A

Dissertation

ON

“DNA Isolation and Amplification with RAPD Marker in an

Endangered Medicinal Plant Species Saraca Asoca”

As partial fulfillment of the degree in

“Master of Science in Biotechnology”

Submitted By

Suraj prakash saket

UNDER THE SUPERVISION OF

SUPERVISOR
Dr. S.K. Tiwari

WORK DONE AT
Forest Genetics Plant Propagation & Biotechnology Branch
State forest research institute Jabalpur (M.P.)
Project Work

Submitted to
Department of Biotechnology

GOVT. THAKUR RANMAT SINGH COLLEGE REWA (M.P.)

1
Dr.AmitTiwari Mailing Address-
M.Sc., Ph.D (Zoology) Civil Lines, In front of TV Tower
H.O.D. Rewa (M.P.) 486001- INDIA
Deptt.of Zoology &Biotechnology. Ph. (Res.) 07662-250159, (off.) 423386
E-mail: draktiwari@rediffmail.com

COLLEGE OF EXCELLENCE
NAAC ‘A’ Grade

CERTIFICATE

This is to certify that Dissertation entitled “DNA Isolation and Amplification with RAND
Marker in an Endangered Medicinal plant Species Saraca asoca” Submitted to Govt.
T.R.S. College Rewa (M.P.) in, Dept. of Biotechnology by Suraj Prakash Saket is a partial
fulfillment of the requirement for the award of the degree of the Master of Science with
specialization in Biotechnology. The matter embodied is the actual work by Suraj Prakash
Saketand this work has not been submitted earlier in past or full for the award of any other
degree.

Date: -
Place-
Dr. AmitTiwari
HOD
Department of Biotechnology

Govt. T.R.S. College Rewa (M.P.)

2
 DECLARATION

I hereby declare that the record submitted to college in partial fulfillment of the requirement

for the award of degree of Master of Science in Biotechnology is of dissertation work

carried out by me in Forest Genetics Plant Propagation & Biotechnology Branch State

forest research institute Jabalpur (M.P.).

Date --------- Suraj prakash saket

Place- Rewa M.Sc Biotechnology IVth Sem.

Govt. T.R.S. College Rewa (M.P.)

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 ACKNOWLEDGEMENT

It is pleasure for me to Acknowledge the help which I received during the work and
thereafter preparing this thesis. This dissertation would not have been possible without the
guidance and the help of several individuals who in one way or another contributed and
extended their valuable assistance in the preparation and completion of this study.
Foremost, I would like to express my sincere gratitude to the director , State forest
research institute Jabalpur (M.P.). Dr. Dharmendra verma (I.F.S) for providing all the
laboratory and other facilities and make me part of this institute.
I am greatly indebted to Dr. S.K. Tiwari, Head forest Genetics Plant Propagation
& Biotechnology Branch not only for suggesting problem but also for his constant
encouragement and guidance during my dissertation period.
I express my profound gratitude to Shri Amit Pandey Senior Research officer
Forest Genetics Plant Propagation & Biotechnology Branch for his stimulating
suggestions and guidance from time to time.
I am deeply grateful to my Mr. Shailendra Singh Yadav (Research Associate)
Forest Genetics Plant Propagation & Biotechnology Branch. SFRI Jabalpur for their
continuous support. The knowledge and logical way of thinking have been of great value for
me. Their understanding, encouraging and personal guidance have provided a good basis for
the present thesis. I am also very thankful to Dr. Amit Tiwari Professor& Head
Department of Biotechnology Govt. TRS. College Rewa (M.P.) for giving me an opportunity
to join this institute.
It is my sincere duty to thanks my parents and my friends for their support and their
faith on me to do the excellent work. And the one above all of us, the omnipresent god, for
answering my prayers for giving me the strength to plod on despite my constitution wanting
to give up and throw in the towel, thank you so much dear lord.

Date: Suraj prakash saket

Place Rewa

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 ABBREVIATIONS

 DDW- Double distilled water.

 EDTA- Ethylene diamine tetra acetic acid.

 CTAB- Cetyl try Methyl ammonium bromide.

 SDS- Sodium dodayl Sulphate.

 PVC- Polyvinylpyrolidone.

 TAE- Tric acetic EDTA

 ETBR- Ethidium bromide.

 BPB- Bromo phenol blue.

 DNA- Deoxyribonucleotide Nucleic acid.

 RNA- Ribo Nucleic acid.

 PCR- Polymarage chain Reaction.

 RFLP- Restriction fragment length polymorphism.

 RAPD- Random Amplified polymorphism DNA.

 BSA- Bovine serum albumin acetylated .

 G.C.% A sequence composition o DNA molecules in

genetics

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 CONTENTS

S.NO. TITLE PAGE NO.

CHAPTER-1 Introduction 7-23

CHAPTER-2 Instrumentation 24-38

CHAPTER-3 Review of Literature 39-42

CHAPTER-5 Material & Methods 43-46

CHAPTER-6 Result & Discussion and Conclusion 47-51

CHAPTER-7 References 52-55

CHAPTER-8 Photo Gallery 56-57

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 Chapter- 1
INTRODUCTION

7
 INTRODUCTION

Saraca asoca
Saraca asoca (Roxb) De Wilde is an increasingly endangered and endemic

medicinal plant in India. Genetic assessment is one of the important parameters

for formulating conservation strategies for endangered species. Randomly

amplified polymorphic DNA (RAPD) profiling was employed to assess the

genetic diversity of 165 individuals representing five natural populations. There

was a relatively low level of genetic diversity in S. asoca at the species level but

a relatively high level of genetic differentiation among populations. Limited

gene flow due to human impact may be the key factor resulting in the observed

genetic structure. These effects may be most pronounced in species that are self-

compatible and/or have limited seed dispersal ability.

