DNA FINGERPRINTING TECHNIQUE FOR IDENTIFICATION OF DRUGS OF NATURAL ORIGIN ANG MICROBIAL CONTAMINATION

DNA FINGERPRINTING TECHNIQUE FOR IDENTIFICATION OF DRUGS OF NATURAL ORIGIN ANG MICROBIAL CONTAMINATION

• 1.
  SRIKRUPA INISTITUTE OFPHARMACEUTICAL SCIENCES (Approved by AICTE; PCI) (Affiliated to Osmania University) ASSIGNMENT ON DNA fingerprinting technique in identification of drugs of natural origin, Microbial contamination in herbal formulation SUBMITTED BY HUZAIFA NAAZ H.TNO:636217885002 DEPARTMENT OF PHARMACEUTICAL ANALYSIS
• 2.
  DNA Fingerprinting  Bar-codelike patterns generated by amplification of chromosomal DNA of an individual which can distinguish the uniqueness of this individual from another.  Also called DNA typing, genetic fingerprinting, DNA profiling and DNA typing.  DNA fingerprinting important tool in herbal drug standardization useful for the identification of genuine drug from substituted or adulterated drug.  DNA fingerprint genome remain the same irrespective of the plant part used while the phytochemical constituents will vary with the part of plant  DNA sequences are highly specific, and can be identified with molecular markers, which can find out a particular sequence of DNA from a group of unknown. Types of DNA Based Markers: These are hybridization based methods, polymerase chain reaction (PCR) based methods and sequencing based methods. Hybridization based:  Restriction Fragment Length Polymorphism (RFLP)  Variable Number Tandem Repeat (VNTR) PCR based:  Inter Simple Sequence Repeat (ISSR)  Random Amplification Polymorphic DNA (RAPD)/Arbitrary Primed PCR  Amplified Fragment Length Polymorphism (AFLP)  DNA Amplification Fingerprinting (DAF)  Simple Sequence Repeats (SSR) Sequence based:  Sequence Characterized Amplified Region (SCAR)  Cleaved Amplified Polymorphic Sequence (CAPS)  Single Nucleotide Polymorphism (SNP). Hybridization-based methods: It uses cloned DNA elements or synthetic oligonucleotides as probes to hybridize the DNA of interest. The probes are labeled with radioisotopes or with conjugated enzymes which catalyze a color reaction to detect hybridization.
• 3.
  Hybridization based methodsinclude following steps  DNA is first extracted from plant cell by various methods of extraction such as Cetyl triethyl ammonium bromide (CTAB), Phenol/ Chloroform extraction, DNA trap methods etc.  DNA is then cleaved with restriction enzyme then it is subjected to gel electrophoresis.  As DNA molecules have net negative charge, at neutral PH, these molecules migrate towards the positive terminal when placed in an electric field in a process known as electrophoresis which is performed in an agarose or polyacrylamide gel.  Nucleic acids are loaded into slots in the gel and allowed to migrate towards the positive terminal. The pores in the gel act to sieve the molecules. So that mobility of a particular nucleic acid species depends on its length.  All the molecules of a particular size move at approximately the same rate through the gel, forming a band, which gradually increases in width during electrophoresis because of diffusion. After gel electrophoresis, DNA sequence is determined by Southern blotting technique. DNA molecules separated by gel electrophoresis are transferred to nitrocellulose or nylon membrane  The DNA is denatured either prior to or during transfer by placing the gel in an alkaline solution, after which it is immobilized on the membrane by drying or UV induced cross linking to the filter.
• 4.
   A radioactiveDNA known as probe which are of two types (Tandem repeats which occurs as clusters among chromosomes and Dispersed repeats which are scattered all over chromosomes) containing the sequence of interest are then hybridized or annealed with the immobilized DNA on the membrane.  The probe will anneal to form a double helix only with complementary DNA molecule on the membrane  Nonannealed probe is then washed off the membrane, and the washed membrane is exposed to X-ray film that detects the presence of the radioactivity in the bound probe, after which autoradiogram is developed where the dark bands show the positions of DNA sequences that has hybridized with the probe. Hybridization-based methods include a) Restriction Fragment Length Polymorphism (RFLP) b) Variable Number Tandem Repeats (VNTR) Restriction Fragment Length Polymorphism (RFLP):  RFLPs involves fragmenting a sample of DNA by a restriction enzyme, which can recognize and cut DNA wherever a specific short sequence ( 4-6 base pair recognition site) occurs, in a process known as a restriction digest.  The resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis, and transferred to a membrane via the Southern blot procedure.  Hybridization of the membrane to a labeled DNA probe then determines the length of the fragments which are complementary to the probe.
• 5.
