Ayurveda The Ancient Indian Therapeutic Measure Biology Essay

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Ayurveda, the ancient Indian therapeutic measure is renowned as one of the major systems of alternative and complimentary medicine. As other herbal systems, greater parts of its medicaments are based on indigenous herbals. The thorough and fractionate knowledge about the medicinal plant is mandatory for all who is working in the field of ayurveda, in order to identify and select the appropriate plant for a specific disease. In the recent years, the interest in medicinal plants has increased a great deal. Apart form this people from the west have also taken this matter seriously by conducting various researches on plant based medicines.

"A medicinal plant is any plant which, in one or more of its organ, contains substance that can be used for therapeutic purpose or which is a precursor for synthesis of useful drugs." (Sofowora, 1982, Medicinal Plant and Traditional Medicine in Africa). This definition of Medicinal Plant has been formulated by World Health Organization (WHO).

The plants that possess therapeutic properties or exert beneficial pharmacological effects on the animal body are generally designated as "Medicinal Plants". Although there are no apparent morphological characteristics in the medicinal plants growing with them, they possess some special qualities or virtues that make them medicinally important. It has now been established that the plants which naturally synthesis and accumulate some secondary metabolites, like alkaloids, glycosides, tannins, volatile oils and contain minerals and vitamins, possess medicinal properties.

Medicinal plants constitute an important natural wealth of a country. They play a significant role in providing primary health care services to rural people. They serve as therapeutic agents as well as important raw materials for the manufacture of traditional and modern medicine. Substantial amount of foreign exchange can be earned by exporting medicinal plants to other countries. In this way indigenous medicinal plants play significant role in the economy of a country.

Medicinal plants, herbs, spices and herbal remedies are known to Ayurveda in India since long times. History of herbal remedies is very old. Since olden times before modern medicine, people became ill and suffered from various ailments. In the absence of modern medicinal remedies people relied on herbal remedies derived from herbs and spices. There are many medicinal herbs and spices, which find place in day-to-day uses, many of these are used as herbal remedies. Many cooked foods contain spices. Some minor ailments like common cold, cough, etc. may be cured by herbal remedies with use of medicinal properties of spices. Herbal remedies can be taken in many forms. Infusions are steeping herbs or spices, with parts like leaves and flowers with boiling water for some time. Filtered or unfiltered use this water extracts of spices as herbal remedies. Decoction is boiling roots, bark and hard parts of herbs and spices with water for along time. Infusion and decoction both are known as herbal teas. Some times essential oil of herbs and spices are also used as herbal remedies. Action of herbal remedies may vary from human to human and care should be observed in using it.

One such prominent medicinal plant genus is Solanum. Solanum, the nightshades, horsenettles and relatives, is a large and diverse genus of annual and perennial plants. They grow as forbs, vines, sub-shrubs, shrubs, and small trees and often have attractive fruit and flowers. Many formerly independent genera like Lycopersicon (the tomatoes) or Cyphomandra are included in Solanum as subgenera or sections today. Thus, the genus nowadays contains roughly 1,500-2,000 species.

Most parts of the plants, especially the green parts and unripe fruit, are poisonous to humans (albeit not necessarily to other animals), but many species in the genus bear some edible parts, such as fruits, leaves or tubers. Several species are cultivated, including three globally important food crops:

Tomato, S. lycopersicum

Potato, S. tuberosum

Eggplant, S. melongena

While most medical relevance of Solanum is due to poisonings which are not uncommon and may be fatal, several species are locally used in folk medicine, particularly by native peoples who have long employed them. Giant Devil's fig (S. chrysotrichum) has been shown to be an effective treatment for seborrhoeic dermatitis in a scientific study.

The tomato (Solanum lycopersicum, syn. Lycopersicon lycopersicum & Lycopersicon esculentum) is a herbaceous, usually sprawling plant in the Solanaceae or nightshade family that is typically cultivated for the purpose of harvesting its fruit for human consumption. Savory in flavor, the fruit of most varieties ripens to a distinctive red color. Tomato plants typically reach to 1-3 meters (3-10 ft) in height, and have a weak, woody stem that often vines over other plants. The leaves are 10-25 centimeters (4-10 in) long, odd pinnate, with 5-9 leaflets on petioles, each leaflet up to 8 centimeters (3 in) long, with a serrated margin; both the stem and leaves are densely glandular-hairy. The flowers are 1-2 centimeters (0.4-0.8 in) across, yellow, with five pointed lobes on the corolla; they are borne in a cyme of 3-12 together. It is a perennial, often grown outdoors in temperate climates as an annual.

