Chloroform Extraction Method Research Articles

Diversity Analysis, Batch Fermentation and Characterization of Nisin in Identified Strains of Lactococcus lactis spp. lactis

Thirty-one strains of Lactococcus lactis spp. lactis were identified out of 89 isolates of lactic acid bacteria (LAB) from dairy and nondairy sources. Of the 31 strains, 24 (46.1%) were obtained from dairy and seven (18.9%) from non-dairy sources. The cluster analysis of rep-PCR showed that (GTG)5-PCR followed by ERIC-PCR exhibited more discriminating potential than BOX-PCR. The obtained banding patterns characterized the polymorphism among strains. The strain level polymorphism was also obtained by the combined cluster analysis of (GTG)5, ERIC and BOX-PCR which exhibited a level of heterogeneity among strains but not with the sources of isolation. Among 31 strains, 17 strains were able to produce zones of inhibition against Lactobacillus acidophilus NCDC 015 and therefore considered as nisin producers. Nisin production by strains was further confirmed by PCR amplification of nisA/Z of 174 bp size. The nisin activity and cell growth observed to be higher in pH controlled batch fermentation than in uncontrolled fermentation. The nisin activity, cell concentration, and acidity were high in immobilized cell system than free cell batch fermentation. The hot acid and chloroform extraction method was found to be the efficient way for the partial purification of nisin from fermented broth.

Engineering Hydrogen Gas Production from Formate in a Hyperthermophile by Heterologous Production of an 18-Subunit Membrane-bound Complex

Biohydrogen gas has enormous potential as a source of reductant for the microbial production of biofuels, but its low solubility and poor gas mass transfer rates are limiting factors. These limitations could be circumvented by engineering biofuel production in microorganisms that are also capable of generating H2 from highly soluble chemicals such as formate, which can function as an electron donor. Herein, the model hyperthermophile, Pyrococcus furiosus, which grows optimally near 100 °C by fermenting sugars to produce H2, has been engineered to also efficiently convert formate to H2. Using a bacterial artificial chromosome vector, the 16.9-kb 18-gene cluster encoding the membrane-bound, respiratory formate hydrogen lyase complex of Thermococcus onnurineus was inserted into the P. furiosus chromosome and expressed as a functional unit. This enabled P. furiosus to utilize formate as well as sugars as an H2 source and to do so at both 80° and 95 °C, near the optimum growth temperature of the donor (T. onnurineus) and engineered host (P. furiosus), respectively. This accomplishment also demonstrates the versatility of P. furiosus for metabolic engineering applications.

Methylenetetrahydrofolate reductase C677T variant in Indian children with craniosynostosis: Its role in the pathogenesis, risk of craniosynostosis.

BACKGROUND:677C to T allele in the 5, 10-methylenetetrahydrofolate reductase (MTHFR) gene has been implicated in the etiology of various syndromes and nonsyndromic diseases but till date no direct studies have been reported with craniosynostosis.OBJECTIVES:The aim was to study the family-based association of MTHFR polymorphism in different categories of craniosynostosis patients.MATERIALS AND METHODS:This was a cross-sectional study in which 30 patients classified as Apert syndrome, Pfeiffr syndrome and nonsyndromic craniosynostosis patients with their family were recruited. A sample of 3 ml intravenous blood was taken from patients and from their family members (father and mother) in ethylenediaminetetraacetic acid-anticoagulated vacutainer for the purpose of the study. Genomic DNA was extracted from peripheral blood lymphocytes by phenol chloroform extraction method. Primers for MTHFR gene were designed. The polymerase chain reaction was carried out. After successful amplification, a small aliquot (5 μl) of the MTHFR reaction mixture was treated with 1 units of Hinf I restriction enzyme (NEB). Results were obtained and compiled.RESULTS:A total of 30 patients/participants with craniosynostosis of Indian descent and their parents formed the study group. The genotyping did not confirm an association between the MTHFR 677C to T polymorphism and between different categories of craniosynostosis. When comparing the offspring of mothers statistically significant differences were found.CONCLUSION:C667T polymorphism of the MTHFR gene is unlikely to play a role in the pathogenesis of craniosynostosis though maternal MTHFR C677T polymorphism may be a genetic risk factor.

