CELL AND MOLECULAR BIOLOGY PPT.pdf

CELL AND MOLECULAR BIOLOGY Chromatography is a combination of two words; * Chromo – Meaning color * Graphy – representation of something on paper.
Chromatography, literally "color writing", was first employed by Russian scie...

CELL AND MOLECULAR BIOLOGY Chromatography is a combination of two words; * Chromo – Meaning color * Graphy – representation of something on paper.
Chromatography, literally "color writing", was first employed by Russian scientist Mikhail Tswett in 1903/1906. He continued to work with chromatography in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls. Since these components have different colors (green, orange, and yellow,respectively) they gave the technique its name.
 DEFINITION It is a physical method of separation in which the components of a mixture are separated by differences in their distribution between two phases, one of which is stationary (stationary phase) while the other (mobile phase) moves through it in a definite direction. The substances must interact with the stationary phase to be retained and separated by it. The stationary phase may be a solid, or a liquid supported on a solid or gel, the mobile phase may be either a gas or a liquid.
CHROMATOGRAPHY , Analyze Identify Purify Quantify Separate Components
Mixture. The molecules in the mixture to be seperated are the solutes. Components of Chromatography
Mobile Phase – gas or liquid that carries the mixture of components through the stationary phase. It is the phase which moves in a definite direction. Analyte (Sample):It is the substance to be separated during chromatography. Eluate:It is the mobile phase leaving the column. Stationary Phase – the part of the apparatus that holds the components as they move through it, separating them, those with strong attraction to the support move more slowly than those with weak attraction(this is how the components are seperated) Stationary phase (bounded phase):It is a phase that is covalently bonded to the support particles or to the inside wall of the column tubing examples silica gel and alumina.
 Chromatogram: It is the visual output of the chromatograph. Chromatograph: It is equipment that enables a sophisticated Separation. Retention time ; It is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions.Eluent:It is the solvent that will carry the analyte. Eluent: Fluid entering a column. Eluate: Fluid exiting the column. Elution: The process of passing the mobile phase through the column. Flow rate: How much mobile phase passed / minute (ml/min). Linear velocity: Distance passed by mobile phase per 1 min in the column (cm/min).
Uses for Chromatography
Chromatography is used by scientists to: Analyze – examine a mixture, its components, and their relations to one another Identify – determine the identity of a mixture or components based on known components Purify – separate components in order to isolate one of interest for further study Quantify – determine the amount of the a mixture and/or the components present in the sample. Real-life examples of uses for chromatography: Pharmaceutical; determine amount of each chemical found in new product. Company Hospital; detect blood or alcohol levels in a patients blood stream Law Enforcement; to compare a sample found at the crime scene to samples from suspects Environmental Agency; determine the level of pollutants in the water supply TYPES OF CHROMATOGRAPHY
Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase) Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase) Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase) Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase).  RADIOIMMUNOASSAY Radioimmunoassay (RIA) is a laboratory technique used to measure the concentration of a specific antigen or hormone in a sample. It's a highly sensitive and specific method that uses radioactivity to detect and quantify the binding of antibodies to antigens..PRINCIPLE 1. Preparation: A known amount of radiolabeled antigen (tracer) is added to a sample containing the antigen of interest. A specific antibody that binds to the antigen is also added. 2. Incubation: The mixture is incubated, allowing the antibody to bind to the antigen and the radiolabeled tracer. 3. Separation: The bound antibody-antigen complex is separated from the unbound tracer using techniques like centrifugation or filtration. 4. Measurement: The radioactivity in the bound fraction is measured using a scintillation counter or gamma counter. 5. Calibration: A standard curve is created by plotting the radioactivity against known concentrations of the antigen. 6. Interpolation:The unknown sample's antigen concentration is determined by interpolating its radioactivity measurement on the standard curve.. RIA is widely used in various fields, including: - Endocrinology (hormone measurement) - Immunology (antibody detection) - Infectious disease diagnosis (viral and bacterial antigen detection) - Cancer research (tumor marker detection) RIA offers high sensitivity and specificity, but it also has some limitations, such as: - Radioactive material handling requirements - Limited shelf life of radiolabeled tracers - Potential for non-specific binding Enzyme-linked immunosorbent assay (ELISA) has largely replaced RIA in many applications, but RIA remains a valuable technique in certain fields. PCR

