HERBAL DRUG STANDARDISATION,Role of biomarkers, Techniques of DNA Fingerprinting

2. PREPARED BY,
ROSHNI ANN BABY
M.PHARM PART I

3. 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.

4. Structural relationship among chromosomes, DNA
and genes
4
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.

6.  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.

7. 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

8. 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.

9.  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.

10. Chemoprofiling and biomarkers
Using 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.

11. 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.

12. 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.

13. Polymerase Chain Reaction
(PCR)
 Invented by KaryMullis 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.

15. Techniques used in DNA Fingerprinting
Microsatellites
 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.

16.  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.

17. 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.

18.  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.

21. 21
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.

22. 22
Outline of Electrophoresis
1. 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.

24. 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.

25. 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.

26.  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.

27.  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.

29. 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, germplasmcan 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.

30. 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 Panax
quinquefolius using amplified fragment length
polymorphism (AFLP) and directed amplification of
minisatellite region DNA (DAMD) J Agric Food Chem
50(7): 1871-1875.