Tomato plants are dicots, and grow as a series of branching stems, with a terminal bud at the tip that does the actual growing. When that tip eventually stops growing, whether because of pruning or flowering, lateral buds take over and grow into other, fully functional, vines. Most tomato plants have compound leaves, and are called regular leaf (RL) plants. But some cultivars have simple leaves known as potato leaf (PL) style because of their resemblance to that close cousin. Of regular leaves, there are variations, such as rugose leaves, which are deeply grooved, variegated, angora leaves, which have additional colors where a genetic mutation causes chlorophyll to be excluded from some portions of the leaves. Tomato fruit is classified as a berry. As a true fruit, it develops from the ovary of the plant after fertilization, its flesh comprising the pericarp walls. The fruit contains hollow spaces full of seeds and moisture, called locular cavities. These vary, among cultivated species, according to type. Some smaller varieties have two cavities, globe-shaped varieties typically have three to five, beefsteak tomatoes have a great number of smaller cavities, while paste tomatoes have very few, very small cavities.

The potato is a starchy, tuberous crop from the perennial Solanum tuberosum of the Solanaceae family (also known as the nightshades). The potato contains vitamins and minerals that have been identified as vital to human nutrition, as well as an assortment of phytochemicals, such as carotenoids and polyphenols. A medium-sized 150 g (5.3 oz) potato with the skin provides 27 mg of vitamin C (45% of the Daily Value (DV)), 620 mg of potassium (18% of DV), 0.2 mg vitamin B6 (10% of DV) and trace amounts of thiamin, riboflavin, folate, niacin, magnesium, phosphorus, iron, and zinc. The fiber content of a potato with skin (2 g) is equivalent to that of many whole grain breads, pastas, and cereals. Nutritionally, the potato is best known for its carbohydrate content (approximately 26 grams in a medium potato). The predominant form of this carbohydrate is starch. Potatoes contain toxic compounds known as glycoalkaloids, of which the most prevalent are solanine and chaconine. These compounds, which protect the plant from its predators, are generally concentrated in its leaves, stems, sprouts, and fruits.

The eggplant, aubergine, or brinjal (Solanum melongena), is a plant of the family Solanaceae (also known as the nightshades) and genus Solanum. It bears a fruit of the same name, commonly used as a vegetable in cooking. As a nightshade, it is closely related to the tomato and potato and is native to Bangladesh, Pakistan, Sri Lanka and India. It is a delicate perennial often cultivated as an annual. It grows 40 to 150 cm (16 to 57 in) tall, with large coarsely lobed leaves that are 10 to 20 cm (4-8 in) long and 5 to 10 cm (2-4 in) broad. The stem is often spiny. The flowers are white to purple, with a five-lobed corolla and yellow stamens. The fruit is fleshy, less than 3 cm in diameter on wild plants, but much larger in cultivated forms.

Solanum nigrum (European Black Nightshade or locally just "black nightshade", Duscle, Garden Nightshade, Hound's Berry, Petty Morel, Wonder Berry, Small-fruited black nightshade or popolo) is a species in the Solanum genus, native to Eurasia and introduced in the Americas and Australasia. Black nightshade is a fairly common herb or short-lived perennial shrub, found in many wooded areas, as well as disturbed habitats. It has a height of 30-120 cm (12-48"), leaves 4-7.5 cm (1 1/2-3") long) and 2-5 cm wide (1-2 1/2"); ovate to heart-shaped, with wavy or large-toothed edges; both surfaces hairy or hairless; petiole 1-3 cm (1/2-1") long with a winged upper portion. The flowers have petals greenish to whitish, surrounded with prominent bright yellow anthers. The berry is mostly 6-8 mm (1/4-3/4") diam., dull black or purple-black. The black ripe berry can be poisonous, but low toxicity variants are directly consumable and the leaves are cooked and consumed.

