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In case of studies associated with human genetics, genomics, and pharmacogenetics the genomic DNA is extracted from the buccal cells, whole blood etc. Several methods are exploited by the researchers to extract DNA from the whole blood. One of these methods, which utilizes cell lysis and proteolytic properties of sodium dodecyl sulfate (SDS) and proteinase K respectively, might also be called SDS-PK method. It does not include any hazardous chemicals such as phenol or chloroform and is inexpensive. However, several researchers report the same method with different formulas and conditions. During our experiments with whole blood DNA extraction we experienced problems such as protein contamination, DNA purity and yield when followed some SDS-PK protocols reported elsewhere. A260/A280 and A260/A230 ratios along with PCR amplification give a clear idea about the procedure that was followed to extract the DNA. In an effort to increase the DNA purity from human whole blood, we pointed out some steps of the protocol that play a crucial role in determining the extraction of high quality DNA.

Isolation of mitochondrial DNA (mtDNA) from milk offers an effective way to monitor aspects of quality control and traceability to ensure food safety. A few methods of DNA isolation from milk have been reported, but many of them are time consuming and expensive. Here, we report a rapid, simple, and efficient method of mtDNA extraction from raw and processed milk (pasteurized, retorted, and UHT milk) to generate substrate for analysis using any PCR analysis platform. Various techniques used for the separation of mitochondria were explored and combined with a sodium dodecyl sulfate method for mtDNA extraction from raw and processed milk. The optimized protocol supports the efficient amplification of mtDNA independent of sample origin and is sufficiently straightforward to allow its widespread adoption by industry., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

We compared the reliability of visual diagnostic criteria to DNA diagnostic techniques, including newly designed primers, to discriminate Japanese marten (Martes melampus) feces from those of other sympatric carnivore species. Visual criteria proved > 95% reliable for fresh, odoriferous scats in good condition. Based upon this verification, we then examined if and how Japanese marten diet differs among seasons at high elevation study site (1500-2026 m). We also considered how intra-specific competition with the Japanese red fox (Vulpes vulpes japonica) may shape marten feeding ecology. From 120 Japanese marten fecal samples, high elevation diet comprised (frequency of occurrence) 30.6-66.0% mammals, 41.0-72.2% insects and 10.6-46.2% fruits, subject to seasonal variation, with a Shannon-Weaver index value of 2.77. These findings contrast substantially to seasonal marten diet reported in adjacent lowland regions (700-900 m), particularly in terms of fruit consumption, showing the trophic adaptability of the Japanese marten. We also noted a substantial dietary overlap with the red fox (n = 26 scats) with a Shannon-Weaver index of 2.61, inferring little trophic niche mutual exclusion (trophic niche overlap: 0.95), although some specific seasonal prey selection differences were likely related to relative differences in body size between foxes and martens. This additional information on the feeding ecology of the Japanese marten enables a better assessment of the specific risks populations face in mountainous regions.

We describe six novel ungulate-specific conserved primers for sequencing the complete mitochondrial cytochrome c oxidase I (COI) gene of selected threatened species from degraded samples for effective conservation planning. These primers amplified 301-1599 bp DNA fragments in various combinations. The method described may assist in the sequencing of either complete gene from moderate to good quality DNA or shorter fragment from degraded DNA., Competing Interests: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article., (© 2017 Wildlife Institute of India. Published by Informa UK Limited, trading as Taylor & Francis Group.)

The introduction of two alternative glycolate catabolic pathways in the chloroplasts of Arabidopsis thaliana rendered plants with increased biomass. To introduce these synthetic pathways, the selected genes were stepwise integrated in the nuclear genome of wild-type plants. These plants were transformed by Agrobacterium tumefaciens carrying the binary vectors using the floral dip method. Selection of transformants was conducted using different selection agents and the expression of the transgenes was confirmed by PCR and enzyme activity measurements.