Mutational identification of fibroblast growth factor receptor 1 and fibroblast growth factor receptor 2 genes in craniosynostosis in Indian population

OBJECTIVE:The Objective of this study was to identify the association of mutation of fibroblast growth factor receptor 1 (FGFR1), FGFR2 genes with syndromic as well as non-syndromic craniosynostosis in Indian population.MATERIALS AND METHODS:Retrospective analysis of our records from January 2008 to December 2012 was done. A total of 41 cases satisfying the inclusion criteria and 51 controls were taken for the study. A total volume of 3 ml blood from the patient as well as parents was taken. Deoxyribonucleic acid extracted using phenol chloroform extraction method followed by polymerase chain reaction-restriction fragment length polymorphism method.RESULTS:There were 33 (80.4%) non-syndromic cases of craniosynostosis while 8 (19.5%) were syndromic. Out of these 8 syndromic cases, 4 were Apert syndrome, 3 were Crouzon syndrome and 1 Pfeiffer syndrome. Phenotypically the most common non-syndromic craniosynostosis was scaphocephaly (19, 57.7%) followed by plagiocephaly in (14, 42.3%). FGFR1 mutation (Pro252Arg) was seen in 1 (2.4%) case of non-syndromic craniosynostosis while no association was noted either with FGFR1 or with FGFR2 mutation in syndromic cases. None of the control group showed any mutation.CONCLUSION:Our study proposed that FGFR1, FGFR2 mutation, which confers predisposition to craniosynostosis does not exist in Indian population when compared to the western world.

The effect of the DNA preparation method on the sensitivity of PCR for the detection of Trypanosoma evansi in rodents and implications for epidemiological surveillance efforts

Trypanosoma evansi is responsible for the most largely distributed animal trypanosomosis, affecting a wide range of wild and domestic animals. Its surveillance requires the implementation of standardized and reliable diagnostic tools. Although the development of polymerase chain reaction (PCR) tools has greatly improved our diagnostic capacity, factors affecting their sensitivity need to be acknowledged and accounted for in the interpretation of results. The targeted gene and the primer design have already been shown to greatly affect the sensitivity of a PCR, and the best-performing sets of primers have been previously identified. However, the sensitivity of the PCR is also largely influenced by the DNA extraction or sample preparation method. In this paper, we selected 6 DNA extraction or blood sample preparation methods representative of what would be used in a budget-constrained setting: phenol–chloroform, Chelex®, Flexigen (Qiagen®) kit, Genekam® kit and two original protocols using sodium hydroxide. We studied the effects of the preparation method on the detection limit of the subsequent PCR. Our results show that the extraction method strongly affects the PCR sensitivity. The classical phenol–chloroform extraction method allowed for the PCR with the lowest detection limit. Some combinations of extraction method and primer set had detection limits that were not compatible with their use as a reliable diagnostic test, and would severely reduce the performance of a surveillance program. Therefore, we encourage laboratories to carefully select their sample preparation and PCR protocols, depending on the aimed sensitivity, cost, safety, time requirement and objectives.

Association between prevalence of pyrimethamine resistance and double mutation in pfdhfr gene in West Bengal, India

Objective To find whether antifolate drug (pyrimethamine) resistance has occurred in the two malaria endemic zones (Kolkata and Purulia) of West Bengal, India. Methods Parasitic bloods were collected from patients of Kolkata and Purulia, in vitro susceptibility test were performed in those 90 isolates. Now parasitic DNA was isolated by phenol chloroform extraction method and then polymerase chain reaction and restriction fragment length polymorphism analysis of different codons of pfdhfr gene (51, 59, and 108) were assessed in Plasmodium falciparum isolates from patients in India. Results Among 45 isolates from Kolkata dhfr mutant isolates at codons 108, 51 and 59 were found in 71.11%, 100% and 15.55% isolates respectively while in Purulia mutation found in those codons were 42.22%, 57.77% and 0%, respectively. In Kolkata, the isolate having double mutation (N108 + 51I) were resistant to pyrimethamine (P < 0.01) whereas in Purulia N108 mutation is occurred with both mixed and mutant 51I mutation. Conclusions Our present findings implicate that due to enormous drug (pyrimethamine) pressure double mutation with dhfr S108N/T and N51I was highly correlated (P < 0.01) with in vitro pyrimethamine resistance in two malaria endemic region of West Bengal. The increase in the number of dhfr mutations was strongly correlated to resistance to pyrimethamine.

