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Semester 3: Molecular Biology, Bioinstrumentation and Biotechniques
Process of Transcription: gene structure, RNA polymerases, transcription factors, steps in transcription
Process of Transcription
Gene Structure
Genes are segments of DNA that encode for proteins or functional RNA. Each gene consists of regulatory regions and coding sequences. The regulatory regions include promoters, enhancers, and silencers that control the gene's expression. The coding sequence is transcribed into mRNA.
RNA Polymerases
RNA polymerases are enzymes responsible for synthesizing RNA from a DNA template. In eukaryotes, there are three main types: RNA polymerase I synthesizes rRNA, RNA polymerase II synthesizes mRNA and some snRNA, and RNA polymerase III synthesizes tRNA and other small RNAs.
Transcription Factors
Transcription factors are proteins that bind to specific DNA sequences to regulate the transcription of genes. They can act as activators or repressors, facilitating or inhibiting the recruitment of RNA polymerase to the promoter regions.
Steps in Transcription
Transcription occurs in several stages: 1. Initiation - RNA polymerase binds to the promoter region, aided by transcription factors. 2. Elongation - RNA polymerase unwinds the DNA and synthesizes RNA complementary to the template strand. 3. Termination - RNA polymerase reaches a termination signal, leading to the release of the newly synthesized RNA molecule.
Process of Translation: genetic code, ribosomes, translation factors, stages of translation
Process of Translation
Genetic Code
The genetic code is the set of rules by which information encoded within genetic material is translated into proteins. It consists of sequences of nucleotides arranged in triplet codons, where each codon corresponds to a specific amino acid or a stop signal. The universal nature of the genetic code allows for the transfer of genetic information across different organisms.
Ribosomes
Ribosomes are the cellular machinery responsible for protein synthesis. They are composed of ribosomal RNA and proteins, forming two subunits: large and small. Ribosomes read the mRNA template and facilitate the assembly of amino acids into polypeptide chains, functioning as the site of translation.
Translation Factors
Translation factors are various proteins that assist in the process of translation. They play crucial roles in the initiation, elongation, and termination stages. Initiation factors help assemble the ribosome and recruit mRNA, while elongation factors aid in the addition of amino acids to the growing polypeptide chain, and termination factors signal the end of protein synthesis.
Stages of Translation
Translation occurs in three main stages: initiation, elongation, and termination. During initiation, the ribosome assembles around the start codon of mRNA. In elongation, tRNA molecules bring amino acids to the ribosome, where they are added to the growing polypeptide chain. Finally, termination occurs when a stop codon is reached, prompting the ribosome to release the completed protein.
Regulation of Gene Expression I: gene regulation in prokaryotes and eukaryotes, chromatin role, post-transcriptional modifications
Regulation of Gene Expression
Gene Regulation in Prokaryotes
Gene Regulation in Eukaryotes
Role of Chromatin
Post-Transcriptional Modifications
Regulation of Gene Expression II: translational regulation, post-translational modifications, gene silencing, RNA interference
Regulation of Gene Expression II
Translational Regulation
Translational regulation is the control of the levels of protein synthesis from mRNA. Mechanisms include the availability of ribosomes, initiation factors, and the stability of the mRNA. Specific sequences in the mRNA, known as untranslated regions (UTRs), play a crucial role in modulating translation efficiency.
Post-Translational Modifications
Post-translational modifications are chemical changes that occur after protein synthesis. Common modifications include phosphorylation, glycosylation, methylation, and ubiquitination. These alterations can influence protein function, localization, stability, and interactions with other molecules.
Gene Silencing
Gene silencing refers to the mechanisms that block gene expression. This can occur through various processes such as DNA methylation, histone modification, and RNA interference. Epigenetic factors can play a significant role in gene silencing, affecting heritable changes in gene expression.
RNA Interference
RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation. This mechanism involves small interfering RNAs (siRNAs) and microRNAs (miRNAs). RNAi serves as a crucial regulatory mechanism in various organisms, playing roles in development, cell differentiation, and defense against viruses.
Principle and Types of Microscopes: microscopy types, applications
Principle and Types of Microscopes
Basic Principles of Microscopy
Microscopy involves the use of instruments to view small objects that cannot be seen with the naked eye. The basic principles include light or electrons interacting with the sample, magnification, resolution, and contrast. Microscopes can be classified based on their illumination source and the type of lenses used.
Types of Microscopes
Light Microscopes
Use visible light and optical lenses to magnify specimens. They include compound microscopes, stereo microscopes, and phase contrast microscopes. They are widely used in biological sciences.
Electron Microscopes
Use a beam of electrons for illumination, providing higher resolution than light microscopes. The two main types are transmission electron microscopes (TEM) and scanning electron microscopes (SEM). TEM provides internal details, while SEM provides 3D surface images.
Fluorescence Microscopes
Utilize fluorescence instead of reflected light. They are used to study specimens that fluoresce naturally or have been stained with fluorescent dyes, enabling visualization of specific components.
Confocal Microscopes
Use a laser to scan specimens and create high-resolution images, allowing for 3D reconstruction. They are valuable in biological research for studying the structure and function of cells.
