Genetic Engineering Lecture Notes 2024-2025 PDF

College of Applied Science Department of Biotechnology Genetic Engineering 4th stage Dr. Noor Thaer Adnan Lecture -3 2024-2025 Genetic Engineering - Dr.Noor Thaer 1 Isolation of RNA Isolation of RNA molecules is...

College of Applied Science Department of Biotechnology Genetic Engineering 4th stage Dr. Noor Thaer Adnan Lecture -3 2024-2025 Genetic Engineering - Dr.Noor Thaer 1 Isolation of RNA Isolation of RNA molecules is crucial to many molecular biology applications, such as cloning, reverse transcription for complementary DNA (cDNA ). Synthesis There are various approaches to RNA extraction and purification including: o phenol-chloroform extraction o spin column purification o magnetic bead-based methods Methods are selected based on: o sample characteristics o contaminants types o target RNA length o abundance o scale of sample processing o downstream analysis. Genetic Engineering - Dr.Noor Thaer 2 Isolation of RNA Requires STRICT precautions to avoid sample degradation. RNA especially labile. RNases are naturally occurring enzymes that degrade RNA Common Laboratory Contaminant (From Bacterial And Human Sources) Also released from cellular compartments during isolation of RNA from biological samples and can be difficult to inactivate RNases are ubiquitous, highly stable, and active in virtually any aqueous environment, and they can regain their activity after denaturation. RNAis therefore subject to rapid degradation by RNases, and scrupulous care must be taken in the laboratory setting to overcome the lability of RNA. RNA isolationrequires steps to inhibit or degrade cellular RNases and prevents their reintroduction into the isolated RNA Genetic Engineering - Dr.Noor Thaer 3  RNases act without cofactors and are stable enzymes.. Small amounts of RNase are sufficient to destroy RNA If RNA isolation is delayed, samples should be stored at −86° C or less temporarily in buffer with RNase inhibitors. Addition of Guanidinium thiocyanate or β-mercaptoethanol to the RNA isolation reagents inhibits or denatures RNase. Purified RNA is rehydrated in nuclease-free water and stored at −86° C to further inhibit any remaining RNase activity. Total RNA : are all types of RNA extracted from a cell which is composed of: rRNA 80% recognizing and binding to the mRNA molecule to help translate the information in (mRNA) into protein mRNA 1-5% messenger RNA functions as a means to transfer genetic information encoded by the DNA in a cell's nucleus to protein-building machinery tRNA The remaining mediating the Transfer of the appropriate amino acid to the ribosome during protein synthesis via the anticodon 4 Genetic Engineering - Dr.Noor Thaer RNA Isolation Unlike genomic DNA, which can be isolated from both active and inactive cells in a sample, the isolation of RNA allows one to view the genes that are being actively transcribed. The isolation of RNA is necessary for a variety of experiments such as reverse transcription complementary DNA (cDNA) synthesis, cloning, RNA sequencing (RNA- seq), and gene expression analysis. Where DNA analyses identify the presence of a gene, RNA analysis measure gene expression and can be used to indicate the function or role of a gene or taxon. The transcriptome of an individual cell is (the full repertoire of RNA transcripts at a given point in time) The isolation of RNA, a process that refers to the extraction of RNA molecules from biological material, is an integral aspect of molecular biology and genetics. RNA purification, also called RNA cleanup, involves the removal of salts, excess reagents, enzymes, or other impurities from the isolated RNA samples Genetic Engineering - Dr.Noor Thaer 5  Mechanical homogenization: The mechanical homogenization step in many of isolation protocols can be further substituted by tissue disassociating enzymes for increased yield and purity. To obtain sufficient yield and quality RNA, mechanical homogenization is considered necessary in industrial, medical, and agricultural applications. Chemical cell lysis: Many of these methods are designed for high-throughput phenol-free extraction of total RNA from tissue, cells, Genetic Engineering - Dr.Noor Thaer 6 Trizol -Chloroform Extraction Method Genetic Engineering - Dr.Noor Thaer 7  Trizol works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components Genetic Engineering - Dr.Noor Thaer 8  Silica Membrane-Based RNA Isolation The principle of using RNA silica-membrane technology to achieve RNA separation and purification is mainly to utilize the binding of nucleic acid to silica matrix material under high salt conditions and detachment under low salt conditions. Genetic Engineering - Dr.Noor Thaer 9 Isolation of mRNA from total RNA Genetic Engineering - Dr.Noor Thaer 10 Eukaryotic mRNA Genetic Engineering - Dr.Noor Thaer 11 mRNA purification using mRNA purification using oligodT matrix magnetic oligodT beads Genetic Engineering - Dr.Noor Thaer 12 Gel Electrophoresis electrophoresis is a basic biotechnology technique that separates macromolecules according