It is distributed in evergreen forests of India up to an elevation of about 750

meters. It is found throughout India. Specially in Himalaya, Kerala, Bengal and

whole south region. In Himalaya it is found at Khasi, Garo and Lussi hills and

in Kerala region it is found in Patagiri, Kaikatty & Pothundi of Palakkad

district, Thrisur, Kollam and Kannur districts

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 Scientific classifications

Kingdom - plantar

Division - Magnoliaphyta

Class - Magnoliopsida

Order - Fabalea

Family - Caesalpinacea.a

Genus - Saraca

Species - Asoca

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 Morphological characteristics

Leaves:It is a peripinnate, alternate, distichous and 7-30 cm long and its

petiolule 0.1-0.6 cm long and opposite leaflets, 4-6 pairs, narrow elliptic-oblong
or lanceolate, and its apex acute to acuminate, base acute to rounded or
subcordate, glabrous, midrib raised above and tertiary nerves reticulate 7.

Bark: The bark is dark brown or grey or almost black with warty surface. Stem
bark are rough and uneven due to the presence of rounded or projecting lenticles
and channeled, smooth with circular lenticles and transversely ridged 7

Flowers: It is inflorescence dense corymbs, orange colour and sometimes white

and fragrant. Flowers are with fragrant, numerous, in dense axillary corymbs
7.5-10 cm. across; peduncles stout; pedicles 8-13 mm. long, red, glabrous;
bracts ovate subacute; bracteoles 2, appearing like a calyx, 4 mm. long,
spathulate-oblong subacute ciliolate, amplexicaul, coloured. Calyx passing from
yellow to orange and finally red; tube 1.3-2 cm. long, cylindric, solidat the base;
segments 7 or 8, oblong or obovate-oblong, 1 cm. long. Petals 0. Stamens 7-03-
8, much exerted; filaments filiform, thrice as long as the calyxsegments; anthers
purple. Ovary pubescent, especially on the sutures; anthers purple. Style curved
into a ring.

Pods: are black, 10-25 by 4.5-5 cm., Linear-oblong, tapering to both ends,
compressing, glabrous, veined. Seeds-:are 4-8 ellipsoid-oblong, 3.8 cm.,slightly
compressed.

Wood: is soft, reddish brown. Bark is distinguishing by the presence of warty

protruberances and transverse lenticels on the its outer surface, septate and
nonseptate crystal fibres and prism of calcium oxalate. Flowering and fruiting
time was from spring to autumn seasons.Photochemicalconstituents of Ashoka

10
Fruits: It is a pod; flat, oblong and fruits have been previously reported for the
presence of various fatty acids such as oleic, linoleic, palmitic and stearic acids;
sterols like catechol and epicatechol, and a flavonoid, leucocyaanidin

Uses of Saraca Asoca

Medicinal use of Saraca asoca

The old Ayurvedic texts combined with numerous ethnobotanical uses of this
plant have inspired several research groups in India and elsewhere to
systematically investigate its beneficial claims and test any novel
pharmaceutical properties that it may possess. Following section reviews the
modern, literature-based reports on the biological and pharmacological
properties of S. asoca.

Antimennorhagic, oxytocic and uterine tonic

The use of S.asoca dried bark, root and flowers to manage uterine
abnormalities, menorrhagia (excessive menstrual bleeding), ammenorhea,
painful periods, endometrosis and disorders of the menstrual cycle is well
known in India. The root decoction of S. asoca is also consumed after delivery
for enhanced lochial discharge.

Antibacterial

Numerous literature reports establish the antibacterial properties of methanolic,

ethnolic, acetone and aqueous extracts of bark, dried flower buds and leaves of
S. asoca These have been tested against many pathogenic bacteria such as
Bacillus subtilis, Escherichia coli, Salmonella typhosa, S. typhimurium, S.
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typhii, S. viballerup, S. enteritis, Staphylococcus aureus, Bacillus cereus,
Klebsiella pneumonia, K. aerogenes, Shigella boydis, S. sonnei, S. flexneri, S.
dyserteriae, Pseudomonas aeruginosa, P. vulgaris, Vibro cholerae, Proteus
vulgaris, etc. Antifungal

Traditionally fungal infections have been attributed to compromised immune

response of and individual and not posing a very serious danger to the
population at large; however, there have been increasing incidences of fungal
disease outbreaks in the past. Finding new antifungal agents is therefore a
priority of the clinical microbiology community. The antifungal activity of
methanolic and hot aqueous extracts of S. asoca leaves, flowers and bark
against Alternaria alternata, Colletotrichum gloeosporioides, Drechlera
specifera, Alternaria cajani,

Anticancer

Natural products have provided some of the most effective anticancer leads in
the past, several already being into successful commercial production3.
Ethnobotanical studies of S. asoca have revealed its flavonoid fraction (from
flowers) to prevent two-stage skin carcinogenesis and preferentially act against

Dalton’s lymphoma ascites and Sarcoma-180 tumour cells, while being non-

toxic to normal lymhpocytes. The ethanolic extract of S. indica was shown to

inhibit breast cancer

Anti-inflammatory,

antiarthritic and cardioprotective effect Chronic arthritis and cardiovascular

diseases are generally attributed to the inflammatory response mediated by
proinflammatory cytokines. The ethanolic and methanolic extracts of the leaf,
bark and root of S. asoca have been shown to exhibi ant-inflammatory potential
by significantly inhibiting the binding of various transcription factors such as

12
NF-κB, AP-1, GATA-1, etc. to their target DNA sequences, thereby lowering

the levels of proinflammatory cytokines11. S. asoca extract has also been shown

to reduce the levels of pro-inflammatory cytokines IL-1 and TNF-α (ref. 40).

