Genetic Engineering Lecture Notes 2024-2025 PDF

College of Applied Science Department of Biotechnology Genetic Engineering 4th stage Dr. Noor Thaer Adnan Lecture -4 2024-2025 Genetic Engineering - Dr.Noor Thaer 1 Southern Blotting Genetic Engineering - Dr.Noor Thaer 2 Genetic Engineeri...

College of Applied Science Department of Biotechnology Genetic Engineering 4th stage Dr. Noor Thaer Adnan Lecture -4 2024-2025 Genetic Engineering - Dr.Noor Thaer 1 Southern Blotting Genetic Engineering - Dr.Noor Thaer 2 Genetic Engineering - Dr.Noor Thaer 3 Steps involved in northern blotting 1. The first step is to denature, , the RNA within the sample, which ensures that the strands are unfolded and that there is no bonding between strands. 2. The RNA molecules are then separated according to their sizes using electrophoresis. 3. Following separation, the RNA is transferred from the gel onto a blotting membrane. Once the transfer is complete, the blotting membrane carries all of the RNA bands originally on the gel. 4. Next, the membrane is treated with a small piece of DNA or RNA called a probe, which has been designed to have a sequence that is complementary to a particular RNA sequence in the sample; this allows the probe to hybridize, or bind, to a specific RNA fragment on the membrane. In addition, the probe has a label, which is typically a radioactive atom or a fluorescent dye. 5. Thus, following hybridization, the probe permits the RNA molecule of interest to be detected from among the many different RNA molecules on the membrane. Genetic Engineering - Dr.Noor Thaer 4  3. Western blotting, also known as immunoblotting or protein blotting, is a technique used to detect the presence of a specific protein in a complex protein mixture It is a core technique in cell biology, molecular biology, virology and others Western blot technique uses three elements to achieve its task of separating a specific protein from a complex: a) separation by size, b) transfer of protein to a solid support c) marking target protein using a primary and secondary antibody to visualize Once detected, the target protein will be visualized as a band on a blotting membrane, X-ray film, or an imaging system Genetic Engineering - Dr.Noor Thaer 5  Steps involved in western blotting 1. Sample preparation 2. Gel Electrophoresis separation by a technique known as sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE, 3. Blotting (or transfer) onto a piece of membrane which traps the proteins in their respective locations 4. Antibody Probing: iimmunoblotting uses antibody-protein and antibody-antibody binding through specific recognition sites, providing the high specificity required for identifying a single protein. 5. Detection: he detection of antibodies takes place using reporter systems which includes the use of enzymes. Enzymes can be attached to the end of an antibody and react with substrates to produce changes in color or light. These signals can then be imaged and quantified using a process called densitometry. Genetic Engineering - Dr.Noor Thaer 6 Genetic Engineering - Dr.Noor Thaer 7 Genetic Engineering - Dr.Noor Thaer 8 Polymerase Chain Reaction (PCR) The polymerase chain reaction (PCR) is a laboratory nucleic acid amplification technique used to denature and renature short segments of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences using DNA polymerase I enzyme, an isolate from Thermus aquaticus, known as Taq DNA In 1985, PCR was introduced by Mullis and colleagues for which they received a Nobel prize. It is a monumental tool used in biomolecular sciences for its profound ability to examine and detect amplified components of DNA. Taq polymerase is a thermostable DNA polymerase I named after the thermophilic microorganism Thermus aquaticus. Genetic Engineering - Dr.Noor Thaer 9  PCR primers are short pieces of single-stranded DNA, provides a starting point for DNA synthesis Two primers are utilized, one for each of the complementary single strands of DNA released during denaturation. The forward primer attaches to the start codon of the template DNA (the anti- sense strand), while the reverse primer attaches to the stop codon of the complementary strand of DNA (the sense strand). Homework How to choose the correct primer set? Genetic Engineering - Dr.Noor Thaer 10 Genetic Engineering - Dr.Noor Thaer 11 Genetic Engineering - Dr.Noor Thaer 12 A REAL-TIME POLYMERASE CHAIN REACTION (REAL-TIME PCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real time), not at its end, as in conventional PCR Two common methods for the detection of PCR products in real-time PCR are (1)non-specific fluorescent dyes that intercalate with any double-stranded DNA and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary sequence. Genetic Engineering - Dr.Noor Thaer 13 Non-specific detection: real-time PCR with double-stranded DNA-binding dyes as reporters A DNA-binding dye binds to all double-stranded (ds) DNA in PCR, increasing the fluorescence quantum yield of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity measured at each cycle. However, dsDNA dyes such as SYBR Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as Primer dimer). This can potentially interfere with, or prevent, accurate monitoring of the intended target sequence. Genetic Engineering - Dr.Noor Thaer 14 Genetic Engineering - Dr.Noor Thaer 15 Genetic Engineering - Dr.Noor Thaer 16