Solanum torvum (Turkey Berry), is a bushy, erect and spiny perennial plant. It is also known as Devil's Fig, Prickly Nightshade, Shoo-shoo Bush, Wild Eggplant, Pea Eggplant etc. The plant is usually 2 or 3 m in height and 2 cm in basal diameter, but may reach 5m in height and 8 cm in basal diameter. The shrub usually has a single stem at ground level, but it may branch on the lower stem. The stem bark is gray and nearly smooth with raised lenticels. The inner bark has a green layer over an ivory colour. The fruits are berries that grow in clusters of tiny green spheres that look like green peas. They become yellow when fully ripe. They are thin-fleshed and contain numerous flat, round, brown seeds. Turkey berry contains a number of potentially pharmacologically active chemicals including the sapogenin steroid, chlorogenin. Aqueous extracts of turkey berry are lethal to mice by depressing the number of erythrocytes, leukocytes and platelets in their blood. A related chemical, cholecalciferol, is the active ingredient in a number of commercial rodentacides. Extracts of the plant are reported to be useful in the treatment of hyperactivity, colds and cough, pimples, skin diseases, and leprosy. Turkey berry is being crossed with eggplant in an attempt to incorporate genes for resistance to Verticillium wilt into the vegetable.

These species in the Solanum genus was compared to study the genetic variability by employing the technique of DNA fingerprinting or DNA profiling. DNA fingerprinting, unlike the usual fingerprinting which is based on the morphological features and primarily restricted to humans is revealing the identity of an organism at the molecular level. In fact this is the technique of finding the genetic identity. This is primarily based on the polymorphisms occurring at the molecular level that is on the base sequences of the genome. The fundamental techniques involved in genetic fingerprinting were discovered serendipitously in 1984 by geneticist Alec J. Jeffreys of the University of Leicester in Great Britain while he was studying the gene for myoglobin. The technique crossed the arena of the scientific frontiers mainly with the application in the forensics. With advent of time, development of various techniques paved way for the use of this technique in different fields giving newer dimensions to this Technique. The DNA profiling is primarily used in plants for protection of biodiversity, identifying markers for traits, identification of gene diversity and variation etc. The most popular or widely used techniques used with relevance to plants are RFLP, RAPD, ISSR, SSR etc.

The basic methodology of DNA profiling in plants involve first the extraction of DNA from plant cells, quantification and quality assessment of extract. The further steps are of two types:

1) PCR based  -   RAPD, ISSR, SSR

2) Non PCR based - RFLP.

The PCR based techniques diluted DNA is mixed with a master mix comprising the PCR buffer, DNTPS, primer, water and the Taq polymerase enzyme in a PCR eppendorf tube. The mixture is loaded into the PCR. The PCR is pre-programmed for appropriate number of cycles and temperature variations depending on the technique.  After required cycles, the samples are subjected to electrophoresis, either AGE or PAGE, depending on the technique. The staining is done for revealing the banding pattern.


Sample collection and storage

Isolation of genomic DNA

Qualitative estimation by Agarose Gel Electrophoresis using Gel Documentation

Quantitative estimation by Nano Drop - Spectrophotometer

RAPD - PCR using random primers

Molecular marker analysis


The most often tested are those based on PCR primed with random oligonuclotides (RAPD), or with specific oligomers designed to simple sequence repeats (SSR) (Baird et al., 1992).

Another PCR based system with semi-random primers targeting the intron-exon splice junction (ISJ), proposed by Weining and Langridge and developed by Rafalski et al., also proved to be very useful for fingerprinting (Weining et al., 1991).

Total genomic DNA was extracted from fresh leaf tissue according to the modified CTAB method of Murry and Thompson (Murray et al., 1980).

RAPD markers are generally inherited as dominant/null alleles (Williams et al., 1990)

Solanum torvum Sw., a wild species related to eggplant (Solanum melongena L.), is an autogamous diploid species (2n = 2x = 24), native to India (Deb, 1979).

In most areas, S. torvum is considered as an invasive species. Its leaves and fruits, which are rich in alkaloids, can however be used for medicinal or ritual purposes (Lans et al., 2001).

S. torvum has also been identified as a potential source of resistance to bacterial wilt, caused by Ralstonia solanacearum, one of the most important plant bacterial diseases (Hébert, 1985).

Bacterial wilt is found on all five continents, and in almost all tropical, subtropical and temperate zones (Hayward, 1991).