Background: Regucalcin (RGN), a calcium regulating protein having anti-prolific, antiapoptotic functions, plays important part in the biosynthesis of ascorbic acid. It is a highly conserved protein that has been reported from many tissue types of various vertebrate species. Employing its effect of regulating enzyme activities through reaction with sulfhydryl group (-SH) and calcium, structural level study believed to offer a better understanding of binding properties and regulatory mechanisms of RGN, was performed., Material and Method: Using sample from testis of Bubalus bubalis, amplification of regucalcin (RGN) gene was subjected to characterization by performing digestion using different restriction endonucleases (RE). Alongside, cDNA was cloned into pPICZαC vector and transformed in DH5α host for custom sequencing. To get a better insight of its structural characteristics, three dimensional (3D) structure of protein sequence was generated using in silico molecular modelling approach. The full trajectory analysis of structure was achieved by the Molecular Dynamics (MD) that explains the stability, flexibility and robustness of protein during simulation in a time of 50ns. Molecular docking against 1,5-anhydrosorbitol was performed for functional characterization of RGN., Results: Preliminary screening of amplified products on Agarose gel showed expected size of ~893 bp of PCR product corresponding to RGN. Following sequencing, BLASTp search of the target sequence revealed that it shares 91% similarity score with human senescence marker protein-30 (pdb id: 3G4E). Molecular docking of 1,5-anhydrosorbitol reveals information regarding important binding site residues of RGN. 1,5-anhydrosorbitol was found to interact with binding free energy of - 6.01 Kcal/mol. RMSD calculation of subunits A, B and D-F might be responsible for functional and conserved regions of modeled protein., Conclusion: Three dimensional structure of RGN was generated and its interactions with 1,5- anhydrosorbitol, demonstrates the role of key binding residues. Until now, no structural details were available for buffalo RGN proteins, hence this study will broaden the horizon towards understanding the structural and functional aspects of different proteins in cattle., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Ageratum conyzoides, Croton bonpladianum and Malvastrum coromandelianum are common weeds found around agricultural fields. In several cases these were found to exhibit vein yellowing and yellow mosaic symptoms. Using degenerate primers specific for whitefly-transmitted geminiviruses (WTGs), and total DNA isolated from such infected plants (exhibiting the above symptoms) as a template, 1.2kbp fragments were amplified and were shown to have homology to DNA-A of Indian tomato leaf curl virus (ITLCV) by Southern hybridization. In control experiments the same primers failed to amplify any DNA fragments from the total DNA isolated from healthy plants (no symptoms as above). These results show that Ageratum, Croton and Malvastrum harbour geminivirus(es). [ABSTRACT FROM AUTHOR]

Objectives: Shigellosis is an acute infection of the intestine caused by bacteria in the genus Shigella and also an important cause of diarrhea in developing countries. This study was carried out to find the extent and nature of the emerging resistance in north part of Karnataka, India, and surrounding region with huge population, and also focused on the molecular mechanism of development of resistance against different generations of fluoroquinolones and explored the diversity of restriction endonucleases; we also tried to establish the significance of reduced minimal inhibitory concentrations (MIC) values., Methods: A total of 32 multidrug-resistant Shigella species (isolated from infants' stools) were subjected to MICs of fluoroquinolone-resistant isolates done by both broth dilution and E-test method. The genes implicated in resistance to fluoroquinolone generations ciprofloxacin, ofloxacin, and gatifloxacin (gyrA, gyrB, parC, and parE) were amplified using polymerase chain reaction (PCR) method and restriction digestion analysis of PCR product were performed using PvuI and HaeII enzymes., Findings: Fluoroquinolone-resistant Shigella species (n = 32) comprising S dysenteriae, S flexneri, and S sonnei were selected for MIC; 90.6% (29/32), 93.75% (30/32), and 93.75% (30/32) of isolates were ciprofloxacin, ofloxacin, and gatifloxacin resistant and showed the MIC range from 4-128 μg/mL. The PCR amplification results were positive for all species and asserted the presence of gyrA, gyrB, parC, and pare and sizes of the amplified products. The restriction banding patterns of amplified resistant genes were employed to detect differences among the Shigella species., Conclusions: The present study found that the genetic basis and its characterization of fluoroquinolone resistance in Shigella isolates was considered for the common resistant genes, namely, gyrA, gyrB, parC, and pare, and had mutations at position 83 of gyrA and at position 80 of parC of the quinolone-resistant determining regions and associated molecular mechanism. Our study beneficial in identification of the causative agents of the infections, careful control and cautions use of antibiotics must be promoted, particularly to monitor the emergence of isolates that are fully resistant to fluoroquinolones., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Pseudomonas aeruginosa infections have emerged as a major infectious disease threat in recent decades with infection particularly in immunocompromised hosts. P. aeruginosa possesses several virulence factors with involvement in pathogenesis. The aim of this study was to examine the prevalence of virulence genes of toxA and toxS and to analyze their relation to antimicrobial resistance of the isolates., Methods: In total 185 clinical isolates of P. aeruginosa were collected from burn patients. Antimicrobial susceptibility testing was done by disk diffusion method. PCR amplification was performed on extracted DNA from the isolates and the presence of encoding genes for exotoxin A (toxA) and exoenzyme S (toxS) were investigated by using specific primers., Results: In disk diffusion method, the isolates showed high sensitivity to colistin sulfate (100%) followed by imipenem (41.9%). The most prevalent resistance was seen against ceftazidime (90.5%) and gentamicin (88.5%). Multidrug resistance (MDR) demonstrated in 113 isolates (76.35%). According to PCR amplification, 133 (89.8%) and 127 (85.8%) isolates possessed toxA and toxS genes respectively. The frequencies of genes among MDR strains were 102 (76.6%) for toxA and 98 (77.1%) for toxS. Eighty five MDR isolates possessed both genes (73.9%). The non-MDR strains (23.65%), harbored lower prevalence of simultaneous toxA and toxS genes (26%) compared to MDR strains., Conclusion: The present study established a higher frequency of MDR among P. aeruginosa isolates from burn patients. It was found that the frequency of both toxA &S genes were significantly higher in MDR strains P. aeruginosa strains., (Copyright © 2016 Elsevier Ltd and ISBI. All rights reserved.)

