Traditional laboratory tests for the diagnosis of genetic diseases are mostly based on the estimation of metabolites and/or enzymes. This is usually done after the onset of symptoms.
The laboratory tests based on DNA analysis can specifically diagnose the inherited diseases at the genetic level. DNA-based tests are useful to discover, well in advance, whether the […]
Polymerase chain reaction and screening for human immunodeficiency virus
The use of the polymerase chain reaction (PCR) to amplify minute quantities of DNA has revolutionized the ability to detect and analyze DNA species. With PCR it is possible to synthesize sufficient DNA for analysis. Conventional methods for detection and identification of the human immunodeficiency virus (HIV), […]
The use of DNA analysis is a novel and revolutionary approach for specifically identifying the disease-causing pathogenic organisms. This is in contrast to the traditional methods of disease diagnosis by detection of enzymes, antibodies etc., besides the microscopic examination of pathogens. Although at present not in widespread use, DNA analysis may soon take over the […]
Defects in mismatch repair cause hereditary nonpolyposis colon cancer (HNPCC) and probably are important in other cancers. This was initially suggested by the finding that some colon tumors showed frequent mutations in microsatellites (short repeating sequences, particularly mono- and dinucleotides), a phenotype called microsatellite instability.
Unexplained weight loss may be a sign of malignancy, and weight loss is common in advanced cancer. Decreased appetite and food intake contribute to but do not entirely account for the weight loss. The weight loss is largely from skeletal muscle and adipose tissue, with relative sparing of c (i.e., liver, kidney, and heart). Although […]
Breast carcinoma is moderately sensitive to multiple antitumor agents and combinations of medicines produce higher response rates and longer durations of response and survival than single-agent therapy. Within the past decade a number of new agents have been developed and proved effective in the treatment of breast cancer. The most prominent are the taxanes, including […]
Cancer is defined as the excessive division of cells. It is recognized pathologically by a higher fraction of cells actively in the cell cycle than is expected for the normal tissue from which it arose. This includes a higher fraction of cells in mitosis, recognizable by microscopy, and a higher fraction of cells in S-phase.
Coomassie brilliant blue Staining
This is the most often used protein stain. It is extremely sensitive and becomes even more sensitive in the presence of sodium dodecyl sulphate. It can be carried out by two variants of what is principally one single method.
The first step is to fix the separated proteins. This is achieved by soaking […]
Hyperlipidemias are disorders of the rates of synthesis or clearance of lipoproteins from the bloodstream. Usually they are detected by measuring plasma triacylglycerol and cholesterol and are classified on the basis of which class of lipoproteins is elevated.
In general, gene therapy is carried out by introducing a therapeutic gene to produce the defective or the lacking protein. But there are certain disorders (cancer, viral and parasitic infections, and inflammatory diseases) which result in an overproduction of certain normal proteins. It is possible to treat these diseases by blocking transcription using a single-stranded […]
DNA chips or DNA microarrays are recent developments for DNA sequencing as result of advances made in automation and miniarization. A large number of DNA probes, each one with different sequence, are immobilized at defined positions on the solid surface, made up of either nylon or glass. The probes can be short DNA molecules such […]
Some groups of research workers have developed alternate methods to Sanger method for sequencing of DNA. Unfortunately, despite the initial excitement, most of these methods have disappeared from the scene. There are at least two methods with some promise for DNA sequencing pyrosequencing, and gene chips (microarrays).
It is expected that the sequencing of human genome and the genomes of other organisms will dramatically change our understanding and perceptions of biology and medicine. Some of the benefits of human genome project are given.
The most important features of a DNA molecule are the nucleotide sequences, and the identification of genes and their activities. Since 1920, scientists have been working to determine the sequences of pieces of DNA. This was further extended for the complete sequence determination of genome of certain lower organism’s example plasmid pBR 322 in 1979. […]
Determination of nucleotide sequence in a DNA molecule is the basic and fundamental requirement in biotechnology. DNA sequencing is important to understand the functions of genes, and basis of inherited disorders. Further, DNA cloning and gene manipulation invariably require knowledge of accurate nucleotide sequence.