  Variable Number TandemRepeats (VNTR)  This technique is similar to RFLP only the probe used for southern blotting exists as tandem repeats which occur as cluster among chromosomes.  They show variations in length between individuals and each variant acts as an inherited allele Polymerase chain reaction based methods:  PCR enzymatically multiplies particular DNA sequence or loci of a template DNA with the help of arbitrary or specific oligonucleotide primers PCR based method involve following steps: Primers, original DNA (extracted from the plant cell) which is to be amplified, a specific type of DNA polymerase, and the necessary chemicals for DNA synthesis are mixed. Then following steps are carried out. 1. Denaturation: DNA fragments are heated at high temperature (950C for 30 seconds or 970C for 15 seconds) which reduces DNA double helix to single helix to single strand which become accessible to primer. 2. Annealing: Here the temperature is lowered until the primers can hybridize, or bind to complementary regions on the DNA. 3. Extension: The primers are used by DNA polymerase to initiate synthesis and new complementary strand of DNA are made.  The enzyme read opposing strand sequence and extends the primer by adding nucleotide in order in which they can pair Once this synthesis is finished, the steps are repeated i.e. denaturation, renaturation, and synthesis. However problems can occur when the plant material of interest contains compounds such as phenolics which can interact with DNA and directly inhibit DNA polymerase or damage the structural integrity of DNA.
• 6.
  Various PCR basedtechniques include: a) Randomly Amplified Polymorphic DNA (RAPD) b) Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) c) Amplified Fragment Length Polymorphism (AFLP) d) Simple Sequence Repeats (SSR) Randomly Amplified Polymorphic DNA (RAPD) and Arbitrarily Primed Polymerase Chain Reaction (AP- PCR):  These methods are most commonly used for primary assay which helps in screening the differences in DNA sequence of two species of plants.  In this method single arbitrarily chosen oligonucleotide is used as both the forward and reverse primer in PCR reaction.  This sequence consists of about 10 nucleotide in case of RAPD and about 20 nucleotides in case of AP-PCR. Product is produced when the primer binds on opposite strands, in the reverse orientation and within an amplifiable distance. PCR fragments are generated from different locations of the genome, because there are multiple sites within the genome for the primer to bind.  Thus, multiple loci may be examined simultaneously. Use of series of different primers, shows the generation of genetic fingerprint.
• 7.
  Amplified Fragment LengthPolymorphism (AFLP):  This fingerprinting technique is based on the detection of multiple DNA restriction fragments by means of PCR amplification and has capacity to detect thousands of independent loci.  Genomic DNA is digested by appropriate restriction enzymes, which cut DNA at defined sequence sites.  A subset of the resultant fragments is then ligated to synthetic double stranded adaptors (DNA segments) at each end and subsequently amplified using two specific adaptor homologous primers.  The amplified and labeled restriction fragments are separated on denaturing gels or by capillary electrophoresis.  The complexity of the AFLP profile depends on the primers and restriction enzymes chosen and on the level of sequence polymorphism between the tested DNA samples.  The number of amplified bands of the preselected PCR is usually so high that a second round of PCR (selective PCR with fluorescent dyes) has to be performed to reduce the number of amplified products.  This is done by using primers that possess 1-3 additional bases at the 3‘end. Several combinations have to be tested for optimization.
• 8.
  Simple Sequence Repeats(SSR)  For this technique, prior sequence information is required. SSR markers or microsatellites also termed simple sequence length polymorphism (SSLP) or sequence tagged microsatellites (STMS) are tandem repeats scattered throughout the genome.  They can be amplified using primers that flank these regions.  The technique has been successfully used to construct detailed genetic maps of several plant species and to study genetic variation within populations of the same species.  As the markers are usually species specific, their development is rather costly, but once they have been developed the method becomes quite inexpensive Sequence based methods:  Concerning this technique, DNA sequences from the nuclear and chloroplast genomes are used for identification of plants at several taxonomic levels.  Certain sequences such as the 5s rDNA spacer and internal transcribed spacer (ITS) regions between the 16S and 26S rDNA are highly variable and can thus be used for authentication at species level.  Sequences such as nuclear 18S or plastidal rbcL, ndhF or matK however, are conserved amongst species and therefore suitable for discrimination at genus or family level.  DNA sequenced based techniques have widely been used for authentication of herbs. Various sequence based methods include: a. Single nucleotide polymorphism (SNP) b. Short tandem repeats (STR) Single nucleotide polymorphism (SNP)  SNP is a DNA sequence variation occurring when a single nucleotide — A, T, C or G— in the genome (or other shared sequence) differs between members of a species. Short tandem repeats (STR)  STR is a class of polymorphisms that occurs when a pattern of two or more nucleotides are repeated and the repeated sequences are directly adjacent to each other.  The pattern can range in length from 2 to 10 base pairs (for example (CATG) n in a genomic region) and is typically in the non-coding intron region, making it junk DNA.
• 9.