The authentication of food components is one of the key issues in food safety. Similarly taxonomy, population and conservation genetics as well as food web structure analysis, also rely on genetic analyses including the DNA barcoding technology. In this scenario we developed a fast DNA extraction method without any purification step from fresh and processed seafood, suitable for any PCR analysis. The protocol allows the fast DNA amplification from any sample, including fresh, stored and processed seafood and from any waste of industrial fish processing, independently of the sample storage method. Therefore, this procedure is particularly suitable for the fast processing of samples and to carry out investigations for the authentication of seafood by means of DNA analysis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

As with natural nucleic acids, pairing between artificial nucleotides can be influenced by tautomerism, with different placements of protons on the heterocyclic nucleobase changing patterns of hydrogen bonding that determine replication fidelity. For example, the major tautomer of isoguanine presents a hydrogen bonding donor-donor-acceptor pattern complementary to the acceptor-acceptor-donor pattern of 5-methylisocytosine. However, in its minor tautomer, isoguanine presents a hydrogen bond donor-acceptor-donor pattern complementary to thymine. Calculations, crystallography, and physical organic experiments suggest that this tautomeric ambiguity might be "fixed" by replacing the N-7 nitrogen of isoguanine by a CH unit. To test this hypothesis, we prepared the triphosphate of 2'-deoxy-7-deazaiso-guanosine and used it in PCR to estimate an effective tautomeric ratio "seen" by Taq DNA polymerase. With 7-deazaisoguanine, fidelity-per-round was ∼92%. The analogous PCR with isoguanine gave a lower fidelity-per-round of ∼86%. These results confirm the hypothesis with polymerases, and deepen our understanding of the role of minor groove hydrogen bonding and proton tautomerism in both natural and expanded genetic "alphabets", major targets in synthetic biology.