Several reports describe the antiarthritic potential of S. asoca using the model
system

Antiulcer

The aqueous suspension extract of S. Asoca flowers, dried flower buds, bark
and seeds were shown to curtail ulcers in albino rats. The anti-ulcer effect of the
aqueous extract of S. Asoca flowers was demonstrated in albino rats by
employing two models, namely pyloric.

Ayurvedic actions

Vedana sthapana- Useful in management of all painful conditions.

Varnya- Ashoka improves complexion of the body.

Grahi- Ashoka improves digestion and assimilation.

Trishanashnam- Ashoka alleviates excessive thirst.

Daha shamanam- Ashoka alleviates burning sensation.
Krimighna- Ashoka kills all infectious agents.
Shothajit- Ashoka is useful in management of all edematous conditions.
Vish asrajit- Ashoka is useful in toxicities and all blood disease.
Apachijit- It useful in management of inflammation of lymph nodes.
Asrigdara nashanam- In management of excessive bleeding during
menstruation

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Other medicinal uses

Dried root of S. asoca has been reported useful in paralysis, haemiplegia and
visceral numbness showing its effect through the parasympathetic and
autonomous nervous systems. The extracts of S. asoca seeds and dried flowers
have shown properties of being an osteoid tissue promoter such as in treatment
of rickets, delayed bone consolidation and calcium deficiency. Antipyretic
action of methanolic extracts of S. ascoa leaves has been recently
reported79,80. Similarly, many other biological/ pharmacological actions of its
various extracts have been reported such as an antidote in snake bite, an
antileucorrhea agent, a contraceptive, and in the treatment of dysentery, worm
infestations and stomach pain

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 Biotechnology

The term biotechnology was coined in 1919 by KarlEreky, a Hungarian

engineer. At that time, the term included all the processes by which products are
obtained from raw materials with the aid of living organisms. Ereky envisioned
a biochemical age similar to the stone and iron ages. Nowadays, according to
the Convention on Biological Diversity (CBD),

Biotechnology is defined as “ any technological application that uses

biological systems, living organisms, or derivatives thereof, to make or modify

products or processes for specific use” (CBD, 1992). The living organisms or

derivatives thereof most frequently used include micro-organisms, animals and

plants (or their isolated cells) as well as enzymes. They can be utilized to
process substances, usually other natural, renewable materials, or serve
themselves as sources for valuable substances or goods. Several branches of
industry rely on biotechnological tools for the production of food, beverages,

pharmaceuticals and biomedicals. The CBD definition is applicable to both “

traditional” or “old” and “new” or “modern” biotechnology Long before the

term biotechnology was coined for the process of using living organisms to
produce improved commodities, people were utilizing living micro-organisms
to obtain valuable products, Modern use of similar terms includes genetic
engineering as well as cell and tissue culture technology.

Definition: Biotechnology is defined as the controlled use of biological agents

such as microorganisms or its cellular components for beneficial use.
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 Fields of biotechnology
Molecular biology Animal biotechnology

Bioinformatics
Plant biotechnology
Energy and environment

Genetic engineering Biotechnology

Healthcare/Pharmaceuticals

Biochemistry
Genomics

Immunology

Applications of biotechnology:

 Medical Biotechnology: Concerns with production of monoclonal

antibodies, synthetic vaccines, valuable drugs (insulin, interferon), gene
therapy, DNA finger printing etc.
 Industrial Biotechnology: Concern with production of ethanol, lactic
acid, glycerine, citric acid, acetone, antibiotic enzymes, single cell
protein, fuel and extraction of minerals.
 Animal biotechnology: Concern with production of transgenic animals,
test tube baby and human induced super ovulation and embryo splitting
etc.
 Environmental biotechnology: Concern with sewage treatment,
deodarization of human excreta, degradation of petroleum and other oil
spills, detoxification of wastes, bio control plant diseases and pests.
 Plant Biotechnology: Concerns with embryo culture clonal
multiplication, recovery of virus or pathogenic free stock, germplasm
conservation, isolation of homozygous lines, isolation of stable

16
somaclonal variants, gene transfer for insect resistant and molecular
marker for linkage mappin

History of Biotechnology

The term biotechnology was coined in 1919 by karl Ereky, a hungarian

engineer, At that time the term meant all lines of work by which products
are produced form raw materials with the aid of living organism.

Year NAME OF SCIENTIST IMPORTANT CONTRIBUTION

1902 Haberlandt First attempt of plant tissue culture

(Father of PTC)

1953 Whatson & Crick DNA double helix model

1956 Kornberg et al In vitro synthesis of DNA

1869 Friedrich Miescher First DNA isolate (is called Nuclein)

1968 H.G. Khurana Awarded noble prize for deciphering

genetic code

1984 Alec jeffreys Development of DNA fingerprinting

technique

1983 Carrymulieus PCR

1990 Walliams et al Development of RAPD technique

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 1990 Doyle and Android DNA extraction method (best method)

Molecular Biology

Molecular Biology or Molecular genetics is the study of the biochemical

mechanisms of inheritance; It is the study of the biochemical nature of the
genetic material and its control of phenotype. It is the study of the connection
between genotypes and phenotypes. The connection is a chemical use.

Applications of molecular Biology

1. Use of detection of genetic diversity.

2. Use of Marker technology.
3. Use of PCR electrophoresis and DNA finger printing.
4. Forensic study.

Importance of molecular Biology

Molecular biology is the most advance branch of biology. They are
very important is modern era. That is many use of modern technology.
Human life is the very important in Molecular Biology.

Deoxyribo nucleic acid (DNA)

DNA is found in the cells of all living organisms except plant

viruses,WhereRNA forms thegenetic material and DNA is absent .The number
of DNA molecules is equivalent to the number of chromosomes per cell .DNA
isfound is the combination with proteins forming nucleoproteins.

