PREPARED BY,

ROSHNI ANN BABY

M.PHARM PART I

Introduction Deoxyribonucleic acid (DNA) is the fundamental

building component of all living cells.

Our characteristics, traits and physical features are determined by the specific arrangement of DNA base-pair sequences in the cell.

It is this distinct arrangement of adenine, guanine, thymine and cytosine (called DNA nucleotides) that regulates the production of specific proteins and enzymes via the Central Dogma Theory.

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Structural relationship among chromosomes, DNA and genes

1. Cells are the basic building blocks of all living things.

2. In a cell, DNA (deoxyribonucleic acid) is packaged in chromosomes within the nucleus.

3. DNA is a large, polymeric molecule.

4. A gene is a segment of DNA molecule of a chromosome. It is the basic unit of heredity. Genes determine the body characteristics of an organism.

5. Each inherited characteristic is controlled by one or several genes.

DNA arrangement is uniform throughout the organism, irrespective of the organ. If the DNA from the hair, organs or any body fluid such as blood, saliva or semen, of a particular organism were analyzed, the result would be similar profiles from each.

This profile is as unique as a fingerprint; it is specific to that individual.

APPLICATIONS To identify and indict suspects in criminal

investigations.

As a legal tool to determine parentage.

Used in combination with forensic and medical evidence.

To determine the ancestry of plants, animals and other microorganisms and to identify variation between strains.

Eg:fraudulent adulteration of Chianti wines with inferior quality grapes

In standardization of herbal drugs The varying drug content of different species of herbal

plants.

A particular plant from a region can be linked to a specific drug content and thus have a therapeutic value assigned to it.

Factors such as soil, climate and adaptability affect its drug content.

In such cases, there are observed variations in the genetic composition of the plant.

Two factors affect the final drug quality:

(1) The variability with respect to strain-specific drug content;(2) The potential adulteration of plant drugs with extracts from plants that have lower drug content.

Typical Eg: The bark of Cinchona grown in the plains contains quinine, which is therapeutically active. The same species of tree grown on hilltops and slopes looks morphologically similar but has no active quinine.

Chemoprofiling and biomarkersUsing chemical fingerprinting, plants can be demarcated

on the basis of their species, strain and geographical origin.

Using chromatographic techniques like (HPTLC) and (HPLC), a profile of their various chemical constituents is obtained. This is called chemoprofiling.

Chemical constituents are isolated based on their affinities for particular organic solvents in an increasing order of polarity. They are resolved using suitable colouring reagents, resulting in characteristic patterns.

The compound specific to that species (sterol, terpenoid, alkaloid, etc.) is characterized as a chemical marker. Some examples of this are ginsengosides for ginseng and hypericin for St. John’s wort.

Demerits of chemical fingerprinting Not all plants contain a unique chemical compound.

Even if there is a unique marker, it may not be biologically active. There is a significant overlap of many molecules, especially phenolics and sterols.

Additional techniques are required to profile natural drugs, particularly when profiling the genotypic differences.

Factors affecting DNA fingerprinting sequence or restriction site data

taxonomic level of study

the level at which the study is being done (species, genera, etc.)

robustness and reproducibility of the method

effectiveness in terms of cost and time

availability of DNA.

Polymerase Chain Reaction (PCR) Invented by Kary Mullis in 1983

PCR is a method used to generate billions of copies of genomic DNA within a very short time. This amplification is useful in criminal cases where there are miniscule amounts of DNA available.

Today PCR finds application in almost all aspects of biomedicine. PCR has been used for the detection of many pathogenic organisms, from bacteria to viruses.

Techniques used in DNA FingerprintingMicrosatellites

Simple sequence repeats (SSRs), 1 to 6 nucleotides in length, which show a high degree of polymorphism. Specific microsatellites can be isolated using hybridized probes followed by their sequencing.

Like any DNA fragment, SSRs can be detected by specific dyes or by radiolabelling using gel electrophoresis.

The advantage of using SSRs as molecular markers is the extent of polymorphism shown, which enables the detection of differences at multiple loci between strains .Coupled with chemical and morphological data, we can identify the plant species or strain of interest.

The main advantage of using SSRs for fingerprinting is that small amounts of DNA are required compared to the restriction fragment length polymorphisms (RFLP) method. This is due to the large amounts of SSRs present in any genome.

Further, assays involving SSRs are more robust than random amplified polymorphic DNA (RAPDs), making them up to seven times more efficient.