The family B DNA polymerase gene from the euryarchaeon Thermococcus barophilus Ch5 (Tba5) contains an open reading frame of 6198 base pairs that encodes 2065 amino acid residues. The gene is split by three inteins that must be spliced out to form the mature DNA polymerase. A Tba5 DNA polymerase gene without inteins (genetically intein-spliced) was expressed under the control of the pET-28b(+)T7lac promoter in E. coli Rosetta 2(DE3)pLysS cells. The molecular mass of the purified Tba5 DNA polymerase was about 90kDa consistent with the 90,470Da molecular mass calculated based on the 776 amino acid sequence. The optimal pH for Tba5 DNA polymerase activity was 7.5 and the optimal temperature was 70-75°C. The enzyme possessed 3'→5' exonuclease activity and was activated by magnesium ions. PCR amplification using Tba5 DNA polymerase enables high-yield for 1- to 6-kb target DNA products, while 8- to 10-kb target DNA products were amplified at low or inefficient levels. To simultaneously improve product yield and amplification fidelity, Tba5 plus DNA polymerase mixtures were constituted with various amounts of Tba5 DNA polymerase mixed with Taq DNA polymerase. The Tba5 plus DNA polymerase mixtures robustly amplified up to 25-kb λ DNA fragments. In addition, the PCR error rate of Tba5 plus3 and Tba5 plus4 mixtures were much lower than those of wild-type Tba5 DNA polymerase, Pfu DNA polymerase, Taq DNA polymerase, and Pfu plus DNA polymerase., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Streptococcus pyogenes is a clinically important pathogen consisting of various serotypes determined by different M proteins expressed on the cell surface. The M type is therefore a useful marker to monitor the spread of invasive S. pyogenes in a population. Serotyping and nucleic acid amplification/sequencing methods for the identification of M types are laborious, inconsistent, and usually confined to reference laboratories. The primary objective of this work is to develop a technique that enables generation of aptamers binding to specific M-types of S. pyogenes. We describe here an in vitro technique that directly used live bacterial cells and the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) strategy. Live S. pyogenes cells were incubated with DNA libraries consisting of 40-nucleotides randomized sequences. Those sequences that bound to the cells were separated, amplified using polymerase chain reaction (PCR), purified using gel electrophoresis, and served as the input DNA pool for the next round of SELEX selection. A specially designed forward primer containing extended polyA20/5Sp9 facilitated gel electrophoresis purification of ssDNA after PCR amplification. A counter-selection step using non-target cells was introduced to improve selectivity. DNA libraries of different starting sequence diversity (10(16) and 10(14)) were compared. Aptamer pools from each round of selection were tested for their binding to the target and non-target cells using flow cytometry. Selected aptamer pools were then cloned and sequenced. Individual aptamer sequences were screened on the basis of their binding to the 10 M-types that were used as targets. Aptamer pools obtained from SELEX rounds 5-8 showed high affinity to the target S. pyogenes cells. Tests against non-target Streptococcus bovis, Streptococcus pneumoniae, and Enterococcus species demonstrated selectivity of these aptamers for binding to S. pyogenes. Several aptamer sequences were found to bind preferentially to the M11 M-type of S. pyogenes. Estimated binding dissociation constants (Kd) were in the low nanomolar range for the M11 specific sequences; for example, sequence E-CA20 had a Kd of 7±1 nM. These affinities are comparable to those of a monoclonal antibody. The improved bacterial cell-SELEX technique is successful in generating aptamers selective for S. pyogenes and some of its M-types. These aptamers are potentially useful for detecting S. pyogenes, achieving binding profiles of the various M-types, and developing new M-typing technologies for non-specialized laboratories or point-of-care testing., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We previously reported that Neq A523R DNA polymerase is more efficient in PCR than wild-type Neq DNA polymerase, and amplifies products more rapidly. Neq A523R DNA polymerase also amplifies templates more rapidly than Pfu DNA polymerase, but has a lower fidelity than Pfu DNA polymerase. To improve product yield and the fidelity of amplification simultaneously, we constructed and characterized the double mutant Neq A523R/N540R. The yield of PCR products was greater for Neq A523R/N540R DNA polymerase than wild-type and other mutant DNA polymerases, and the Neq double mutant catalyzed amplification of a 12-kb PCR product from a lambda template with an extension time of 3 min. The PCR error rate of Neq A523R/N540R DNA polymerase (6.3×10(-5)) was roughly similar to that of Pfu DNA polymerase (4.8×10(-5)), but much lower than those of wild-type Neq DNA polymerase (57.2×10(-5)), Neq A523R DNA polymerase (13.1×10(-5)), and Neq N540R DNA polymerase (37.7×10(-5)). These results indicated that A523R and N540R mutations of Neq DNA polymerase had synergistic effects on its fidelity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Target enrichment is commonly used in next generation sequencing (NGS) workflows to eliminate genomic DNA regions that are not of interest for a particular experiment. By only targeting specific regions such as exons, one can obtain greater depth of DNA sequencing coverage for regions of interest or increase the sampling numbers of individuals, thereby saving both time and cost. This overview of target enrichment strategies provides a high-level review of distinct approaches to capture specific sequences: (a) hybridization-based strategies, (b) transposon-mediated fragmentation (tagmentation), (c) molecular inversion probes (MIPs), and (d) singleplex and multiplex polymerase chain reaction (PCR) target enrichment. Strategies for assay design and performance criteria are also discussed. Other platforms currently in development are also briefly described., (Copyright © 2015 John Wiley & Sons, Inc.)