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 DISCOVERY OF DNA

DNA was first isolated by the Swiss physician, Friedrich Miescher in 1869
while working in the laboratory of the biochemist Felix Hoppe- Seyler. This he
did as part of a project to determine the chemical composition of cells which he
saw as the means to unraveling the fundamental principles of the like of cells.
Mierscher decided to call the new substance ‘nuclein’ by virtue of its presence
in the nuclei of the cell.The first isolation of DNA was done in 1869 by
Friedrich miescher currently it is a routine procedure in molecular analysis
biology or forensic study.
In 1953 Whatson and Crick the model proposed DNA double helix model.
The DNA contains two polynucleotide chains running antiparallel and twisted
around each other in the form of regular double helix.

DNA Extraction

DNA extraction is difficult in a variety of plants because of the presence of

metabolites that interfere with DNA isolation procedures and downstream
applications such as DNA restriction, amplification, and cloning. Here we
describe a modified procedure based on the hexadecyltrimethylammonium
bromide (CTAB) method to isolate DNA from tissues containing high levels of
polysaccharides. The procedure is applicable to both dry and fresh leaves of
Pennisetum glaucum. This modified CTAB (2%) protocol include the use of 1.4
M NaCl, 1% polyvinylpyrrolidone (PVP), 1% -mercaptoethanol and 100%
ethanol in the extraction as well as reducing the centrifugation times during the
separation and precipitation of the DNA. This method solved the problems of

19
DNA degradation, contamination, and low yield due to binding and/or
coprecipitation with starches and polysaccharides. The isolated DNA proved
amenable to PCR amplification and restriction digestion. The technique is fast,
reproducible, and can be applied for SSR-PCR markers identification.

Genetic marker

A DNA sequence at a unique physical location in the genome, which varies

sufficiently between individuals that its pattern of inheritance can be tracked
through families and it can be used to distinguish among cell types, A Marker
may or may not be part of a gene. Markers are essential for use in linkage
studies and genetic maps to help scientists to narrow down the possible location
of new genes, and to discover the associations between genetic mutations and
disease.

1.RAPD (Random amplified polymorphic DNA) Marker

RAPD markers are decamer (10 nucleotide length) DNA fragments from PCR
amplification of random segments of genomic DNA with single primer of
arbitrary nucleotide sequence and which are able to differentiate between
genetically distinct individuals, although not necessarily in a reproducible way.
It is used to analyze the genetic diversity of an individual by using random
primers. In this paper, the principles, working mechanism, differences between
standard PCR and RAPD-PCR, characteristics, laboratory steps, data analysis
and interpretation, advantages and disadvantages and several of the most
common applications of RAPD markers in biology are discussed.

Uses:

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This technique can be used in various ways such as for varietal identification,
DNA fingerprinting, gene tagging and construction of linkage maps. It can also
be used to study phylogenetic relationship among species and sub-species and
assessment of variability in breeding populations.

2. RFLP Marker
Developing sets of RFLP probes and markers is labour intensive. This technique
requires large amount of high quality DNA. The multiplex ratio is low, typically
one per gel. The genotyping throughput is low. It involves use of radioactive
chemicals. RFLP finger prints for multi-gene families are often complex and
difficult to score. RFLP probes cannot be shared between laboratories.

Uses:
They can be used in determining paternity cases. In criminal cases, they can be
used in determining source of DNA sample. They can be used to determine the
disease status of an individual. They are useful in gene mapping, germplasm
characterization and marker assisted selection. They are useful in detection of
pathogen in plants even if it is in latent stage.

3. Amplified Fragment Length Polymorphism (AFLP):

AFLPs are differences in restriction fragment lengths caused by SNPs or
INDELs that create or abolish restriction endonuclease recognition sites. AFLP
assays are performed by selectively amplifying a pool of restriction fragments
using PCR. RFLP technique was originally known as selective restriction
fragment amplification.It provides very high multiplex ratio and genotyping
throughput. These are highly reproducible across laboratories. No marker
development work is needed; however, AFLP primer screening is often
necessary to identify optimal primer specificities and combinations.

Uses:
This technique has been widely used in the construction of genetic maps
containing high densities of DNA marker. In plant breeding and genetics, AFLP
markers are used in varietal identification, germplasm characterization, gene
tagging and marker assisted selection.

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4. Cleaved Amplified Polymorphic Sequences (CAPS):
CAPS polymorphisms are differences in restriction fragment lengths caused by
SNPs or INDELs that create or abolish restriction endonuclease recognition
sites in PCR amplicons produced by locus-specific oligonucleotide
primers.CAPS assays are performed by digesting locus-specific PCR amplicons
with one or more restriction enzymes and separating the digested DNA on
agarose or polyacrylamide gels.CAPS analysis is versatile and can be combined
with single strand conformational polymorphim (SSCP), sequence-
characterized amplified region (SCAR), or random amplified polymorphic
DNA (RAPD) analysis to increase the chance of finding a DNA
polymorphism.Michaels and Amasino (1998) proposed a variant of the CAPS
method called dCAPS based on SNPs.

Uses:
This is straightforward way to develop PCR-based markers from the DNA
sequences of previously mapped RFLP markers. It is a simple method that
builds on the investment of an RFLP map and eliminates the need for DNA
blotting.

5. Simple Sequence Repeats (SSRs):

Simple sequence repeats (SSRs) or microsatellites are tandemly repeated mono-
, di-, tri-, tetra-, penta-, and hexanucleotide motifs. SSR length polymorphisms
are caused by differences in the number of repeats. SSR loci are individually
amplified by PCR using pairs of oligonucleotide primers specific to unique
DNA sequences flanking the SSR sequence. Jeffreys (1985) showed that some
restriction fragment length polymorphisms are caused by VNTRs. The name “
mini satellite” was coined because of the similarity of VNTRs to larger satellite
DNA repeats.