EFFECT OF IMMUNOGLOBULIN Y PURIFIED FROM IMMUNIZED HEN EGGS ON THE GROWTH OF STAPHYLOCOCCUS AUREUS

A novel purification method of egg yolk immunoglobulin (IgY) based on precipitation using agarPEG was developed. This me thod was compared with chloroform extraction and polyethylene glycol (PEG) precipitation methods. The results showed the protein contents were high with chloroform method followed by agarPEG then PEG method. The purity of resultant I gY was homogeneous with agar� PEG method followed by PEG method then chloroform extraction method. The IgY purified by agar -PEG method, obtained from hens immunized by formalintreated S. aureus , showed a significant reduction in bacterial growth and the growth inhibition was dependent on specificIgY concentration.

Prevalence of severe chloroquine resistance associates the point mutation in pfcrt and pfmdrI gene in eastern India

Objective To evaluate the molecular mechanism of chloroquine resistant Plasmodium falciparum malaria in West Bengal, eastern part, India. Methods Parasitic bloods were collected from patients in Kolkata and Purulia, in vitro drug susceptibility test were performed in those 179 isolates. Now parasitic DNA was isolated by phenol chloroform extraction method and then polymerase chain reaction and restriction fragment length polymorphism analysis of different codons of pfcrt gene (76) and the pfmdrI gene (86, 1246) were assessed. Results The response of 179 patients to chloroquine was determined. The prevalence of both pfcrt K76T (68.72%) and pfmdrI N86Y (43.1%) mutation was found here. But most importantly severe in vitro chloroquine resistance has been found (53.07%) here, with either double 76T + 86Y mutation or triple 76T + 86Y + 1246Y mutation. Conclusions Our present findings implicate that due to enormous drug (chloroquine) pressure, double mutation with pfcrt 76T + pfmdrI 86Y and triple mutation with pfcrt 76T + pfmdrI 86Y + pfmdrI 1246Y was highly correlated ( P = 0.001) with in vitro chloroquine resistance for the first time in eastern India.

Recovery and purification of intracellular polyhydroxyalkanoates from recombinant Cupriavidus necator using water and ethanol

A new halogen-free and environmental-friendly method using water and ethanol is developed as an alternative for the recovery of polyhydroxyalkanoates (PHA) from recombinant Cupriavidus necator in comparison to the established chloroform extraction method. After optimisation, our results showed that the halogen-free method produced a PHA with 81% purity and 96% recovery yield, in comparison to the chloroform extraction system which resulted in a highly pure PHA with 95% yield. Although the purity of the PHA using the new method is lower, the molecular weight of the extracted PHA is not compromised. This new method can be further developed as an alternative and more environmental-friendly method for industrial application.

Development of a quantitative PCR assay for residual mouse DNA and comparison of four sample purification methods for DNA isolation

Reliable and sensitive assays are required to determine whether a pharmaceutical product meets current regulatory guidelines for residual host cell DNA. In this study, the sensitivity of the qPCR assay was significantly improved by targeting the repetitive elements of mouse genome. This improved method allowed for sensitive and accurate quantitation of mouse genomic DNA in the range of 1 to 10 6 pg/mL. In addition, four sample purification methods for DNA isolation (Wako DNA extractor kit, MasterPure™ DNA purification kit, PrepSEQ™ residual DNA sample preparation kit, and phenol–chloroform extraction method with addition of glycogen), each representing a different strategy for DNA isolation from proteinaceous solutions, were evaluated by isolating DNA from a mouse monoclonal IgG antibody. Among these methods, Wako DNA extractor kit and MasterPure™ DNA purification kit demonstrated superior DNA recovery, repeatability, and sensitivity, with quantitation limits of 1 pg/mL. To further evaluate these two DNA isolation methods, six replicates of an unspiked mouse polyclonal IgG antibody sample were tested by both methods, and both methods demonstrated a good degree of precision. Therefore, the residual mouse DNA quantitation methods described here represented rapid, accurate, precise, and sensitive procedures that can be used in quality control testing for regulatory compliance in the pharmaceutical industry.