Glutamate synthase (GOGAT) from Chlamydomonas reinhardtii is able toform functional covalent complexes with its substrate ferredoxin (Fd), either wild-type (WTFd) or recombinant form (rFd). However, when Fd carboxyl groups were chemically modified (mdFd), no complexes were detected and its ability to serve aselectron donor for glutamate synthase activity was also decreased. By site-directed mutagenesis, we have demonstrated that Fd glu91 and anegative core in the helix Alpha?1 are critical for Fd interaction with this enzyme and its functionality as electron carrier for glutamate synthase. As a previous step to elucidate the specific positive charged residues involved in glutamate synthase interaction with Fd, wehave isolated a cDNA, CrFG-3, encoding Fd-GOGAT from C. reinhardtii.The cDNA comprised about 60% of the protein and sequence comparison showed that CrFG-3 was structurally more similar to higher plant enzymes than to the corresponding prokaryotic GOGAT. Two conserved domains were present in this protein fragment, the FMN-binding domain and the cysteines involved in the iron-sulfur cluster binding. [ABSTRACT FROM AUTHOR]

Twa DNA polymerase from hyperthermophilic archaeon Thermococcus waiotapuensis has exceedingly high fidelity among family B DNA polymerases. However, Twa DNA polymerase has significant shortcomings in terms of a low extension rate and poor processivity. To resolve these weaknesses, we focused on two amino acid residues (N565 and H633) in the palm and thumb subdomains of the Twa DNA polymerase. These two residues were replaced by site-directed mutagenesis and the enzymatic properties of the mutants were analyzed. Here, Twa H633R DNA polymerase showed significantly improved polymerase function compared to wild-type Twa DNA polymerase in terms of processivity (2-fold), extension rate (1.5-fold) and PCR efficiency. Kinetic analysis using DNA as a template revealed that the kcat value of the Twa H633R mutant was similar to that of wild-type, but the Km of the Twa H633R mutant was about 1.6-fold lower than that of the wild-type. These results showed that the Arg residue substitution at H633 located in the thumb subdomain has a positive effect on processivity, extension rate and PCR efficiency, suggesting that the Twa H633R mutant allows a conformational change for easy access of the primer-template to the binding site of the polymerase domain., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Among the family B DNA polymerases, the Twa DNA polymerase from T. wiotapuensis, a hyperthermophilic archaeon, has exceedingly high fidelity. For applications in PCR, however, the enzyme is limited by its low extension rate and processivity. To resolve these weaknesses, we focused on two amino acid residues (A381 and N501) located at the palm subdomain of Twa DNA polymerase. Following replacement of these residues by site-directed mutagenesis, Twa N501R DNA polymerase showed significantly improved polymerase function compared to the wild-type enzyme in terms of processivity (3-fold), extension rate (2-fold) and PCR efficiency. Kinetic analysis using DNA as template revealed that the kcat value of the Twa N501R mutant was similar to that of wild-type, but the Km of the Twa N501R mutant was about 1.5-fold lower than that of the wild-type. These results suggest that a positive charge at residue 501 located in the forked-point does not impede catalytic activity of the polymerase domain but stabilizes interactions between the polymerase domain and the DNA template., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Corynebacterium species other than Corynebacterium diphtheriae rarely cause infections in human but rather reside in flora, however they have been reported to cause opportunistic infections in both immunocompromised and immunecompetent patients. Here we report for the first time a case of an elderly female patient presenting with a fatal urosepsis caused by a recently defined pathogen, Corynebacterium riegelii, identified on second day after patient hospitalization leading to a progressive worsening and death of the patient on 6th day.