Outbreaks of the disease bacterial wilt have moreover recently been observed in colder climates, such as in Europe (Olsson, 1976).

In Reunion Island, 3 different populations of R. Solanacearum have been described: race 1 biovar 1 (the rarest); race 1 biovar 3 (the most common type), and race 3 biovar 2 (Girard et al., 1993).

Over 50 botanical families, represented by more than 200 plant species are concerned by this disease (Hayward, 1994).

The wild species S. phureja and S. stenotomum were identified as possible sources of resistance for potato (S. tuberosum), Lycopersicon pimpinelifolium for tomato (L. esculentum), and S. torvum and S. aethiopicum for eggplant (S. melongena) (Rowe et al., 1972).

Grafting of susceptible tomato or eggplant varieties onto S. torvum rootstocks has been reported as a means of controlling bacterial wilt disease (Peregrine et al., 1982).

Embryo rescue was used to circumvent sexual incompatibility (Bletsos et al., 1998).

Traits of bacterial wilt resistance have successfully been introduced from S. torvum into eggplant by using somatic fusion of protoplasts (Collonnier et al., 2003).

Therefore, this study was initiated to determine the levels of resistance against three local races of R. solanacearum in S. torvum accessions collected in Reunion Island. Moreover, RAPD markers were used to assess genetic diversity for these accessions, in reference to accessions collected in Java Island (Indonesia) (Williams et al., 1993).

Low levels of polymorphism were reported using RAPDs in tomato or eggplant (Williams et al., 1993).

The use of AFLP markers to discriminate two tomato lines revealed that AFLP markers appeared more efficient than RAPD markers, as also shown in eggplant, but did not reveal more polymorphism (Mace et al., 1999).

Given the strong homologies between the genomes of Solanaceae species, AFLP markers might therefore not be more polymorphic than RAPDs in S. torvum but should improve resolution power by providing more scorable markers (Doganlar et al., 2002).

I-SSR markers have also been shown to be powerful polymorphic markers, as they benefit from the length polymorphism of the SSR loci used as primers (Zietkiewicz et al., 1994).

I-SSR failed to distinguish clementine varieties and could therefore be unsuccessful in revealing polymorphism in S. torvum, as shown from preliminary experiments (Sihachakr D, pers. com.) (Bretò et al., 2001).

Weedy species introduced by man to new areas often exhibit reduced genetic variation due to marked founder effects (Husband et al., 1991).

A genetic study using 491 AFLP markers showed the complete absence of intra and inter area variability in these introduction areas, whereas high levels of genetic diversity were revealed in its area of origin (Asia) (Amsellem et al., 2000).

In many countries, S. torvum fruits are dispersed by birds (D'Arcy, 1974).

A total of 198 scorable fragments were amplified using 10 random amplified fragment length polymorphism and Inter Simple Sequence Repeat-Polymerase Chain Reaction have been developed in the last decade; they do not require prior investments in terms of sequence analysis, primer synthesis or characterization of DNA probes (Zietkiewicz et al., 1994).

RAPD and ISSR-PCR are the most favoured methods for DNA fingerprinting largely because of the relative ease with which the techniques can be practiced in any molecular biology laboratory (Milbourne et al., 1997).

Several other factors including concentration of primer, template DNA and Mg -~§ ions in the reaction mixture, type of thermal cycler and PCR tubes are found to influence RAPD profiles (Weising et al., 1995).

However, high repeatability can be achieved by standardization of the above components (Smith & Williams, 1994).

Total genomic DNA from 20 commercial Indian potato cultivars was extracted from young leaf samples by a modified CTAB procedure (Doyle & Doyle, 1987).

Quantity and quality of DNA preparations were checked by standard spectrophotometry and the samples were diluted to 25 ng DNA/lal concentrations (Ausubel et al., 1995).

The polymerase chain reaction was performed in a reaction volume of 25 lal containing lx Taq DNA polymerase buffer with 1.5 mM MgCI~ (Perkin Elmer), 100 /aM of each dNTP (Promega), 25 pmole random primer (Operon Technology, USA), 100 ng genomic DNA and 1.0 unit Taq DNA polymerase (AmpliTaq, Perkin Elmer) (Nadeau et al., 1992).

