therapeutic agents at a
tumor site through conjugation/fusion [1,
2]. Antibodies are Therapeutic proteins
used for the treatment of malignancy as
well as auto-immune diseases.
Production of Therapeutic Proteins by
Gene Transfer
There are numerous proteins fundamental
to great wellbeing that a few people can't
produce due to genetic defects. These
proteins incorporate different blood-
clotting components bringing about
hemophilia, insulin (bringing about
diabetes), development hormone (bringing
about absence of appropriate
development), and different proteins, the
organization of which revises neurotic
conditions or results in other Therapeutic
advantages.
The early work in this field utilized
bacteria. Some bacteria in a bacterial
culture may contain little round DNA
particles called plasmids. These plasmids
are not part of the chromosomal DNA that
is controlled that is possessed by all the
bacteria of the culture. As these
outstanding bacterial cells replicate by
binary fission or cell division, the plasmids
are transmitted to the Daughter cells. They
can likewise be transmitted to different
cells by conjugation. Researchers have
figured out how to use plasmids to
exchange human genes to bacterial cells. If
the gene inserted into the plasmid of
bacteria into the human gene for insulin,
for instance, the bacteria into which this
gene is inserted produces human insulin.

IJMB (2016) 26-31© JournalsPub 2016. All Rights Reserved Page 26
Vol. 2: Issue 2
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Rapid DNA Isolation Protocol for ISSR and SSR Markers
Analysis in Trans-Himalayan Cabbage Species
S. Allen, S. Kapoor, M.K. Patel, V. Kumar, O.P. Chaurasia*, B. Kumar
Department of Plant Biotechnology, Defense Institute of High Altitude Research DRDO, Chandigarh, India
ABSTRACT
In the present study, we present a simple one day protocol for DNA extraction from cabbage,
with no requirement of RNase and overnight precipitation with isopropanol. Due to high
endogenous phenolics and polysaccharides and other substances in plants, it is difficult to
isolate DNA with high concentration and optimum ratio. With added protein and RNA
contamination, ISSR and SSR based primer amplification pose a challenge for the same. This
method involves modified CTAB protocol with fine crushing by liquid nitrogen. The reagents
required during the extraction are CTAB, NaCl, EDTA, Tris HCl and Ascorbic acid. The
extraction buffer should be freshly prepared on the same day as results indicated low yield
with overnight extraction buffer. The yield of DNA isolated varies from 107.2 to 3855 ng/µl
with ratio ranging from 1.79 to 1.88. The primers developed for the case study were mainly
ISSR (inter Simple Sequence Repeats) and SSR (Simple sequence Repeats) which were first
standardized for PCR reaction mixture such as MgCl2 (1.8 mM), primers (0.2µM) and Taq
Polymerase (0.3 units) with 20 ng/µl of DNA. The gradient PCR showed the annealing
temperature of all the primers ranging from 55 to 64°C. Polymorphism was seen among all
the amplified PCR products run on 2% agarose gel electrophoresis.
Keywords: cabbage, CTAB, DNA extraction, ISSR, polymorphism
Abbreviations: CTAB, cetyltrimethyl ammonium bromide; EDTA, ethylene
diaminetetraacetic acid; NaCl, sodium chloride; TrisHCl, tris hydrochloride
INTRODUCTION
Plants produce secondary metabolites that
interfere not only with extraction of high
quality genomic DNA but also with the
subsequent reactions such as PCR [1, 2].
PCR based DNA marker analysis is now-
a-days, widely used in several crop plants
for genetic diversity analysis, Marker
assisted selection, (MAS), genetic
mapping and varietal identification [3]. An
array of DNA isolation protocols are
available and are being used for the
extraction of nucleic acids from plants [4].
RNA and protein contamination in DNA
may result in no band or smear formation
during PCR amplification. DNA
amplification can be affected by the
presence of certain impurities/ co purifying
inhibitors, which reduces PCR efficiency.
Therefore quality of DNA is of utmost
importance [5–7].
The family Brassicaceae consists of
approximately 340 genera and more than
3350 species [8]. In addition to providing
vegetables, fodder, vegetable oil and
condiment, Brassicas are important
sources of dietary fiber, vitamins and
anticancer compounds [9]. Cabbage
(Brassica oleracea L. varcapitata), family

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