6. Expressed sequence tagged polymorphisms (ESTPs)

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ESTPs are PCR-based genetic markers that are derived from expressed
sequenced tags (ESTs). Expressed sequenced tags are partial cDNA sequences
that have been obtained by automated DNA sequencing methods therefore,
ESTPs are a genetic marker for structural gene loci. The EST databases contain
hundreds of thousands of entries from a variety of organisms, most notably
Arabidopsis thaliana, rice, and maize in plants. In forest trees, there are EST
databases for Pinus, Populus, and Eucalyptus. The ESTs are routinely compared
to DNA sequence databases to determine their biochemical function. It is also a
goal of most genome projects to place the ESTs onto genetic linkage maps.
Expressed sequenced tags can be genetically mapped by a variety of methods,
all of which rely on detecting polymorphism for the ESTs, hence the name
ESTPs for the genetic marker

23
 Chapter -2
Instrumentati
on

24
 Autoclave
Principle
autoclaving in the laboratory, the most agreeable and commonly
used method is to use steam at heat at 121oC and presser and
stylizations zone 15 to 30 minutes depending upon the particular
material to be sterilized.

Types of autoclave
Only autoclaves designed for laboratory work and capable of
dealing with a 'mixed load' should be used. 'Porous load' and 'bottled
fluid sterilizers' are rarely satisfactory for laboratory work.

Horizontal autoclave Vertical autoclave

25
 Hot Air Oven
Principle
The instrument is electrically operated and should be equipped
with a fan to have uniform temperature inside, the required
temperature for sterilization is generally160oC for 1 hour

Operation of Hot air Oven

 Switch on the power supply and control the temperature of the

oven by adjusting thermostat. The time. Time taken for the oven
to reach the desired temperature is called 'heating up period'.
 Hold the load in the oven at this temperature for a definite
period of time. This period known as 'holding up period' is
dependent upon the temperature employed. At 160oC the
holding up period is 60 minutes, at 170oC, 18 minutes, at
180oC, 7.5 minutes and at 190oC it is 90 seconds.

26
 The most common temperature for hot air sterilisation is 160oC
for one hour. When the temperature is raised further, cotton
plugs and paper wrappings get charred.

pH METER
A pH meter consists of an electrode pair which is sensitive to
hydrogen ion concentration due to the development of an
electrical gradient which is directly proportional to the ion
concentration. The electrodes commonly used are one of glass
for the unknown

Precautions while using pH meter are

 The electrodes specially the glass ones should be handled

carefully to prevent breakage due to contact with hard
surface.

27
  Sufficient time should be given to warm up the
instrument before use.
 Frequent standardizations of the pH meter should be
made using standard buffer solution.
 Electrodes are to be washed with a stream of distilled
water between measurements.
 The electrodes should never be removed from the
solution when the measuring circuit is closed.
 When not in use, the electrodes must be kept immersed
in water or electrode solution.

Weighing scale
Weighing scales (or weigh scales or scales) are devices to measure
weight or calculate mass. Spring balances or spring scales measure weight
(force) by balancing the force due to gravity against the force on a spring,

28
whereas a balance or pair of scales using a balance beam compares masses by
balancing the weight due to the mass of an object against the weight of a known
mass or masses. Either type can be calibrated to read in units of force such as
new sensor in units of mass such as kilograms, but the balance or pair of scales
using a traditional balance beam to compare masses will read correctly for mass
even if moved to a place with a different (non-zero) gravitational field strength
but would then not read correctly if calibrated in units of force), while the
spring balance would read correctly in force in a different gravitational field
strength (but would not read correctly if calibrated in units of mass).used in
scientific fields such as chemistry.

RO WATER PURIFIER

29
 Ro water purifier is the purifying normal water to double
distilled water.

Mortar and pestle

Mortar and pestle is used of leaf dis-ruptured.

Micropipetes

30
 It is use of exact measuring different solutions,
chemical & buffer and inject into the tubes.

MICROWAVE OVEN

Microwave oven is use melting agarose for gel electrophoresis

Thermo mixer
31
 Thermo mixer comfort, Thermo mixer compact and can be used
in almost any field of molecular and cell biology. Important methods
are outlined below, and detailed descriptions begin on Determination
which device is most appropriate for your application depends on the
type of reaction tubes being used, the desired temperature range and
whether or not the tubes need to be mixed during incubation. Thermo
mixer comfort offers the highest degree of flexibility, as it can
accommodate 10 different exchangeable thermo blocks as well as
heat, actively cool and mix samples. The smaller Thermo mixer
compact is optimized for heating and mixing samples in 1.5 mL micro
test tubes. The Thermostat plus, which can also be used with different
thermo blocks, is perfect for applications where samples require
heating or cooling to -5 °C but do not require mixing and outline the
key features and technical specifications of these devices and their
exchangeable thermo blocks.

Water Bath

32
 Water bath is a water container having an electrically
operated heating device to provide a fixed and uniform
temperature. A thermometer is inserted inside the water bath for
recording temperature. A mixer immersed inside water is also
desired to maintain uniform temperature throughout the water
bath.

A few applications of water bath are:

 37oC Water bath - required during performance of WIDAL

test;
 44oC Water bath - required in faecal coliform count (water
bacteriology) test.
 56oC Water bath - for inactivating complement in the
serum.
 60oC water bath - 4 ml buffer solution and mix 10 mg
polyvinyalpyrrolidone (PVP)

Centrifuge
33
 For an average laboratory a small table top centrifuge with
maximum rounds per minute of 16000 and capable of accommodating
01-24 tubes of 2 ml capacity is sufficient. The tubes should be placed
exactly opposite to each other, should be of the same weight and
should contain same amount of fluid. The speed is adjusted by a speed
adjusting knob and should be allowed to rise slowly. A timer for fixed
duration of centrifugation is preferred.

