The polymerase chain reaction

The Polymerase Chain Reaction (PCR) is a technique for the in vitro synthesis of billions of copies of a specific nucleic acid sequence by performing successive rounds of in vitro nucleic acid replication. This is achieved by using two oligonucleotide primers that hybridize (annealing) to the opposite strand of the target DNA at positions that flank the region to be amplified through simultaneous extension of both primers

श्वेत चित्ति संलक्षण विषाणु (वाइट स्पोट सिंड्रोम वाइरस) के लिए सी एम एफ आर आइ का पी सी आर किट

कृपया पूरा लेखा पढ

The principles of isolation, purification and analysis of nucleic acids

Advanced Biotechnological research is largely depended on the genome analysis and recombinant DNA technology. Good quality nucleic acid is an essential prerequisite for consistent results in most of the down stream applications in the genome analysis and recombinant DNA technology. The general principle underlying the isolation of nucleic acids is common with few modifications depending on the type of nucleic acids being isolated. The type of the nucleic acid intending to isolate is to be made free from the other biological macromolecules and cell debris. This is achieved by properly lysing the cell wall or cell membrane as the case may be and by selectively denaturing the other macromolecules like proteins

Molecular identification of heat shock protein 70 (Hsp70) gene in the Indian edible oyster Crassostrea madrasensis (Preston) and Indian brown mussel Perna indica Kuriakose & Nair, 1976

Bivalves are constantly exposed to different kinds of stressors as they live in a habitat with frequent changes in environmental parameters. The xenobiotic pollutants also contribute to the stressful routine of bivalves. Studies on the genes which mediate and contribute to the physiological plasticity of bivalves in stressful situations, induced by natural and anthropogenic agents are gaining importance. Among the stress related genes, HSP family genes play an important role in managing stress induced by various factors. Recent reports underline the role of heat shock proteins in thermo tolerance, host defense and even in aging. Here we report the molecular expression and detection of heat shock protein genes (Hsp70) from the Indian edible oyster Crassostrea madrasensis and the Indian brown mussel Perna indica with unique distribution in Indian waters. The c- DNA reverse transcribed from the total RNA of gill was used as template in Polymerase Chain Reaction (PCR) to amplify Hsp70 gene segments with primers designed from the conserved nucleotide sequences of Crassostrea gigas and Perna viridis. PCR products were sequenced, and the similarity search in NCBI-BLAST confirmed the molecular identity of targeted genes. Phylogenetic analysis of the Hsp gene sequence data reveals the unique position of the Indian edible oyster and Indian brown mussel among the other counterparts inhabiting rest of the world. This stands out as the first report on the expression and PCR amplification of stress related genes from Indian bivalves

Morphometric and Genetic Analyzes of Indian Mackerel (Rastrelliger kanagurta) from Peninsular India

A holistic approach, combining one phenotypic and two genotypic methods, was adopted to analyze possible population differences in Indian mackerel (Rastrelliger kanagurta) from selected centers in the East and West coasts of India. Principal component analysis of truss landmark variables revealed that the area encompassing depth between the origin of anal and origin of second dorsal and caudal peduncle depth has high component loadings. Bivariate scatter plots of principal components showed a great degree of morphometric homogeneity between Indian mackerel populations from Mandapam, Kochi and Karwar. Clustering pattern of polypeptide markers revealed relatively greater population homogeneity among Mandapam fish (58%) than Kochi samples (33%). The three random amplified polymorphic DNA (RAPD) primers used in the present study have generated a total of 59 loci varying in size from 560 to 4500 bp. None of the populations from Mandapam, Kochi and Karwar showed RAPD fragments of fixed frequencies, to be treated as population-specific markers. No significant differences were found among the three populations

Identification of antioxidant enzyme genes of the Indian edible oyster, Crassostrea madrasensis (Preston) through polymerase chain reaction