to their charge and size. It is frequently used to analyze and manipulate samples of DNA, RNA, or proteins. Gel electrophoresis has a rich history, dating back to the early 20th century. Initially, in the 1930s, scientists like Lothar Cremer and Alois H. H. F. K. G. Platt began experimenting with electrophoresis to separate proteins in solution. Genetic Engineering - Dr.Noor Thaer 13 In gel electrophoresis, samples to be separated are applied to a porous gel medium made of a material such as agarose. Agarose gels are made by pouring a molten solution of agarose and buffer into a gel mold called a casting tray. Before the agarose solidifies, a comb is placed in the casting tray to create a row of wells into which samples are loaded once the comb is removed from the solidified gel. The casting tray and solidified gel are then placed in an electrophoresis chamber that has wire electrodes at each end. The gel is covered with a buffer that controls the pH of the system and conducts electricity. The comb is then carefully removed from the gel and samples are loaded into the resulting wells using a pipet. Once all the samples have been loaded into the wells, the chamber is connected to a power supply and an electrical current is applied to the gel. The chamber is designed with a positive electrode (anode) at one end and a negative electrode (cathode) at the other end. Molecules with a net negative charge migrate toward the positive electrode and molecules with a net positive charge migrate toward the negative electrode because opposite charges attract. Genetic Engineering - Dr.Noor Thaer 14 loading dyes are an important component in gel electrophoresis and are mixed with samples for use. Loading dyes have three important functions for gel electrophoresis. First, the main function is that the dye visualizes the sample, thus simplifying the loading process. Second, loading dyes are used as color indicators of nucleic acid migration in gel electrophoresis, allowing for the estimation of the distance that nucleic acid fragments have migrated. Finally, loading dyes containing Ficoll or glycerol add density to the sample so that it sinks into the gel and does not diffuse into the buffer Factors Affecting Speed of Molecules Migration ❑ Net Charge ❑ Size ❑ Shape Genetic Engineering - Dr.Noor Thaer 15 Genetic Engineering - Dr.Noor Thaer 16  Genetic Screening the process of testing a population for a genetic disease in order to identify a subgroup of people that either have the disease or the potential to pass it on to their offspring Genetic Screening Methods Cytogenetic (to examine whole chromosomes) Biochemical (to measure protein produced by genes) Molecular (to look for DNA mutations) Genetic Engineering - Dr.Noor Thaer 17 Types of genetic screening 1. Forward genetics (or a forward genetic screen) is an approach used to identify genes (or set of genes) responsible for a particular phenotype of an organism. 2. Reverse genetics (or a reverse genetic screen), on the other hand, analyzes the phenotype of an organism following the disruption of a known gene. forward genetics starts with a phenotype and moves towards identifying the gene(s) responsible, whereas reverse genetics starts with a known gene and assays the effect of its disruption by analyzing the resultant phenotypes. Both forward and reverse genetic screens aims to determine gene function Genetic Engineering - Dr.Noor Thaer 18  Genetic tests look for : a) changes in a individual's genes b) changes in the amount, function,structure of key proteins coded by specific genes. Blotting Techniques Blotting :is a technique by which a macromolecule such as DNA, RNA, or protein is resolved in a gel matrix, transferred to a solid support, and detected using specific probe These powerful techniques allow us to identify and characterize specific molecules in a complex mixture of related molecules Genetic Engineering - Dr.Noor Thaer 19 Genetic Engineering - Dr.Noor Thaer 20 Genetic Engineering - Dr.Noor Thaer 21 Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. Southern Blotting could be used to locate a particular gene within an entire genome. The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing. One picogram is 10 -12 grams Genetic Engineering - Dr.Noor Thaer 22 Genetic Engineering - Dr.Noor Thaer 23 Steps involved in southern blotting 1) Extract DNA 2) Digest the DNA with an appropriate restriction enzyme. 3) Run the digest on an agarose gel. 4) Denature the DNA (usually while it is still on the gel). 5) Transfer the denatured DNA to the membrane. Traditionally, a nitrocellulose membrane is used. 6) Addition labelled prop. This is also known as hybridization. P 32 labelled probe is used 7) Visualize your radioactively labelled target sequence Radioactive probes are DNA or RNA sequences that are labelled with radioactive isotopes. Hybridization is the process in which two complementary single-stranded DNA and/or RNA molecules bond together to form a double-stranded molecule. Genetic Engineering - Dr.Noor Thaer 24 Applications Genetic Engineering - Dr.Noor Thaer 25