Band informativeness (Ib) was represented into a 0-1 scale by the formula: Ib=l-(2 x 10.5-

p!), where p is the proportion of the 20 samples containing the band (Prevost, et al., 1999).

A similarity matrix was generated on NTSYSpc 2.0 h using Dice coefficient (Dice, 1945).

The number of fragments produced by different primers ranged from 6 (OPD-04) to 31 (OPD-03). But more fragments per primer were detected in our experiment in comparison with earlier reports (Hosaka et al., 1994).

Usually 20--50% of the random primers used in RAPD analysis do not give rise to any PCR products (Caetano-Anolles, 1994).

With multiplex RAPD, more amplified fragments are usually expected (Micheli et al., 1993).

Brinjal, eggplant or aubergine (Solanum melongena L.) is widely cultivated as vegetable in both temperate and tropical areas, especially in Asia. In India, it is also used for the treatment of diabetes, bronchitis, asthma, dysuria, dysentery, etc (Daunay, et al., 2000).

In African countries S. aetheopicum group gilo and S. anguri are used for the treatment of many diseases. Many other Solanum species are also used for medicinal Purposes (Bukenya, et al., 1999).

It is particularly useful for characterizing individual accessions and cultivars and as a general guide in the selection of the parents for hybridization. Several workers have contributed to the characterisation of the largest genus of Solanaceae family (Correll, et al., 1962).

Great degree of taxonomic confusion exists as regard to genus Solanum (Daunay, et al., 1988).

DNA-based markers provide powerful tools for discerning variations within crop germplasm and for studying evolutionary relationships (Gepts, 1993).

Among molecular markers, random amplified polymorphic DNAs (RAPDs) have been extensively used in genetic research owing to their speed and simplicity (Williams, et al., 1990).

Most variability/taxonomic affinity studies in eggplant have focused mainly on morphology, crossability, anatomy, isozyme and chloroplast DNA diversity (Welsh, et al., 1989).

Limited work has been done so far with nuclear DNA diversity. Greater DNA polymorphism has been reported in weedy S. insanum than in advanced cultivars of eggplant (Karihaloo, et al., 1995).

In another study AFLP was found to be an excellent tool for the determination of genetic relationship among the species of Solanum (Mace, et al., 1999).

India or Indochina is the centre of eggplant diversity but the affinities of S. melongena to related species are uncertain (Lester, et al., 1991).

Taxonomic uncertainties exist because phylogenetic relationships among taxa have been established considering mainly the morphological features, crossability and F1 fertility (Baksh, et al., 1979).

Establishing genetic affinities on such parameters are insufficient, as S. melongena makes successful cross with putative progenitors as well as distantly related species (Daunay, et al., 1991).

Reactions products were mixed with 2.5 ml of 10X loading dye (0.25% bromophenol blue,

0.25% xylene cyanol and 40% sucrose, w/v) and spun briefly in a micro centrifuge before loading (Sambrook, et al., 1989).

Data were scored as discrete variables, using 1 to indicate presence and 0 to indicate absence of the band. A pairwise different matrix between genotypes was determined using Jaccards similarity coefficient and the average taxonomic distance (Jaccard, 1908).

High degree of diversity of species belonging to Solanum may be attributable to the fact that it is an ancient plant (Whalen, 1979).

RAPD and other discontinuous markers can serve as a means of genetic distances to establish phylogenetic relationships among taxa (Rodriguez, et al., 1999).

The present results and those obtained by others are not in agreement with the earlier workers who studied variation among the cultivated and weedy taxa of S. melongena by allozymes and RAPD analysis (Furini, et al., 2004).

A high degree of variation has also been reported by using AFLP technology for S. melongena with weedy relative of the cultivated eggplant (Mace, et al., 1999).

At the species level S. melongena (cultivable type) is more closely related to S. incanum followed by S. viarum whereas S. surattence and S. nigrum showed a closer association among themselves in comparison with the cultivated type. Wild forms of S. incanum are regarded as belonging to the same species as S. Melongena (Karihaloo, et al., 1994).

Hybridization examples with eggplant show that S. melongena is crossable with several species and to a certain degree, also with other sections (Decondolle, 1904).

It will be worth to investigate specific traits in the wild species and they may be introgressed by sexual crossing or somatic hybridization into commercial varieties of S. melongena (Furini, et al., 2004).