Atomic Force Microscopes
Employ a probe to scan the surface of a specimen, providing atomic-level resolution. Useful for nanotechnology and material science studies.
Applications of Microscopy
In Biological Research
Microscopes are essential for studying cell structures, microorganisms, and tissue samples. They allow researchers to visualize biological processes and diagnose diseases.
In Material Science
Microscopy techniques are used to examine material properties, structures, and defects at micro and nano scales.
In Clinical Settings
Microscopes are utilized in diagnostic labs for analyzing blood samples, smears, and biopsies to identify pathogens or abnormalities.
Centrifugation and Chromatography: principles, types of centrifuges and chromatography
Centrifugation and Chromatography
Principles of Centrifugation
Centrifugation is a technique that involves the application of centrifugal force to separate components of different densities. The sample is placed in a rotor and spun at high speeds, causing denser materials to move outward and lighter materials to remain closer to the center. This process leverages the principles of sedimentation and particle size.
Types of Centrifuges
There are several types of centrifuges, including: 1. **Clinical Centrifuges**: Used primarily in medical laboratories for blood and urine samples. 2. **Refrigerated Centrifuges**: Maintains low temperatures during centrifugation, essential for heat-sensitive samples. 3. **Ultracentrifuges**: Operates at extremely high speeds to separate smaller particles such as viruses and proteins. 4. **Tabletop Centrifuges**: Compact and convenient for various laboratory applications. 5. **Continuous Flow Centrifuges**: Allow the continuous processing of samples, ideal for industrial applications.
Principles of Chromatography
Chromatography is a separation technique based on the distribution of components between two phases: a stationary phase and a mobile phase. Components in the sample interact differently with these phases, leading to separation as the mobile phase moves through the stationary phase.
Types of Chromatography
Chromatography can be classified into various types, including: 1. **Paper Chromatography**: Involves the use of a strip of paper as the stationary phase. 2. **Column Chromatography**: Utilizes a column filled with stationary phase material, allowing separation based on size or polarity. 3. **Thin Layer Chromatography (TLC)**: A modern version of paper chromatography, using a thin layer of adsorbent material coated on a glass plate. 4. **Gas Chromatography (GC)**: Separates volatile substances using a gas as the mobile phase. 5. **Liquid Chromatography (LC)**: Involves liquid mobile phases, used for more complex separations.
Spectrophotometry and Biochemical Techniques: pH measurement, spectrophotometry principles, radio-tracer techniques
Spectrophotometry and Biochemical Techniques
Item
pH measurement is the determination of the acidity or alkalinity of a solution. It is a critical parameter in many biochemical processes.
pH influences enzyme activity, metabolic pathways, and the solubility of compounds in biological systems.
pH meter
pH paper
Glass electrode method
Biochemical assays
Cell culture
Water quality testing
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Spectrophotometry is a technique used to measure the amount of light absorbed by a solution at specific wavelengths.
Based on Beer-Lambert law, it states that the absorption of light is directly proportional to the concentration of the absorbing species.
Light source
Monochromator
Sample cuvette
Detector
Quantifying biomolecules
Determining enzyme kinetics
Analyzing protein concentrations
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Radio-tracer techniques involve the use of radioactive isotopes to study biochemical processes in living organisms.
Radioactive isotopes emit radiation that can be detected, allowing for the tracking of substances within biological systems.
Metabolic studies
Locating tumor sites
Tracing biochemical pathways
Molecular Techniques: nucleic acid detection, DNA sequencing, PCR, PAGE, ELISA, Western blot
Molecular Techniques
Nucleic Acid Detection
Methods to identify nucleic acids (DNA and RNA) in samples include hybridization techniques, fluorescence in situ hybridization (FISH), and real-time PCR. These techniques allow for the visualization and quantification of specific nucleic acid sequences.
DNA Sequencing
DNA sequencing determines the precise order of nucleotides in a DNA molecule. Techniques include Sanger sequencing and next-generation sequencing (NGS). Sanger sequencing relies on chain-termination methods, while NGS allows for massive parallel sequencing, significantly increasing throughput and reducing costs.
Polymerase Chain Reaction (PCR)
PCR is a technique used to amplify specific DNA sequences. It involves repeated cycles of denaturation, annealing, and extension. Variants include quantitative PCR (qPCR) for measuring DNA quantity and reverse transcription PCR (RT-PCR) for amplifying RNA.
Polyacrylamide Gel Electrophoresis (PAGE)
PAGE is used for the separation of proteins and nucleic acids based on size and charge. PAGE provides high resolution and is commonly utilized in analyzing DNA fragments, RNA molecules, and proteins.
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA is a plate-based assay technique used for detecting and quantifying proteins, hormones, antibodies, or antigens. It involves binding the target molecule to a solid surface and using enzyme-linked antibodies for detection, producing a measurable signal.
Western Blot
Western blotting is a technique for detecting specific proteins in a sample. It involves the separation of proteins by gel electrophoresis, transfer to a membrane, and identification using specific antibodies. This technique is widely utilized in research and clinical diagnostics.