Purpose: Detection of the virulence gene is a key component in determining the pathogenicity of any isolates, because these genes act multi-functionally and multi-factorially. A gyrase specific gene primer, in combination with the PCR technology, allows the precise detection of the DNA gyrase subunit B2 gene (gyr-B-2) from different virulent microorganisms. In the present study, forward and reverse primers with lengths of 20bp and 21bp were used for the detection of the gyr-B-2 genes in the clinical isolates of the Klebsiella sp. which were collected from patients who were suffering from pneumonia and urinary tract infections (UTIs)., Materials and Methods: A total of 14 isolates viz., K1, K2, K3, K4, K5, K6, K7, K8, K9, K10, K11, K12, K13 and K14 were isolated from 3 different private medical colleges of Sylhet city., Results: The gyr-B-2 gene which was amplified in 12 isolates viz., K1, K2, K3, K4, K5, K6, K7, K8, K10, K11, K12 and K14 gave the expected 411bp PCR product after its visualization under a gel documentation system in a 1.2% agarose gel., Conclusions: The present study was undertaken to detect the gyrB2 gene from Klebsiella sp, which will be helpful for further scientific studies. This PCR was outstanding in the detection of the gyb-B-2 gene in pneumonia and urinary tract infections in patients, which were caused by the Klebsiella species.

Site-directed mutagenesis is a PCR-based method to mutate specified nucleotides of a sequence within a plasmid vector. This technique allows one to study the relative importance of a particular amino acid for protein structure and function. Typical mutations are designed to disrupt or map protein-protein interactions, mimic or block posttranslational modifications, or to silence enzymatic activity. Alternatively, noncoding changes are often used to generate rescue constructs that are resistant to knockdown via RNAi., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cephalotaxus oliveri is a scarce medicinal conifer endemic to the south central region of China and Vietnam. A small fragmented population presently exists due to anthropogenic disturbance. C. oliveri has been used for its alkaloids harringtonine and homoharringtonine, which are effective against leucocythemia and lymphadenosarcoma. Monoecious plants have been detected in nature, although they were understood to be dioecious. In order to study the mating system, population genetics and the genetic effects of habitat fragmentation on C. oliveri, 15 polymorphic and 12 monomorphic microsatellite loci were developed for C. oliveri by using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol. The polymorphisms were assessed in 96 individuals from three natural populations (32 individuals per population). The number of alleles per locus ranged from two to 33, the observed and expected heterozygosity per locus ranged from 0.000 to 1.000 and from 0.000 to 0.923, respectively. These loci would facilitate a comprehensive understanding of the genetic dynamics on C. oliveri, which will be useful for establishing effective conservation strategies for this species.

Chrysanthemums constitute approximately 30 species of perennial flowering plants, belonging to the family Asteraceae, native to Asia and Northeastern Europe. Chrysanthemum is a natural cosmetic additive extracted from Chinese herb by modern biochemical technology. It has the properties of anti-bacterial, anti-viral, reducing (detoxification) and anti-inflammation. It possesses antioxidant characteristics, which could assist in minimizing free-radical induced damage. Therefore, it is widely used in skin and hair care products. Chemical composition of this herbal remedy includes kikkanols, sesquiterpenes, flavonoids, various essential oils containing camphor, cineole, sabinol, borneole and other elements that interfere with DNA, causing erroneous or no PCR products. In the present study, testing and modification of various standard protocols for isolation of high-quality DNA from leaf tissues and seeds of C. indicum was done. It was observed that the DNA obtained from seeds and leaf tissues with a modified cetyltrimethylammonium bromide buffer protocol was of good quality, with no colored pigments and contaminants. Also, DNA could be extracted from leaf tissues without using liquid nitrogen. Quality of DNA extracted from seeds was much better as compared to that extracted from leaf tissues. The extracted DNA was successfully amplified by PCR using arbitrary RAPD primers. The same protocol will probably be useful for extraction of high-molecular weight DNA from other plant materials containing large amounts of secondary metabolites and essential oils.