Solanum torvum Sw., a wild species related to eggplant (Solanum melongena L.), is an autogamous diploid species (2n = 2x = 24), native to India (Deb, 1979).

In most areas, S. torvum is considered as an invasive species. Its leaves and fruits, which are rich in alcaloids, can however be used for medicinal or ritual purposes (Lans, et al., 2001).

S. torvum has also been identified as a potential source of resistance to bacterial wilt, caused by Ralstonia solanacearum, one of the most important plant bacterial disease (Hébert, 1985).

Bacterial wilt is found on all five continents, and in almost all tropical, subtropical and temperate zones (Hayward, 1991).



The plant species are collected from Department of Horticulture, Gandhi Krishi Vighyan Kendra (GKVK), Bangalore, India. The collected samples were stored in sterilized zip bags and kept at -20oC inside deep freezer.

The plant samples are:

Solanum tuberosum

Solanum nigrum

Solanum melongena

Solanum lycopersicum

Solanum torvum



Mix 350 µl of extraction buffer with 350 µl of 8M LiCl and pre-warm to 65°C.

Weigh 0.1g of the fresh leaf sample. Grind the fresh leaf sample with mortar and pestle. Transfer the sample to micro centrifuge tubes and add the pre warmed isolation buffer.

Vortex thoroughly and place the tube at 65°C.

Hold the tube at 65°C for 10 min and vortex 3-4 times during the incubation.

Add 700 µl chloroform: IAA (24:1) and mix thoroughly.

Centrifuge the tube at 13,000 rpm (13,793 g-force) for 5 min at room temperature.

Transfer the upper phase into a new tube and extract again with 700 µl chloroform-IAA and centrifuge.

Pipet the upper phase into a new tube. Add 0.5 vol (300 µl) 3M potassium acetate (pH4.8), mix by inverting the tube carefully.

Place the tube at -20°C for 30min.

Centrifuge the tube at 13,000 rpm (13,793 g) for 10min at 4°C and pipet the supernatant into a new tube.

Add 0.6 vol (600 µl) cold isopropanol and invert the tube carefully several times to mix the 2 layers.

Place the tube at -20°C for 30 min.

Centrifuge the tube at 13,000 rpm (13,793 g) for 10 min at 4°C.

Discard the supernatant.

Add 2vol (600 µl) cold absolute ethanol and place the tube at -20°C for overnight.

Centrifuge the tube at 13,000 rpm (13,793 g) for 10 min at 4°C and wash the DNA pellet with cold 70% ethanol (300 µl), centrifuge at 13,000 rpm (13,793 g) for 5 min at 4°C.

Dry the DNA pellet and dissolve in 30-50 µl 1X TE buffer.

This DNA sample was stored at 4oC to ensure its viability.

Check the quality and concentration of the DNA with a spectrometer and on a 0.8% agarose gel.



Agarose gel electrophoresis is a procedure used to separate DNA fragments based on their molecular weight and is an intrinsic part of almost all routine experiments carried out in molecular biology.

The technique consists of three basic steps - preparation of agarose gel, electrophoresis of DNA fragments and visualization.

Agarose is a linear polymer extracted from seaweeds. Purified agarose is a powder insoluble in water or buffer at room temperature but dissolves on boiling. Molten solution is then poured in to a mould and allowed to solidify as it cools agarose undergoes polymerization i.e. sugar polymers cross-link with each other and cause the solution to gel, the density or pore size of which I determined by concentration of agarose.

Electrophoresis is a technique used to separate charge in molecule.DNA is negatively charged at neutral pH and electric field is applied across the gel, DNA migrates towards the anode. Migration of DNA through gel depends up on molecular size and conformation of DNA, agarose concentration and applied current.

Matrix of agarose gel acts as a molecular sieve through which the DNA fragments move on application of electric current. Higher concentration of agarose gives firmer gels, i.e. spaces between cross-linked molecules is less and hence smaller DNA fragments easily pass through these spaces. As the length of DNA increases, it becomes harder for the DNA to pass through the spaces, while lower concentration of agarose helps the movements of larger DNA fragments as the spaces between the cross-linked molecules is more. The progress of gel electrophoresis is monitored by observing the migration of a visible dye (tracking dye) through the gel. Two commonly used dyes are xylene cyanol and bromophenol blue that migrate at the same speed as double stranded DNA of size 5000 bp and 300 bp respectively. These stacking dyes are negatively charged, low molecular weight compound that are loaded along with each sample at the start of run, when the tracking dye reaches towards the anode, run is terminated.