Polymerase Chain Reaction. It's a laboratory technique used to make many copies of a specific DNA sequence. Here's a brief overview: 1. Denaturation: DNA double helix is melted into single strands. 2. Annealing: Primers bind to target DNA sequence. 3. Extension: DNA polymerase enzyme synthesizes new DNA strands. 4. Repeat: Steps 1-3 are repeated multiple times, amplifying the target sequence. PCR is a powerful tool in molecular biology, used for: - DNA cloning - Genetic testing - Forensic analysis. - PCR is a laboratory technique used to amplify specific DNA sequences, developed in 1983 by Kary Mullis. Revolutionized molecular biology, genetics, and forensic science. A PCR has five core ‘ingredients’: The DNA template to be copied. primers – short stretches of DNA or RNA, 20 to 30 bases in length, that bind either side of the DNA section of interest and mark the point that PCR starts. DNA nucleotide bases (also known as dNTPs). DNA bases (A, T, C and G) are the building blocks of DNA and are needed to construct the new strand of DNA. an enzyme, called Taq polymerase, which adds the bases to the copied sequence. a buffer to ensure the right conditions for the reaction. The PCR Process 1. Denaturation (94-96°C): DNA double helix is melted into single strands 2. Annealing (50-65°C): Primers bind to target DNA sequence 3. Extension (72°C): DNA polymerase enzyme synthesizes new DNA strands 4. Repeat (steps 1-3): Multiple cycles of denaturation, annealing, and extension  Denaturing The reaction mixture is heated to 94-95⁰C, for between 15 and 30 seconds. The high temperature causes the hydrogen bonds between the bases in two strands of template DNA to break and the two strands to separate. This results in two single strands of DNA, which will act as templates to produce the new copies of each strand of DNA. It is important that the temperature is maintained at this stage for long enough to ensure that the DNA strands have separated completely. Annealing. The reaction is cooled to enable the primers to attach to a specific location on the single-stranded template DNA by way of hydrogen bonding. The temperature depends on the characteristics of the primer, but is usually between 50 and 65⁰C. The two separated strands of DNA are complementary and run in opposite directions (from one end – the 5’ end – to the other – the 3’ end). As a result, there are two primers – a forward primer and a reverse primer. This step is essential because the primers serve as the starting point for DNA synthesis, by providing a short region of double stranded DNA for the polymerase enzyme to work with. Only once the primer has bound can the polymerase enzyme attach and start making the new complementary strand of DNA from the loose DNA bases, in the extending step. The annealing step usually takes about 10-30 seconds. Extending The heat is increased to 72⁰C to enable the new DNA to be made by a special Taq DNA polymerase enzyme which adds DNA bases. Taq DNA polymerase is an enzyme taken from the bacteria Thermus aquaticus (“Taq”): This bacterium normally lives in hot springs so can tolerate temperatures above 80⁰C, but its optimum temperature is 72⁰C. The bacteria’s DNA polymerase is very stable at high temperatures, which means it can withstand the temperatures needed to break the strands of DNA apart in the denaturing stage of PCR. DNA polymerase from most other organisms would not be able to withstand these high temperatures. For example, human polymerase works ideally at 37˚C (body temperature). At 72⁰C, the Taq polymerase begins to build the complementary strand. It attaches to the primer and then adds DNA bases to the single strand one-by-one in the 5’ to 3’ direction. The result is a brand-new strand of DNA and a double-stranded molecule of. Types of PCR 1. Conventional PCR: Traditional PCR method 2. Real-time PCR (qPCR): Monitors amplification in real-time 3. Reverse Transcription PCR (RT-PCR): Amplifies RNA sequences 4. Multiplex PCR: Amplifies multiple sequences simultaneously Applications Genetic testing, Gene expression analysis,Forensic analysis, Cancer research, Forensic analysis, Cancer research,Viral load detection and DNA cloning Advantages - High sensitivity: Detects small amounts of DNA - High specificity: Targets specific DNA sequences - Fast results: Amplification in a few hours Limitations - Contamination risk: Requires strict laboratory protocols Inhibitors: Substances that interfere with PCR efficiency Enzyme Immunoassay An enzyme immunoassay (EIA) is a type of immunoassay that uses an enzyme bonded to an antigen or antibody to detect specific molecules in a sample. There are several types of enzyme immunoassays, including. - Enzyme-linked immunosorbent assay (ELISA) - Enzyme multiplied immunoassay technique (EMIT) - Fluorescent enzyme immunoassays (FEIAs) - Chemiluminescent immunoassays (CLIAs) - Radioimmunoassays (RIAs) These assays rely on the binding of antibodies to specific antigens, and the enzyme label allows for detection through color changes, fluorescence, or chemiluminescence. EIAs are widely used in medical and research applications for measuring protein, pathogens and other molecules in blood samples.