Principle- A centrifuge is a device for separation particles from a

solution According to their size, shape, density, viscosity of the
medium and Rotor speed

rpm - round par minute

rcf - Relative centrifugal force

Deep Refrigerator

34
 Refrigerators are essential for storage of degradable
laboratory substances like media, reagents, antisera, antibiotic
discs etc. Refrigerators can vary in their capacity ranging from
table top to a large walk-in-type. The usual temperature needed
is - 40 oc to – 80oc which is maintained comfortably by
household use refrigerators. Substances to be kept at frozen state
like sera may be kept in the freezer units of the same (-8 oc).
Proper recording of the temperature is very important to avoid
deterioration of biological materials.

Electrophoresis Units

35
 Electrophoresis Units is worked in biomolecules are separated by
applying an electric field to move the charged molecules through an agarose
matrix and the biomolecules are matrix.

UV- TRANSLUMINATOR
36
 Uv transluminator is used to see agarose gel moving to DNA Molecules.

Computer system

A computer system is the most important role play in a molecular

laboratory. They are used operate a PCR and gel Doc system and take gel image
of the DNA.

Gel Documentation System

The System should be able to image fluorescent DNA, RNA &
protein gels, colorimetric gels & blots and colony arrays.
37
1. High resolution CCD camera : 1.44 mega pixel resolution
with 1360x1024 pixel array.
2. Data acquisitions: 12 bit and 4096gray level. Pixel Size :
4.6x4.6 micron.
3. Motorized control for Zoom with numerical feedback and
software acquisition preset integrated into an intuitive and easy
to use interface along with gel alignment templates, aperture &
Iris with f/1.2, 12-75 mm lens with broad range amber filter.
4. Gel alignment templates matched to agarose or protein gel
trays and ready gels.
5 Blue illumination integrated in a light tight darkroom with
software controlled illumination to view DNA gels using blue
excitable DNA fluorescent stains such as Gel screen, SYBR safe
dyes and SYBR Green.
6. UV and white light source : 302 nm illumination source
having 25x26 Tran- illumination area with White Light
Converter screen for viewing protein gels with trans blue
illumination.

PCR

38
 Polymerase chain reaction is techniques are used in molecular biology to
amplify a single copy or a few copies of a segment of DNA across several
orders of millions of copies of a particular DNA Sequence.

39
Chapter - 3
Review of literature

Review of literature

40
 Thakur 1989 Saraca asoca (Roxb.) Wilde (Caesalpiniaceae) is a
medicinally important and globally vulnerable plant species found in
the evergreen forests of India.
 Hattori 1995S. asoca, commonly known as Ashoka tree, is considered
as one of the sacred trees of India and is highly prized for its beautiful
foliage and fragrant flowers. Almost all parts of the tree are known to
have important medicinal properties including antiviral oxytotic.
 Satyavati . 1970 , menorrhagic, anti-HIV.
 Kusumoto 1995 and antibacterial activities.
 Anonymous 1952 Mukherji 1970; and Verghese 1992
Overharvesting of S. asoca due to its high medicinal value along with
high deforestation rates, habitat fragmentation and illegal
encroachments of its natural habitats have resulted in severe reduction
in natural populations of this species.

 Gowda 2002 This species is currently listed as a ‘ globally

vulnerablespecies by the. In this paper, we report identification of

novel microsatellite markers and discuss the utility of these markers in
addressing questions related to the population genetics of this
species.Ten microsatellite markers were identified in this species.

These microsatellite loci have 2–22 alleles per locus; with observed

and expected heterozygosity of 0.001 – 1.00 and 0.273 – 0.964,

respectively. These markers will be invaluable in gaining insights into

the population genetics of the species for formulating sound
conservation and management strategies.
 Doyle and Doyle 1987, Material and methods The leaf samples from
S. asoca (Roxb.) trees in the Western Ghats of India were collected

41
 and genomic DNA was extracted using the CTAB method Doyle and
Doyle. The extracted DNA was purified, dissolved in Tris-EDTA
buffer and used for microsatellite identification through the
enrichment-subtractive hybridization protocol with minor
modifications
 Glenn and Schable 2005 Saraca asoca. bridizing with the DNA
fragments and magnetically captured using Dynabeads (Sigma-
Aldrich, Bangalore, India).The captured DNA was washed, amplified
and cloned to pTZ57R/T plasmid vector using Fermentas TA Cloning
Kit.
 Sadhu 2007 and Pradhan 2009 The species is under severe threat due
to unscientific management practices, ever-increasing demands for its
phytochemicals, poor seed viability and overexploitation of the plant
parts, such as its bark, flowers, seeds, etc., resulting in the dwindling
of populations in the wild (Wiersum et al. 2006; Rama Subbu,
Chandraprabha, and Sevugaperumal 2008).
 The species has also been included in the list of endangered and
threatened species of India (Redd and Reddy 2008). As predicted by
population genetic theory, loss of genetic variation is a major threat
tendangered species with small populations or that are located in
narrow geographic areas (Ellstand and Ela 1993; Segarra-Moragues et
al. 2005). A low level of
 genetic variability often results in lower fitness of individuals (Luijten
et al. 2000; Hansson and Westerberg 2002), reduces the viability or
adaptability
1. In Sushruta Samhita, it is described in rodhradi gana
2. Ashokaghrita prescribes in vatavyadhi
3. InAshtanga Hridaya, Vagbhata mentioned rodhradi gana