Since DNA is not naturally coloured, it will not be visible on the gel. Hence we use an intercalating agent like ethidium bromide (EtBr) is added to the agarose gel and the location of bands determined by examining the gel under UV transilluminator.


Prepare 1X TAE by diluting appropriate amount of 50X TAE buffer.

Weigh 0.24 g of agarose and add to 30 ml of 1X TAE. This gives 0.8% agarose gel.

Boil till agarose dissolves completely and a forms a clear solution.

Meanwhile place the combs carefully in to the boat and make it ready.

Add 10 μl EtBr per 100 ml of agarose gel once the temperature of the agarose solution reaches 60oC.

Pour the agarose gel in the central part of the boat and note that air bubbles are not generated.

Keep the gel undisturbed at room temperature for the agarose to solidify.

Pour 1X TAE buffer into the tank.

Gently lift the comb from the boat, ensuring that the wells remain intact.

Then the solidified agarose with the boat was carefully placed in to the tank. Note that the buffer level stands at 0.5 to 0.8 cm above the gel surface.

Take 6 μl of DNA sample which was mixed with 4 μl of tracking dye. Load 10 μl of this mixture into the wells carefully.

Connect the power cords to the power pack and set the voltage to 50 V for 20 minutes and then change the voltage to 100 V for 30 minutes.

Take out the gel from the tank and was viewed under UV transilluminator for the presence of the DNA



Quantitative analysis was done using Nanodrop - spectrophotometry. The sample showing 1.6 to 1.8 OD indicates the purest form of DNA, increased or decreased OD value indicates contamination (proteins or RNA). The instrument used for this estimation is Nanodrop - 1000.


Switch on the system.

Click ND -1000 software icon.

Select Nucleic acid button on the software.

Place 1 µl of distilled water on the pedestral to initiate the instrument.

Place 1 µl of TE buffer to the pedestral and click re-blank.

Place the DNA samples (1 μl) on the pedestral and select measure button to analyse the results, the concentration profile was obtained from the nanodrop readings.


In 1991 Welsh and McClelland developed a new PCR-based genetic assay namely randomly amplified polymorphic DNA (RAPD). This procedure detects nucleotide sequence polymorphisms in DNA by using a single primer of arbitrary nucleotide sequence. In this reaction, a single species of primer anneals to the genomic DNA at two different sites on complementary strands of DNA template. If these priming sites are within an amplifiable range of each other, a discrete DNA product is formed through thermocyclic amplification. However, due to the stoichastic nature of DNA amplification with random sequence primers, it is important to optimize and maintain consistent reaction conditions for reproducible DNA amplification. RAPDs are DNA fragments amplified by the Polymerase Chain Reaction (PCR) using short (generally 10 bp) synthetic primers of random sequence. Because of the short primers used, the reannealing temperature in the PCR must be low (35-40oC) for the primer to bind. However, due to low temperature, the binding is not very specific, which means that primers will bind also to sequences which are not completely complementary. These oligonucleotides serve as both forward and reverse primer and usually are able to amplify fragments from 3-10 genomic sites simultaneously. Amplified fragments (within the 0.5-5 kb range) are separated by gel-electrophoresis and polymorphisms are detected as the presence or absence of bands of particular size. These polymorphisms are considered to be primarily due to variation in the primer annealing sites.

The application of RAPDs and their related modified markers in variability analysis and individual-specific genotyping has largely been carried out, but is less popular due to problems such as poor reproducibility faint or fuzzy products, and difficulty in scoring bands, which lead to inappropriate inferences.

In RAPD analysis the total DNA from an organism is mixed with (generally) 10 bp single stranded DNA (primer) together with the four different deoxynucleotides and a heat stable DNA polymerase enzyme. The reaction mixture is placed on a thermocycler (PCR-machine) which can change the temperature of the reaction rapidly according to a predefined PCR programme.

Reannealing at low temperature makes the primer molecules attach to complementary site on the DNA. Elongation is the period when the polymerase enzyme elongates the attached primers from their 3' end. Denaturation separates the new formed strands.