Blotting techniques are very widely used analytical tools for the specific identification of desired DNA or RNA fragments from thousands of molecules. Blotting refers to the process of immobilization of sample nucleic acids or solid support. The blotted nucleic acids are then used as targets in the hybridization experiments for their specific detection.
In 1937 Tiselius described his moving boundary electrophoresis; Konig published the first experiment on the use of filter paper as stabilizing medium in electrophoresis. It, however, took ten more years for filter paper electrophoresis to become popular as an efficient, inexpensive, routine technique. This paved the way for several other porous stabilizing media, most of […]
Separation of large chromosomes of eukaryotes is not possible by conventional electrophoresis. Fluorescence-activated cell sorting, also known as flow cytometry or flow karyotyping, can separate the individual chromosomes of eukaryotes.
Fluorescence-activated cell sorting
To carry out FACS, the dividing cells are carefully broken open, and a mixture of intact chromosomes is prepared. These chromosomes are then stained […]
Electrophoresis can be divided into two main techniques: free electrophoresis or electrophoresis without stabilizing media and zone electrophoresis or electrophoresis in stabilizing media.
Free electrophoresis
Free electrophoresis has two main techniques: microelectrophoresis and moving boundary electrophoresis. Both the techniques have now become obsolete and are at best of historical significance.
Both pig (porcine) and cow (bovine) insulin are commonly used in the treatment of human diabetics. Because of the differences in amino acid sequence from the human insulin, some diabetic individuals will have an initial allergic response to the injected insulin as their immunological system recognizes the insulin as foreign, or develop an insulin resistance […]
Almost all the experiments dealing with gene manipulations require pure forms of either DNA or RNA, or sometimes even both. Hence there is a need for the reliable isolation of nucleic acids from the cells. The purification of nucleic acids broadly involves three stages.
1. Breaking or opening of the cells to expose nucleic acids.
2. Separation of nucleic […]
Electrophoresis refers to the movement of charged molecules in an electric field. The negatively charged molecules move towards the positive electrode while the positively charged molecules migrate towards the negative electrodes.
Gel electrophoresis is a routinely used analytical technique for the separation/purification of specific DNA fragments. The gel is composed of either polyacrylamide or agarose. Polyacrylamide […]
In this Pulse Field Gel Electrophoresis technique, electric field is applied alternatively at definite period of time pulse (60 sec pulse).
Principle
DNA molecules are very long molecules. When electric field is applied these molecules stretch out to become a linear molecule before they migrate to the gel. When the direction is changed the smaller molecules realign […]
Principle
Separation of negatively charged nucleic acid molecules based on their charged by mass ratio on agarose gel in the presence of an electric field.
Topoisomerases are emerging as important targets of antimicrobial and antineoplastic agents. These agents share a common principal mechanism of action by interfering with the enzyme-catalyzed rejoining of DNA strands, in effect inhibiting one of two substeps in the action of Topoisomerases. Therefore topoisomerase medicines do not inhibit overall activity of the enzyme, as is the […]
The presence of reiterated three-base-pair DNA sequences has been noted in a number of human genetic diseases including fragile X syndrome, myotonic dystrophy, X-linked spinal and bulbar muscular atrophy (Kennedy’s syndrome), spinocerebellar ataxia, and Huntington’s disease. These diseases are associated with expansion of certain triplet nucleotide repeats that appear to be over represented in the […]
Telomerase, the ends of linear Eukaryotic chromosomes, are critical for maintaining the stability of the genome. Telomeres are progressively shortened during each cycle of cell division. Upon reaching a critical length, programmed cell death (apoptosis) occurs. Ribonucleoprotein Telomerase acts to maintain or lengthen the Telomeres but is not active in normal somatic cells. Telomerase activity […]
A number of diseases result from abnormalities in lipid metabolism. They are due to genetic defects affecting one of the enzymes necessary for their catabolism.