42
4,.Ashoka is mentioned in Nighantus also.In Kaideva Nighantu, described
for dosha, apachi, trisha, daha, krimi, shoshaand vishaghna
5, Bhavaprakash Nighantu, described for dosha, apachi, trisha, daha,
krimi, shosha and vishaghna
6, Shodhala Nighantu, its properties and uses in raktapradara are
described,Dhanvantari Nighantu described for dosha, apachi, trisha, daha,
krimi, shosha and vishaghna. In Raj Nighantu, it is described as hridya,
gulmashulodaradhmanahara and krimihara
7.Ashoka is also mentioned in Chikitsa Granthas like Chakradatta (CD,
Asrigadara chikitsa, 58.5) and Bhavamishra, in his section Bhavaprakash,
madhyamakhanda mentions Ashokakshiram in raktapradara
8. Shivadasa described the seeds of ashoka in mutraghata and ashmari. In
Bhashajya Ratnavali, ashokarishta-ashokaghritam is described

43
 Chapter -4
Materials and method

44
 Materials and method

List of chemicals use in DNA isolation and marker technology.

 Reagent and buffers.

S.No. Name of chemicals Amounts

1 Tric-base 100mM
2 NaCl 1.4 M
3 EDTA 20mM
4 C-TAB 1%

 DNA isolation

1 Polyvinylpyrrolidone(PVP) 10 mg
2 RNase 10𝜇L
3 Phenol 500 𝜇L
4 Chloroform:Isoamyl alcohol 500 𝜇L
5 Propanol 400 𝜇L
6 Ethanol (70% alcohol) 200 𝜇L

 Electrophoresis gel preparation

1 TAE-Buffer 100ml
2 Agarose gel 0.8%
3 ETBR 10 𝜇L
4 Bromophenol blue dye 2 𝜇L
5 DNA ladder + Bromophenol blue 10 𝜇L

45
 Methodology of DNA isolation

The leaf sample of saraca asoca(1.0 g) were grinded in liquid nitrogen using
mortar pestle to fine powder. The powder was then transferred to 1 ml DNA
extraction buffer containing 1M Tris-Cl (pH 8.0), 0.5 M EDTA (pH 8.0), 5M
NaCl and 2% CTAB and add 1.5% PVP (w/v) and 10 mM -mercaptoethanol
(added freshly) and after thorough mixing, solution was incubated at 65ºC for
60 min. then centrifuged at 14,000 rpm and take supernatant in fresh new
centrifuge DNA contained RNA as impurity, Bovine Pancreatic RNase (5 mg/
ml) was added (100 μg RNase /ml DNA) to dissolved in extraction buffer and
incubated for one hour at 37ºC. After this an equal volume of phenol:
chloroform: isoamyl alcohol (25:24:1) was added, mixed and centrifuged at
16,000 rpm for 10 min at room temperature and take supernatant carefully in
fresh new centrifuge tube add equal amount chloroform: isoamyl alcohol (24:1)
was added, mixed and centrifuged at 16,000 rpm for 10 min at room
temperature after that take supernatant carefully in fresh new centrifuge equal
volume of chilled isopropanol were added and kept at –20ºC for one hour. DNA
was pelleted by centrifuging at 10,000 rpm for 10 min at 4ºC followed by 70%
ethanol washing. The pellet was air dried and dissolved in 100 μl of 1X TE
buffer.
DNA Quantification
The purity and concentration was also determined by electrophoresis on 0.8%
agarose gel based on band intensities when compared with lambda DNA/Hind
III digest marker.

Optimization of RAPD Reaction The extracted genomic DNA was tested for
PCR amplification using RADP decamer arbitrary primers (OPA08, OPA04
,OPN06 and OPC15 , Operon Technology, USA). The reactions were carried
46
out in 0.2 ml PCR tubes. The reaction mixture (25 μl) 25-50 ng of DNA, 2.5 U
Taq DNA polymerase enzyme (Promega), 0.4 μM each dNTPs (Promega),
2.5mM MgCl (Promega), 1X Taq DNA polymerase buffer supplied with
enzyme (Promega) and 0.4μM decamer primer (Operon, series USA). The plant
DNA was amplified in a programmable mastercycler epgradient (eppendrof)
using the following conditions: Initial denaturation at 94°C for 5 min, followed
by 45 cycles; denaturation at 94°C for 30 Sec, annealing of primer at 35°C for
45 sec and extending primer at 72°C for 1 min and final extension at 72°C for 7
min. The amplified PCR products were fractionated on 1.2% agarose gel using
1X TAE buffer containing 7μg/ml ethidium bromide. Gel was photographed on
Gel documentation system (GENEVIEW 645SC).

47
 Chapter -5
Result, Discussion and
Conclusion

48
 Result, Discussion and Conclusion

The genomic DNA was extracted by using CTAB method produced. Smeared
bands when subjected to electrophoresis indicated its degradation .In contrast
the DNA extracted from CTAB method resulted in intact bands. Quality of
genomic DNA was also confirmed through gel electrophoresis analysis in 0.8%
agarose gel that showed the DNA is good quality (Fig. 1.). The genomic DNA
isolated by this method produced reliable amplified products showing its
compatibility for RAPD PCR based marker using random decamer primers.
(Fig. 2. ). In this study we used four accession of Saraca Asoca collected from
different State of India Maharastra, Karnataka, Kerla and Madhya Pradesh
.Those accession established in SFRI, Jabalpur nursery. We have tested four
primers OPA-08, OPA-04, OPN-06 and OPC-15 amplified with 4 accessions. In
our finding was that OPN-06 amplified with accession no 1, 2 and 3 ware
positive results and 4 negative results . Then we have tested a second primer
OPA-08 amplified with accession no. 1, 2 and 3 ware positive result and 4
negative result Then we have tested a third primer OPA-04 amplified with
accession no.1, 2 and 3 ware positive results and 4 negative results Then we
have tested a last primer OPC-15 amplified with accession no.1, 2 and 3 ware
positive results and 4 negative results (Table 1 , 2 , 3 , and 4)The presence of
polyphenols, which are powerful oxidizing agents present in many plant
species, can reduce the yield and purity by binding covalently with the extracted
DNA making it useless for most research applications (Katterman and Shattuck,
1983; Peterson et al., 1997; Porebski et al., 1997). Tannins, terpenes and resins
considered as secondary metabolites are also difficult to separate from DNA
(Ziegenhagen and Scholz, 1998). Certain polysaccharides are known to inhibit
RAPD reactions. They distort the results in many analytical applications and
therefore lead to wrong interpretations (Kotchoni et al., 2003). Polysaccharides