   By examiningenough STR loci and counting how many repeats of a specific STR sequence, there are at a given locus, it is possible to create a unique genetic profile of an individual Case study of EXACUM LAWII by DNA fingerprint flow chart: 1. EXACUM LAWII  The Exacum lawii is annual, glabrous, small erect herb rarely reaching 15 cm tall and flowers are bluish-purple.  Whole plant has been used traditionally as the folk remedy for the treatment of kidney disorders, eye diseases and also used as laxative.  The common name is Law’s Persian violet. It is locally known as Lahan chirayata in Maharashtra, Manali in Malayalam, Marukozhunthu in Tamil.  The Exacum lawii is annual, glabrous, small erect herb rarely reaching 15 cm tall. Flowers are bluish-purple. 2. DNA isolation, Quantification.  The fresh plants of Exacum lawii were flashed with liquid nitrogen followed by grinding of frozen tissues using a mortar and pestle.  3mg/ml proteinase k was added to the samples in eppendorf.  These samples were incubated at 37°C for 1h in a water bath shaker.
• 10.
   Pre-warmed extractionbuffer (100 mM Tris-HCl (pH 8.0), 2.5 M NaCl, 20 mM EDTA, 3 % CTAB, 1 % (v/v) 2-mercaptoethanol and 1 % Sarkosyl) was added after incubation kept in shaker water bath at 65°C.  Supernatant (5 ml) were separated from each tube and allowed to cool at room temperature followed with equal volume chloroform: isoamyl alcohol (24:1) solution.  An upper aqueous and a lower organic solvent layer in each tube were formed.  Tubes were slowly inverted without shearing of DNA until homogenous emulsion was formed.  The tubes were centrifuged at 9200 rcf for 15 min at 4°C using 3K30 laboratory centrifuge.  Upper transparent aqueous layer containing nucleic acid was transferred to another 50 mL tube using a 5 mL micropipette with broad bore tips.  The tubes left over night at −20°C for nucleic acid precipitation after adding double volume of pre-chilled ethanol and 3M sodium acetate (pH 5.2).  The tubes were centrifuged at 20,000 rcf for 15 min to pellet the precipitated nucleic acid.  The supernatant were removed and pellets obtained were washed in 70 % ethanol.  The pellets were dried in hot air blower for complete removal of ethanol. These pellets were suspended in 50 μl of TE buffer (Tris-HCl 10 mM pH 8.0 and EDTA 1 mM).  The suspension was incubated with pancreatic RNaseA (final concentration 0.2μg/μl) for 2h at 37°C to remove RNA. The resulting suspensions contained DNA from Exacum lawii plant.  The concentration of DNA was checked by measuring A260/A280 ratio in a Hitachi UV-Vis spectrophotometer. If uv shows 1.8 value DNA is of the highest purity, if more than 1.8 shows the presence of RNA contamination and less than that indicates protein contamination  Isolated DNA was electrophoresed in 0.8 % agarose gel containing 1 μg/ml ethidium bromide using TAE electrophoresis buffer (40 mM Tris-acetate and 1 mM EDTA of pH 8.0) and photographed in the gel documentation system. 3. RAPD-PCR and Electrophoresis:  Six different primers (matK-2.1F, matK-5R, rbcLaF, rbcLaR, rbcL-1F, and rbcL- 724R) were selected to generate reproducible amplification products.  PCR was performed in 25 μl reaction mixtures containing 40 ng DNA, 2.5 μl of 10X PCR buffer (500 μM dNTPs, 4.5 mM MgCl2, 0.5μM of each primer and 0.6 units per μl Taq DNA polymerase in an Icycler PCR thermal cycler.  The thermal cycler profile was as follows: initial denaturation for 1 min at 95°C followed by 35 incubation cycles each consisting of 94°C for 30 sec denaturation, 30 sec annealing at 48°C then 60 sec at 68°C and a final 5 min elongation at 68°C.
• 11.
   Primers werecombined in an equimolar ratio for the multiplex reaction.  PCR products were seperated by electrophoresis in 1.5 % (w/v) agarose gel using TAE buffer (40 mM Tris-acetate and 1 mM EDTA at pH 8.0) containing 0.5 μg/ml ethidium bromide and photographed.  RAPD banding profiles were scored by using an Amersham 100-bp ladder on the basis of presence or absence of the bands of a particular size were determined utilizing fingerprinting II version-3 software. RESULTS Random Amplified Polymorphic DNA (RAPD) fingerprinting profile:  The genomic DNA isolated from Exacum lawii and purity was A260/A280 ratio was found to be 1.6.  The DNA was subjected to gel electrophoresis and image was shown if Figure, The 6 primers pairs were successfully used for amplification of matK and rbcL gene.  The primer pair rbcL_1F and rbcL_724R, rbcLaF and rbcLarR and MatK_2.1F and MatK_5R produced two DNA fragments from 700 to 800 bp. APPLICATIONS: For Identification & Authentication
• 12.