When an organism is exposed to stress by way of environmental fluctuations or pathogenic attack, reactive oxygen species (ROS), which cause severe oxidative damage to the cells and hamper the cellular as well as membrane functions, are produced. In order to counter these effects, the cells activate the production of antioxidant enzymes which play pivotal role in removing ROS and maintaining the homeostasis within the cells. Crassostrea madrasensis is a promising bivalve species, living in intertidal region amidst a variety of stressors. In the present study, the RNA was isolated from gills and cDNA synthesised by Reverse Transcription PCR (RT- PCR) and amplification of these c DNA were carried out using a combination of different primers designed for super oxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPX).. The PCR generated amplicons of 464 bp, 171 bp and 147 bp of Cu/Zn-SOD, CAT and GPX respectively were purified and sequenced. Similarity search in NCBI - BLAST confirmed these sequences as the respective antioxidant enzyme genes. The amino acid profile of Cu/Zn-SOD deduced from the sequences representing original reading frame (ORF) on InterProScan analysis was found to contain the characteristic Cu and Zn binding domains. PCR with the SOD specific primer pair resulted in the amplification of ORF of SOD with both genomic DNA and C-DNA as templates. This indicates the intronless nature of SOD gene, an adaptation to initiate fast expression at times of stress, as observed in certain other stress related genes such as the inducible form of heat shock protein 70 (Hsp 70). This is the first report on the molecular detection and identification of antioxidant enzyme genes of Indian edible oyster

Stress, heat shock proteins & biotechnological interventions. In: Winter School on Impact of Climate Change on Indian Marine Fisheries held at CMFRI, Cochin 18.1.2008 to 7.2.2008

Fish are exposed to stressors in nature, as well as in artificial conditions such as in aquaculture, or in the laboratory. The increasing contamination of bodies of natural freshwater and marine ecosystem around the world by anthropogenic substances is one category of environmental stressor. Various stressors, such as grading, transportation, and vaccination, are necessary components of modern intensive fish culture. The response of the fish to such stressors involves all levels of organization, from the cell, to the individual organism, to the structure of the population. In as much as the responses of the fish to a stressor is the essence of maintaining homeostasis, it is not surprising that fish respond to a variety of stressors in a generalized way at all these levels of organization. Stress is most often associated with a negative perspective. This is natural as the word and concept in common use is generally associated with a system that is severely challenged, and often fatigued. Experimental biologists are all involved in the practice of systematically imposing some perturbation and measuring a response

Morphometric structure of the jumbo tiger prawn, Penaeus monodon Fabricius, 1798 from southeast and southwest coasts of India

Morphometric variables were measured to detect variation among the random population samples of Penaeus monodon from east and west coast - Kochi, Calicut, Mangalore, Karwar, Kakinada and Chennai of India. Among these variables, PCL showed the highest correlation with the tail weight (TLW) in both males (0.9605) and females (0.9639). Truss network analysis of 26 measurements from the six centres were log transformed and were subjected to Principal Component Analysis and accounted a total of 89.15% variations in truss measurements data and showed no separate cluster formation in the plot of sheared PC scores and hence confirm homogeneous stock structure

Changes in serum proteins and immune response in oreochromis mossambicus induced by aflatoxin B

Studies were conducted to evaluate the molecular changes induced by aflatoxin, the most frequently encountered dietary carcinogen in Oreochromis mossambicus. Three sub-lethal test doses of aflatoxin B1 incorporated in feed (1.5, 5.0 and 15.0mg/kg feed) were given to the fishes for a period of 30 days and the changes in serum protein profile were resolved through SDS PAGE at regular intervals. Similarly, the changes in immune responses were also studied. The study revealed significant changes in both protein profile and immune response in the experimental fishes compared to the control, indicating that the toxin exerts its effect even at sublethal levels

Genetic Characterization of Aeromonas hydrophila using Protein Profiling and RAPD PCR

Genetic relationship among Aeromonas hydrophila isolates from fish and traditional brackishwater farms were evaluated through Randomly Amplified Polymorphic DNA (RAPD) analysis as well as Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS PAGE) profile of cellular proteins. PCR amplification of the DNA from the bacterial isolates using five random primers (OPA-01, 03, 04, 05 and 10) produced 46 amplicons, which were scorable as distinct bands on agarose gel electrophoresis. Absolute polymorphism of RAPD profile was evident with a unique pattern for each isolate, indicating its usefulness as an ideal tool for evaluation of genetic heterogeneity within the species, which are not revealed by other methods like morphological and biochemical characterization and cellular protein profile. Cellular protein profile did not reveal significant polymorphism as all the isolates revealed uniform pattern indicating its usefulness as a tool for species level identification. Four protein bands of molecular size viz., 19.5 kDa, 25.6 kDa, 29 kDa and 65.6 kDa were shared by all the isolates in the study