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), […]
Chromatography is one of the most useful and popular tools of biochemistry. It is an analytical technique dealing with the separation of closely related compounds from a mixture. These include proteins, peptides, amino acids, lipids, carbohydrates, vitamins and medicines.
Flow Cytometry has numerous applications and as in case of PCR it might be said that the applications of flow Cytometry are limited only by our own imagination. A detailed discussion of these applications is impossible her. However, major well documented applications have been cursorily dealt with.
There is numerous application of PCR and since the techniques is comparatively recent, new applications are being added with an amazing speed. We will not discuss the applications which are common and which can easily be imagined by the reader. Instead some of the novel applications of PCR are discussed in this section.
PCR in cancer […]
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), […]
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 […]
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).
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.
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.
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
Normally, the red blood cells remain suspended uniformly in circulation. This is called suspension stability of red blood cells. If blood is mixed with an anticoagulant and allowed to stand on a vertical tube, the red cells settle down due to gravity with a supernatant layer of clear plasma. The rate at which the cells […]
Principle
This technique combines the technique of IEF, which separates proteins in a mixture according to charge isoelectric point, with the size separation technique of SDS-PAGE. When combined to give 2D PAGE, the most sophisticated analytical method for separating proteins available is obtained.
Gel preparation of Isoelectric focusing gel electrophoresis
The carrier ampholytes, riboflavin and acrylamide solution with covering suitable pH range mixture is poured over a glass plate (25×10cm) that contains a spacer. The 2nd glass plate is then placed on top of the first to form gel cassette. Gel is polymerized by photo polymerization by placing the […]
Principle
Isoelectric focusing gel electrophoresis is a separation of amphoteric substance such as protein, amino acids and peptides based on their different isoelectric point on supporting medium using electricity.
Introduction
Separation of protein is carried out using cross-linked polyacrylamide gel electrophoresis. This is due to molecular sieving property of polyacrylamide gel electrophoresis that slows the migration of protein according to their molecular weight. Proteins were originally separated on polyacrylamide gels that were polymerized in a glass tubes, approximately 7 mm in diameter and about 10cm […]
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