Intervention of azithromycin on Pseudomonas aeruginosa biofilm and virulence factors

Objective To evaluate the effect of azithromycin on Pseudomonas aeruginosa PAO1 biofilm formation and virulence factors production. Methods Detect the minimum inhibitory concentration of azithromycin against PAO1 by 2-fold dilution method. Crystal violet staining assay was used for initial adhesion assays. The PAO1 biofilm was established in vitro and observed by scanning electron microscope. Viable bacterial counts were determined by serial dilution. LasB elastolytic activity was determined by using Elastin-Congo Red. Protease activity was determined by Azo-casein. Chloroform extraction method was used for pyoverdine assay . The orcinol assay was used to directly assess the amount of rhamnolipids . Results Scanning electron microscope biofilm and viable bacterial counts of PAO1 adhered to the surface of catheter in PAO1 azithromycin group were less than the PAO1 control group after incubated for 3 d and 7 d ( P ＜0.05), and the initial adhesion was weaker ( P ＜ 0. 05 ). The virulence factors production were obviously decreased (P ＜0.01 ). LasB elastolytic activity and pyoverdine were even reduced to the same as with the PA-JP3 group ( P ＞ 0.05 ), but the protease activity and the rhamnolipids concentration were higher than the PA-JP3 group ( P ＜ 0.05 ). Conclusion Azithromycin can inhibit PAO1 bioflim formation in vitro and virulence factors production. Key words: Pseudomonas aeruginosa ; Azithromycin ; Biofilm ; Virulence factors ;

Comparison of methods in the recovery of nucleic acids from archival formalin-fixed paraffin-embedded autopsy tissues

Archival formalin-fixed paraffin-embedded (FFPE) human tissue collections are typically in poor states of storage across the developing world. With advances in biomolecular techniques, these extraordinary and virtually untapped resources have become an essential part of retrospective epidemiological studies. To successfully use such tissues in genomic studies, scientists require high nucleic acid yields and purity. In spite of the increasing number of FFPE tissue kits available, few studies have analyzed their applicability in recovering high-quality nucleic acids from archived human autopsy samples. Here we provide a study involving 10 major extraction methods used to isolate total nucleic acid from FFPE tissues ranging in age from 3 to 13 years. Although all 10 methods recovered quantifiable amounts of DNA, only 6 recovered quantifiable RNA, varying considerably and generally yielding lower DNA concentrations. Overall, we show quantitatively that TrimGen’s WaxFree method and our in-house phenol–chloroform extraction method recovered the highest yields of amplifiable DNA, with considerable polymerase chain reaction (PCR) inhibition, whereas Ambion’s RecoverAll method recovered the most amplifiable RNA.

Hypochlorite digestion method for efficient recovery of PHB from Alcaligenes faecalis

We reported the optimum amount of PHB accumulated by Alcaligenes faecalis during its 24 h growth under nitrogen deficient conditions. After 24 h incubation decrease in the amount of PHB was recorded. Hypochlorite digestion of biomass of organism followed by extraction with a solvent system consisting of 1:1 mixture of ethanol and acetone resulted in efficient recovery of PHB vis-à-vis earlier methods. This solvent system gave a high recovery yield, i.e. 5.6 gL(-1) vis-à-vis earlier reported yield, 1.34 gL(-1) (by same method), 0.63 gL(-1) (by chloroform extraction method) and 1.1 gL(-1) (by dispersion method).

An Efficient Organic Solvent Based Extraction Method for the Proteomic Analysis ofArabidopsisPlasma Membranes

Membrane proteins are involved in diverse cellular processes and are an integral component of many signaling cascades, but due to their highly hydrophobic nature and the complexities associated with studying these proteins in planta, alternative methods are being developed to better characterize these proteins on a proteome-wide scale. In our previous work ( Mitra , S. K. et al. J. Proteome Res. 2007 , 6 , ( 5 ), 1933 - 50 ), methanol-assisted solubilization was determined to facilitate the identification of both hydrophobic and hydrophilic membrane proteins compared to Brij-58 solubilization and was particularly effective for leucine-rich repeat receptor-like kinases (LRR RLKs). To improve peptide identification and to overcome sample losses after tryptic digestion, we have developed an effective chloroform extraction method to promote plasma membrane protein identification. The use of chloroform extraction over traditional solid-phase extraction (SPE) prior to off-line strong cation exchange liquid chromatography (SCXC) and reversed-phase liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis facilitated the removal of chlorophylls, major contaminants of plant tissue preparations that can affect downstream analysis, in addition to the effective removal of trypsin used in the digestion. On the basis of a statistically derived 5% false discovery rate, the chloroform extraction procedure increased the identification of unique peptides for plasma membrane proteins over SPE by 70% which produced nearly a 2-fold increase in detection of membrane transporters and LRR RLKs without increased identification of contaminating Rubisco and ribosomal peptides. Overall, the combined use of methanol and chloroform provides an effective method to study membrane proteins and can be readily applied to other tissues and cells types for proteomic analysis.