A drawback to using SSRs is the need to develop separate SSR primer sets for each species. The latest research suggests that SSRs will be involved in new methods of detection of alterations of specific sequences in the DNA.

Restriction fragment length polymorphisms

Unequal lengths of DNA fragments obtained by cutting Variable Number of Tandem Repeat (VNTRs) sequences up to 30 sequences long with restriction enzymes at specific sites.

VNTRs vary between plant species, as do the number and location of restriction enzyme-recognition sites.

On an agarose gel, RFLPs can be visualized using radiolabeled complementary DNA sequences.

There is no need for PCR amplification of DNA in this method. A routine southern blot experiment is used instead.

Normally, RFLPs are used to identify the origins of a particular plant species, setting the stage for mapping its evolution.

There are some problems with the RFLP method of DNA fingerprinting. First, the results do not specifically indicate the chance of a match between two organisms. Secondly, the process involves a lot of money and labor, which not many laboratories can afford. Finally, unlike the microsatellites, a few loci in the assay must suffice.

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Electrophoresis

DNA fragments of different sizes can be separated by using gel electrophoresis.

DNA fragments carry negative charges. When there is a current flow, DNA fragments move towards the anode.

Source: Science Education Section,

CDI, EDB.

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Outline of Electrophoresis1. DNA fragments are loaded into

“wells” in a gel. The gel floats in a buffer solution within a chamber between two electrodes.

2. When an electric current is passed through the chamber, negatively charged fragments move towards the positive terminal.

3. Shorter DNA fragments (smaller size) move faster than the longer ones (bigger size).

4. In a given time, DNA fragments are separated into bands according to their size.

“wells”

Source: Science Education

Section, CDI, EDB.

Source: Science Education

Section, CDI, EDB.

Amplified fragment length polymorphism (AFLP)

A PCR-based derivative method of RFLP in which sequences are selectively amplified using primers. It is a reliable and efficient method of detecting molecular markers.

DNA is cut with two restriction enzymes to generate specific sequences, which are then amplified suitably. The mere addition or deletion of bases at the 3′ end determines the selectivity and complexity of the amplification 4.

By using AFLP, it is possible to evaluate more loci than with RFLP or RAPD. AFLP is also capable of determining a large number of polymorphisms. Similar to SSRs, AFLP-based assays are cost-effective and can be automated.

Random amplified polymorphic DNA is

One of the most commonly used primary assays for screening the differences in DNA sequences of two species of plants.

RAPD consists of fishing for the sequence using random amplification. Here, plant genomic DNA is cut and amplified using short single primers at low annealing temperatures, resulting in amplification at multiple loci.

By running a 2-dimensional electrophoresis gel, it is possible to determine the change in sequence pattern by superimposing the 2 gels. Once the band of interest is identified, the gel is cut, and the DNA is isolated and sequenced.

Using this target, DNA from other cultivars can be assessed using other techniques such as AFLP or SSRs. It is also more cost effective than RFLPs. RAPDs lack specificity, however, due to low annealing temperatures and easier reaction conditions.

Other Methods include the use of single nucleotide polymorphs (SNPs), DNA amplification fingerprinting (DAF) and their offshoots. Although these techniques vary slightly from each other, they operate on the same principle.

CONCLUSION DNA fingerprinting, apart from identifying alterations

in the genotypes of plant species, is also used for the betterment of drug-yield by tissue culturing.

DNA of interest can be stored as germplasm, which is then used for future cultivation.

In addition, germplasm can be used for the conservation of selected plant species, which are endangered such as Rauwolfia serpentina (Snake Root).

DNA fingerprinting of herbal drugs, though still in its early years, seems to be a promising tool for the authentication of medicinal plant species and for ensuring better quality herbs and nutraceuticals.

References 1. Breithaupt, H. (2003) Back to the roots EMBO Rep

4(1): 10-12.2. Mihalov, J. J., Marderosian, A. D., and Pierce, J. C. (2000) DNA identification of commercial ginseng samples J Agric Food Chem 48(8): 3744-3752.3. Henry, R J. (2001) Plant Genotyping: The DNA fingerprinting of Plants, CABI Publishing, New York.4. Ha, W. Y., Shaw, P. C., Liu, J., Yau, F. C., and Wang, J. (2002) Authentication of Panax ginseng and Panaxquinquefolius using amplified fragment length polymorphism (AFLP) and directed amplification of minisatellite region DNA (DAMD) J Agric Food Chem50(7): 1871-1875.