   Swertia speciesknown as Kiratatikata in Ayurveda are authenticated using AFLP and SCAR markers and identified 483 bp amplicon primer which is specific to Swertia chirayita.  ISSR markers for authentication of Withania somnifera (Ashwagandha) are used.  AFLP markers for identification of Zingiber officinale (Ardraka) from its various species is done For Adulteration Detection  RAPD markers used for authentication and identification of genuine and adulterant samples of Emblica officinalis (Amalaki),Diascoria bulbifera (Varaahikanda,)Andrographis paniculata (Bhunimba),Glycyrrhiza glabra L.(Yashtimadhu) For Species Differentiation  319 bp unique primers is Species-specific SCAR marker for identification of Phyllanthus urinaria (Bhumi amalaki) from its various species. Identification and differentiation of the Taxus baccata aerial parts from its species using technology of Single Nucleotide Polymorphism (SNP). DNA Fingerprinting  RAPD-based SCAR marker was developed to identify Bacopa monnieri (Brahmi) from its adulterants Centella asiatica (Mandukaparni), and Malva rotundifolia (Suvarchala).
• 13.
  Microbial Contamination inherbal formulation  The microbial quality of pharmaceuticals is influenced by the environment and Quality of the raw materials used during formulation.  The incidence of micro flora in non-sterile medicines generally is indicated by the Nature of the ingredients, the quality of the vehicle and the care and attitude of Personnel involved in their handling.  Herbal drugs normally carry a number of bacteria and molds, often originating in the soil.  Poor methods of harvesting, cleaning, drying, handling, and storage may also cause additional contamination, as may be the case with Escherichia coli or Salmonella spp.  Raw materials of herbal formulation are frequently carrier of numerous possibly pathogenic microorganisms which may cause serious infection.  The Permitted numbers of pathogenic bacteria in oral medicaments (103 bacteria /g).  The limit in natural starting material should be (104 bacteria/g) and 100 mould or yeast cell/g. The type and frequency of the tests depends on the product.  The WHO has specified total microbial contamination limits for Contamination crude plant materials the limit adopted for untreated plant material Harvested under acceptable hygienic condition. Pseudomonas aeruginosa:  This rod-shaped bacterium, Pseudomonas aeruginosa, is normally found in Water , soil and other places that contain moisture.  It is a pathogen that takes advantage of the weakened immune system of an ill person and causes different infections.  Thus, these types of pathogen are called 'opportunistic pathogen'. Health effect of Pseudomonas aeruginosa Infection:  The symptoms of pseudomonas aeruginosa infection Fever, muscle and joint pain are common. The following list gives the respective symptoms of each infection Bone infection: Swollen infected part, redness. Ear infection: Pain in the ear, reduced ability to hear, facial paralysis. Eye infection: Pain in the eye, reduced vision, swollen eyelids. Cystic fibrosis: Cough reduced appetite, fast breathing, and enlargement of abdomen. Skin infections: Ulcer that can result in bleeding.
• 14.
  Escherichia coli:  E.coli are a group of bacteria that can cause a variety of illnesses in humans, Including, respiratory illnesses and other problems.  There are also many types of E.coli bacteria that are harmless.  One type of E. coli that often causes illness and outbreaks in humans is known as E. Coli O157.  There are other strains of E. coli that cause illness as well, but O157 is the most notorious. Health effect of E. coli infection: Symptoms of an intestinal E. coli infection vary by person but can include: Stomach cramps Diarrhea (usually bloody) Vomiting Low-grade fever (typically not over 101)  These symptoms usually resolve on their own within 5 to 7 days. E. coli infections are mild in most people, but they can be serious or even life threatening in others.  Symptoms of an E. coli infection typically start 3 to 5 days after exposure to the bacteria, but they can occur as early as one day or as late as 10 days after exposure. Staphylococcal infection: Staphylococcus aureus S. aureus is a spherical bacterium (coccus) which on Microscopic examination appears in pairs, short chains. Health effect of Staphylococcus aureus S. aureus can cause serious infections such as bloodstream infections, pneumonia, or bone and joint infections. According to the Pharmacopoeia, microbial limitation for nonsterile edible products such as powders, tablets, and capsules should be as follows:  For 1 gram or 1 milliliter; total aerobic bacterial count must not exceed 5.0 x 105 colonies.  Enterobacteriaceae count must not exceed 5.0x 103 colonies.  total yeasts and molds count must not be more than 5.0 x 103 colonies,  Escherichia coli (E.coli) must not be presented more than 50 colonies, while Staphylococcus aureus (S.aureus), Salmonella spp. and Clostridium spp. must not be presented at all in the sample of 10 grams or10 milliliters.