Antifungal activity of neutralized wood vinegar with water extracts of Pinus densiflora and Quercus serrata saw dusts.

The major objectives of this study were to find out the mechanism behind the sapstaining fungal hyphae penetration into wood elements using electron microscope and to evaluate the exact role of phenolic compounds existing in wood vinegar towards the sapstaining fungal growth inhibition. To formulate the new environmental friendly wood preservative, we neutralized (to pH 7) wood vinegar with NaOH and hot water extracts of Pinus densiflora and Quercus serrata saw dusts were used in the media instead of sterilized distilled water to enhance the fungal growth inhibition property of neutralized wood vinegar. The initial calibration of wood extract was done by calculating the specific gravity, tar calculation and total organic content. GC-MS analysis was performed to identify the phenolic compounds and other ingredients present in both neutralized wood vinegar and acidic wood vinegar by chloroform extraction method. Eleven major compounds were identified in acidic wood vinegar and 7 major compounds in neutralized wood vinegar. Ophiostoma polonicum, O. ips, O. flexuosum, O. narcissi and O. tetropii were the sapstaining fungi used in this study against neutralized wood vinegar, extracts of Pinus densiflora and Quercus serrata. More than 2.5% neutralized wood vinegar contained agar plates were showed strong antifungal activity against sapstaining fungi. The Scanning Electron Microscopy image of fungal growth on wood samples showed the phenomenon of fungal penetration into wood elements. Pinus densiflora was used as wood element for SEM analysis.

Evaluation Of &lt;i&gt;In-Vitro&lt;/i&gt; Antimicrobial Activities And Phytochemical Constituents Of &lt;i&gt;Cassia occidentalis &lt;/i&gt;

The research was carried out to evaluate the in-vitro antimicrobial activity and phytochemical constituents of Cassia occdentials. Cassia leaves were collected from Kacha town in Niger State and extracted using methano, hexane, chloroform and water extraction methods. Serial concentrations: 50 60, 70, 80, 90 and 100 % methanol, hexane, chloroform and aqueous extracts were prepared and sterilized. The bacterial isolates used; E. coli, P. multocida, S. typhi, S. typhimurium, S. pyogenes, S. pneumoniae and K. pneumoniae were authenticated using biochemical and serological methods. The suspenson (0.5) of each bacterial isolate was prepared in isotonic sodium chloride. The disc agar diffusion method was performed on 70 Mueller-Hinton agar pates, 10 per microorganism , using serial diffusion concentraton: 500, 600, 700, 800, 900 and 1000 mg o hexane, methanol, chloroform and water. The results showed that all the extracts of Cassia occidentalis have antimicrobial activity on E col at concentrations between 900 – 1000 mg. E. coli was most susceptible to hexane extract at concentration ranges between 500 – 1000 mg, there was no antimicrobial activity exhibited against the other tested microorganisms Phytochemical analyses showed the presence of alkaloid, tannin, saponin, glycoside and flavonoid, steroid was absent. Keywords: Evaluation, In-vitro, Antimicrobial activity, Phytochemical properties, Cassia occidentalisAnimal Research International Vol. 3 (3) 2006 pp. 566-569

Removal of phenol and chlorophenols from water by new ozone generator

Presence of phenol in river water occasionally disturbs the production of drinking water. Phenol often originated from coal power plants or pesticides. New ozone generator [Kuraica et al., Vacuum, 73 (2004) 705] was used for removal of phenol and chlorophenols from water. The ozone generator was based on coaxial dielectricbarrier-discharge and operated in the air at an atmospheric pressure. Flowing water was one of dielectric layers. Ozone and ozonized water were generated in the same volume of the discharge. This type of ozone generator has considerable advantages in comparison with the classical ones. In this paper five experiments were described, three with bidistilled water and two with different water samples from river Danube. In all cases, 5L of water containing 6mg/L of corresponding phenol was passed once through the ozonizer. The concentration of dissolved ozone in water was 7–40mg/L. The ozonization lasted 1 h, but the total contact time was up to 94 h. In all cases, before and after the treatment of water with ozone, the concentration of corresponding phenol [Standard Methods for Examination of Water and Wastewater, method 510 B, Chloroform Extraction Method, American Public Health Association, Washington, 1985, p. 558], dissolved and residual ozone [Standard Methods for Examination of Water and Wastewater, Methods 2-41, 2-42 (dissolved ozone) Iodometric Method; method 4-104 (ozone residual) Indigo Colorimetric Method, American Public Health Association, Washington, 1995] were determined by standard methods. In experiments with distilled water most of the phenol was removed during ozonization (89.5%). After 24 and 96 h the percentage of removed phenol increased to 93.3 and 98.9%, respectively. With 4-chlorophenol and 2,4-dichlorophenol, 99.8 and 98.9% of corresponding phenols were removed. In experiments with two different water samples from Danube, the phenol was also mostly removed during ozonization, 76.7 and 80.0%, respectively.