Acyl CoA dehydrogenase deficiency:
The most common genetic defect in lipid metabolism is an acyl CoA dehydrogenase deficiency. There are four different isoenzymes of acyl CoA dehydrogenase and each acts on fatty […]
Abnormal amounts of glycogen are deposited in various diseases due to inherited deficiencies of the enzymes involved in glycogen metabolism. In addition to this they produce deleterious effects on the growth and well being of the individual. The details of different types of glycogen storage diseases are given below:
Hexokinase (HK) deficiency:
Hexokinase catalyses the first chemical reaction in glycolysis and it exits as isoenzymes. A genetic defect of HK leads to low concentrations of glycolytic intermediates, including 1,3-BPG, the precursor of 2,3-BPG, which binds to hemoglobin and lowers its affinity for oxygen. This effect allows hemoglobin to release oxygen normally in tissue capillaries. Patients […]
Hereditary persistence of fetal hemoglobin (HPFH) is a group of conditions in which fetal hemoglobin synthesis is not turned off with development but continues into older stage. The homozygous form of the disease is extremely uncommon, being characterized by changes in red blood cells similar to those found in heterozygous beta thalassemia. HPFH, in either […]
The local three-dimensional structure of DNA is important in interactions with proteins involved in repair, transcription, recombination, and chromatin condensation. Recently, it has been proposed that antibiotics can induce formation of DNA structures that can recruit these proteins with cytotoxic results. The best-studied example of this phenomenon is the antitumor medicine cisplation, a tetra coordinate […]
With pending completion of the human genome project, a wealth of genetic information is rapidly becoming available. Application of this knowledge to medicine requires development of new techniques to monitor gene expression and rapidly evaluate genes for mutations and other sequence variations. Oligonucleotide arrays have shown great promise for these applications. Such arrays consist of […]
Peroxisomes are responsible for a number of important metabolic reactions, including synthesis of glycerol ethers, shortening very-long-chain fatty acids so that mitochondrial can completely oxidize them, and oxidation of the side chain of cholesterol needed for bile acids synthesis.
Clinical correlation
Human Lysosomal acid lipase (hLAL) hydrolyzes triacylglycerol to free fatty acids and glycerol, and cholesteryl esters to free cholesterol and fatty acids. It is a critical enzyme in cholesterol metabolism, serving to make available free cholesterol for the needs of cells. Cholesteryl ester storage disease (CESD) and Wolman’s disease are two distinct phenotypic forms […]
Catabolism of purines, nitrogen-containing heterocyclic compounds found in nucleic acids, leads to formation of uric acid, which is excreted in urine normally. Gout is an abnormality in which uric acid is produced in excess, leading to an increase of uric acid in blood and deposition of urate crystals in joints.
Introduction
Traditional procedures of vaccination have used purified components of an infectious organism, dead or attenuated intact cells or viruses, to provide individuals with active immunity by eliciting production of specific antibodies. Many have been successful in providing protection against diseases such as polio, smallpox, whooping cough, typhoid fever, and diphtheria.
Apoptosis: programmed cell death
Eukaryotic cells have genetically regulated mechanisms for programmed cell death, termed apoptosis. Programmed cell death is important during embryogenesis and throughout older life and occurs when a cell has fulfilled its biological function. It is distinct from necrotic death of a cell caused by injury due to radiation or anoxia. Initiation of […]
Electrophoretic analysis of the plasma proteins is commonly used in diagnosis of disease. Electrophoresis of plasma buffered at pH 8.6 separate the major plasma proteins as they migrate to an anode in the electric field into bands or peaks, based on their charge differences.
Introduction
The endocrine system consists of ductless glands, which secrete hormones. The hormones are directly poured into circulation without the help of a duct. Hormone is the secretion of an endocrine gland, which is transported to a site distant from its origin where it produces it action.
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