49
like contaminants, which are undetectable by most criteria, can cause
anomalous re-association kinetics. Polysaccharide co-precipitation is avoided by
adding a selective precipitant of nucleic acids, i.e. cetyltrimethylammonium
bromide (CTAB) Addition of PVP may bind to the polyphenolic compounds by
forming a complex with hydrogen bonds and may help in removal of impurities
to some extent. Long-term chloroform: isoamylalcohol treatment ensured
removal of chlorophyll and other colouring substances such as pigments, dyes,
etc.
DNA isolated by this method yielded strong and reliable amplification products
showing its compatibility for RAPD-PCR using random decamer primers
(Figures 1b and c, Figures 2a and b). Almost all the tested parameters for
RAPDs were also effect on amplification, banding patterns and reproducibility.

The whole study was conclude that many factors affecting in amplification
process like that DNA quality, DNA quantity, applied primers, PCR
temperature and most important for RAPD primers concentration of genomic
DNA, primers, DNTPs, Taq DNA polymerase, MgCl2, annealing temperature
etc.

Table 1.-Amplification with OPA08 RAPD primer four accessions of saraca

asoca
Primer name Sequence GC %
OPA08 5'-GTGACGTAGG-3' 60
Sample No. Accession Name Amplification
(+/- )
1. Maharastra +
2. Karnataka +
3. Kerla +
4. Madhya Pradesh -

50
Table 2.-Amplification with OPA04 RAPD primer four accessions of saraca
asoca

Primer name Sequence GC %

OPA04 5’-AATCGGGCTG-3’ 60
Sample No. Accession Name Amplification
(+/- )
1. Maharastra +
2. Karnataka +
3. Kerla +
4. Madhya Pradesh -

Table 3.-Amplification with OPN06 RAPD primer four accessions of saraca

asoca
Primer name Sequence GC %
OPN06 5’-GAGACGCACA-3’ 60
Sample No. Accession Name Amplification
(+/- )
1. Maharastra +
2. Karnataka +
3. Kerla +
4. Madhya Pradesh -

Table 4.-Amplification with RAPD primer four accessions of Saracaasoca

Primer name Sequence GC %
OPC15 5’- GACGGATCAG-3’ 60
Sample No. Accession Name Amplification
(+/- )
1. Maharastra +
2. Karnataka +
3. Kerla +
4. Madhya Pradesh -

51
 Figure: 1. Isolated genomic DNA of Saraca asoca

Figure: 2. Amplification of RAPD primers profile of Saraca asoca

52
Chapter - 6
Reference

53
 Reference
 Katterman FRH, VI Shattuck VI (1983). An effective method of DNA
isolation from the mature leaves of Gossypium species that contain large
amounts of phenolic terpenoids and tannins. Prep. Biochem. 13: 347-359.

 Peterson DG, Boehm KS, Stack SM (1997). Isolation of milligram

quantities of nuclear DNA from tomato (Lycopersicon esculentum), a
plant containing high levels of polyphenolic compounds. Plant Mol. Biol.
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 Porebski S, Baily LG, Baum BR (1997). Modification of a CTAB DNA

extraction protocol for plants containing high polysaccharide and
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 Ziegenhagen B, Scholz F (1998). Methods for difficult plant species.In:

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35

 Kotchoni SO, Gachomo EW, Betiku E, Shonukan OO (2003). A home

made kit for plasmid DNA mini-preparation. Afr. J. Biotechnol. 2: 88-
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 Doyle,J.J. and Doyle,J.L.(1990) isolation of plant DNA from fresh tissue.
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 Mueller. U.G. and Wolfenbarger, L. (1999) RAPD genotyping and
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 Trivedi, P.C (2006) Medicinal plants – Traditional knowledge .Ik

international Pvt. Ltd. New Delhi

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preparation version II. Plant Mol Biol reptr.1:19-21.

 Doyle JJ and Doyle JL (1987) A rapid DNA isolation procedure from

small quantities of fresh leaf tissue phytochen bull. 19:11-15.

 Khanuja SPS,Shasany AK, Dhawan S and kumar S (1998) Rapid

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55
 Loomis MD (1974) Overcoming problems of phenolics and quinines in
the isolation of plant enzymes and organelles. Methods Enzymol 31:528-
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 Murry MG and Thompson WF (1980) Rapid isolation of high molecular

weight plant DNA . Nucl Acids Res.8:4321-4325.

 Porebski S, Bailey LG and Baum BR(1997) Modification of a CTAB

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 Weishing K, Nybom H, wolff K and Meyer W (1995) DNA isolation and

purification in: DNA fingerprinting in plants and fungi ,PP 44-59.CRC
press. Boca Raton, Florida.

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 Chapter -8
Photo Gallery

57
DNA in UV-transluminatre

58
GOVT.T.R.SCOLLEGE
REWA
(M.P.)
(INDIA)
2017
Reported by
Name- Suraj Prakash Saket

Course-Master of Science in

Biotechnology

Roll No. 15233020

59
60