A rapid and cost-effective method of genomic DNA isolation from cyanobacterial culture, mat and soil suitable for genomic fingerprinting and community analysis

This study presents a phenol and lysozyme free protocol for genomic DNA isolation of cyanobacteria from culture, mats and soil. For an efficient and pure DNA isolation from cyanobacteria having tough cell wall, extra steps of glass beading and Sepharose 4B purification were added. The modified method gave a higher yield of DNA than the phenol: chloroform extraction method. Four parameters selected for purity testing of the isolated DNA were: (i) restriction digestion with Hind III, (ii) randomly amplified polymorphic DNA-PCR of axenic culture of cyanobacteria to assess phylogenetic relatedness, (iii) denaturing gradient gel electrophoretic (DGGE) analysis of cyanobacterial mat and soil to ascertain the applicability of the isolated DNA for community analysis, and (iv) sequencing of partial 16S rDNA of Hapalosiphon intricatus BHULCR1, Anabaena doliolum LCR1, Anabaena oryzae LCR2, Aulosira fertilissima LCR4, and Tolypothrix tenuis LCR7 and BLAST analysis to confirm their cyanobacterial identity. Data generated from above analyses lead us to conclude that the modified method in question is rapid, cost effective, health and time conscious and promising for genetic fingerprinting and community analysis of cyanobacteria from diverse habitats.

Fast Analysis of Nicotine in Tobacco Using Double-Shot Pyrolysis−Gas Chromatography−Mass Spectrometry

A simple, rapid, and sensitive assay of nicotine in tobacco has been developed using pyrolysis-gas chromatography-mass spectrometry (DSP-GC-MS). The optimum pyrolyzer (desorption) conditions, using Korean tobacco grade B1O (0.4 mg) and n-heptadecane as an internal standard, were found to be heating from 50 to 300 degrees C at a rate of 60 degrees C/min. Replicate determinations (n = 5), on the same tobacco and using n-heptadecane as the internal standard, resulted in a nicotine content of 1.96%, with a relative standard deviation (RSD) of 1.9%. This result was in good agreement with those from established methods: the Cai ether extraction method, a chloroform extraction method, and the CORESTA recommended method. However, the DSP method requires less than half the time of the solvent extraction methods, requires less sample, is almost solvent-free, and is less labor-intensive. The DSP method was used to determine the nicotine content of eight flue-cured tobaccos from Brazil, China, Korea, and the United States, which were found to have contents ranging from 1.21 to 2.19%.

Sex identification from exfoliated primary teeth - a PCR study

Teeth endure postmortem degradation and extreme changes in ambient temperature and pressure better than most human tissues. In the present day scenario the growing number of crime against children in the form of battering, physical/sexual abuse, abduction and kidnapping, the use of exfoliated primary teeth, become many times the only evidence available at the crime scene. Despite exposure of the body to burial, mutilation, explosion or incineration, it is usually possible to extract DNA from pulp tissue of tooth with sufficient quality and quantity. Hence the present study was undertaken to find out the sex of a child from exfoliated/extracted deciduous teeth using a Polymerase Chain reaction (PCR) based analysis. Tooth samples were stored in room temperature after double coding for various periods. Dental pulp tissue was collected from each sample and DNA was isolated by proteinase-k digestion and phenol chloroform extraction methods. PCR amplification was done with two sets of oligonucleiotide primers. Amplification of X (131bp) and Y-specific sequences (172bp) in males and that of the X-specific sequence in females was observed and compared with the template DNA showing male and female controls. Determination of sexes of all freshly collected samples within 24 hours and after 1 month of extraction respectively gave 100% result. However, PCR was not found to be an effective method for sex determination after 6 months post extraction.