After a few PCR cycles DNA pieces with well defined length similar to the one between the two original primer sites dominate the mixture. This is because they amplify exponentially. In most RAPD reactions the result will be amplification of 5 to 10 different DNA segments whose lengths depend on the primer recognition sites available in the genome. The DNA after amplification is visualized for bands using Agarose Gel Electrophoresis (AGE). The band scoring is done by comparing with the marker bands.

Polymerase Chain Reaction (PCR):

The DNA amplification by thermal cycling called Polymerase Chain Reaction is in vitro method that can be used to amplify a specific DNA segment from small amounts of DNA template or duplex into millions of copies.  It is invented by Kary Mullis et al., (1985).

Steps involved in PCR are:

Heat Denaturation


Primer Extension

Heat Denaturation:

This temperature denatures the double stranded DNA into two individual strands. Denaturation temperature is 95oC for 30 seconds or is 97oC for 15 seconds, however higher temperature may be appropriate, especially for G+C rich nucleotides. 


During this time one primer binds with the 5 prime end of one DNA strand and the other primer binds with 3 prime end of its complementary strand. Annealing is hybridization of primers to single stranded DNA and the length of time required for primer annealing depends on the basic composition, length and concentration of primers.

Primer Extension:

This temperature is varies for Taq DNA polymerase which adds complementary nucleotides one by one to the 3' OH group of the primer. Estimates for the rate of nucleotide incorporation at 72oC vary from 35-100 nucleotides per second depending upon the buffer, pH salt concentration and nature of DNA template.

Number of PCR Cycles:

The optimum number of cycles depends mainly upon the starting concentration of target DNA, when other parameters are optimized.  A common mistake is to execute too many cycles. The increase in the number of cycles will increase the amount and complexity of non-specific background products.


In the present study, random primers such as OPU (5, 6, 7, 8, & 9) and OPU (15, 16, 17, 18 &19) were used.


The components were taken to match the final concentration of 25 μl. The components include:

Assay buffer (10X)

dNTP's (10mM)

Taq (3U/μl)


DNA (100 ng/μl)

Nuclease free water


All the above mentioned components were taken in a PCR tube and placed in a thermocycler by following conditions were set to run the PCR

Step-1: (1 repeat)

Initial denaturation at 94°C for 4 minutes

Step-2: (30 repeats)

Final denaturation at 94°C for 1 minute

Annealing temperature is 35°C for 50 seconds

Initial Extension at 72°C for 1 minute

Step-3: (1 repeat)

Final extension at 72°C for 5 minutes

Storage at 4 °C


The amplified products were resolved by electrophoresis in 1.5% of agarose gel using 1x TAE buffer (appendix 5) at 50volt for 2 ½ hours .A 100 basepair ladder was included as molecular size marker. Gels were visualized by staining with Ethidium bromide (1µl/10ml) and banding patterns were photographed over UV light.


Prepare 1X TAE by diluting appropriate amount of 50X TAE buffer.

Weigh 0.90 g of agarose and add to 60 ml of 1X TAE. This gives 1.5% agarose gel.

Boil till agarose dissolves completely and a forms a clear solution.

Meanwhile place the combs carefully in to the boat and make it ready.

Add 10 μl EtBr per 100 ml of agarose gel once the temperature of the agarose solution reaches 60oC.

Pour the agarose gel in the central part of the boat and note that air bubbles are not generated.

Keep the gel undisturbed at room temperature for the agarose to solidify.

Pour 1X TAE buffer into the tank.

Gently lift the comb from the boat, ensuring that the wells remain intact.

Then the solidified agarose with the boat was carefully placed in to the tank. Note that the buffer level stands at 0.5 to 0.8 cm above the gel surface.

The reaction mixture has a total volume of 25 μl, to this mixture add 10 μl of tracking dye and mix well. Load 15 μl of this mixture into the wells carefully.

To the first well 2 μl ladder was loaded and DNA samples were loaded in the remaining wells respectively.

Connect the power cords to the power pack and set the voltage to 50 V for 180 minutes.

Take out the gel from the tank and was viewed under UV transilluminator for the presence of the DNA.

Phylogenetic variation were determined by converting RAPD data into a frequency similarity and analysed by Unweighted Pair Group Method with Arithmetic mean (UPGMA) cluster analysis to produce a phylogenetic tree.