Laboratory Techniques in Genetics Overview.pdf

🧬 9/3/24, 5:03 PM Laboratory Techniques in Genetics Overview Laboratory Techniques in Genetics Cell Biology Lab Techniques Hemocytometers Count...

🧬 9/3/24, 5:03 PM Laboratory Techniques in Genetics Overview Laboratory Techniques in Genetics Cell Biology Lab Techniques Hemocytometers Count samples by loading them onto a grid, allowing for division of the view into fields under a microscope. Flow Cytometry A more advanced technique that funnels samples (e.g., cells) through a laser light source, allowing for the inference of different properties (e.g., granularity, density, size, markers) by measuring the fluorescence emitted. Cell Fractionation Use a centrifuge to spin down a suspension, forming a precipitate, and separate out components of different densities. Centrifugation Speed Pellet Low speed Whole cells, nu Medium speed Mitochondria, l High speed Sma Lab Techniques for Nucleic Acids and Proteins Karyotyping Visualize and analyze the chromosomal number and structure of an organism's chromosomes. Sanger Sequencing "A laboratory technique used to determine the order of the four chemical building blocks (called "bases") that make up an organism's DNA." 💡 Sanger sequencing works by synthesizing a new DNA strand and terminating the reaction at random points using dideoxynucleotides, which lack a 3' hydroxide group. The resulting fragments are then separated by size using gel electrophoresis, and the order of the bases is determined by the length of the fragments. Next Generation Sequencing (NGS) "A high-throughput method of DNA sequencing that allows for the simultaneous analysis of millions of DNA sequences in a single reaction." 💻 NGS works by synthesizing a new DNA strand and detecting the incorporation of fluorescently labeled nucleotides in real-time, allowing for the simultaneous sequencing of millions of DNA molecules. Recombinant DNA DNA that has had a new sequence added to it, often using a plasmid vector. Restriction Fragment Length Polymorphisms (RFLPs) A technique used to identify specific genotypes or polymorphisms by cutting DNA with restriction enzymes and analyzing the resulting fragments. Restriction Enzyme Cut Present Absent Southern Blot A technique used to detect DNA in a sample (to be discussed further in the next section). https://www.turbolearn.ai/content/f2b8e2bc-f9ab-4a6e-a07e-277d516c98a4 1/5 9/3/24, 5:03 PM Laboratory Techniques in Genetics Overview DNA Fingerprinting A technique used to identify an individual's unique DNA profile by analyzing a set of DNA samples.## DNA Analysis Techniques 🧬 Restriction Fragment Length Polymorphism (RFLP) RFLP is a technique used to detect polymorphisms in DNA. It involves cutting DNA into smaller fragments using enzymes, and then analyzing the fragments using gel electrophoresis. This allows for the detection of genetic variations between individuals. Polymerase Chain Reaction (PCR) 🔬 PCR is a laboratory technique used to amplify specific DNA sequences. The process involves: Denaturation: The DNA double helix is unwound, and the two strands are separated. Annealing: Primers bind to the target sequence, which is the specific DNA sequence we want to amplify. Extension: New DNA strands are synthesized, and the target sequence is duplicated. This process is repeated multiple times, resulting in an exponential amplification of the target sequence. Molecular Cloning 🧬 Molecular cloning is a laboratory technique used to create multiple copies of a specific DNA sequence. This is achieved by: Isolating the DNA sequence of interest Inserting the sequence into a plasmid (a small, self-replicating circular DNA molecule) Transforming the plasmid into bacteria Selecting for bacteria that have taken up the plasmid using antibiotics Screening for the presence of the inserted sequence using the lacZ gene Step Description Isolation Isolate the DNA sequence of interest Insertion Insert the sequence into a plasmid Transformation Transform the plasmid into bacteria Selection Select for bacteria that have taken up the plasmid using antibio Screening Screen for the presence of the inserted sequence using the lacZ Gel Electrophoresis 🌊 Gel electrophoresis is a laboratory technique used to separate DNA fragments based on their size. The process involves: "DNA is negatively charged, and because it's negatively charged, we're able to run it on an electric current." Running the DNA through an agarose gel, which acts as a resistance layer The DNA fragments migrate towards the positive electrode, with smaller fragments moving faster than larger ones The resulting gel shows a gradient of DNA fragment sizes, with smaller fragments closer to the positive electrode Southern Blotting 🌊 Southern blotting is a laboratory technique used to detect specific DNA sequences. The process involves: "We can then use nitrocellulose, which is sticky to DNA... We can then take probes which are complementary to that DNA sequence." Running the DNA through a gel to separate the fragments https://www.turbolearn.ai/content/f2b8e2bc-f9ab-4a6e-a07e-277d516c98a4 2/5 9/3/24, 5:03 PM Laboratory Techniques in Genetics Overview Transferring the DNA to a nitrocellulose membrane Using labeled probes to hybridize with the target DNA sequence Detecting the presence of the target sequence on the membrane Northern Blotting (RNA Analysis) 🌊 Northern blotting is a laboratory technique used to detect specific RNA sequences. The process is similar to Southern blotting, but uses RNA instead of DNA. Note: The teacher's lecture transcript has been interrupted, and the rest of the content will be covered in the next section.## Northern Blotting 🧬 Northern blotting is a laboratory technique used to study gene expression by detecting specific RNA molecules in physiological samples. It is similar to Southern blotting, but instead of detecting DNA, it detects RNA. Importance of RNA stability: RNA is intrinsically very unstable and is easily degraded by RNases present in the environment. This makes it essential to handle RNA samples with care to maintain their integrity. Western Blotting 🧬 Western blotting is a laboratory technique used to detect specific proteins in a sample. It involves denaturing proteins, separating them by size using SDS-PAGE, transferring them to a membrane, and detecting them using antibodies. Steps in Western Blotting: Prepare protein sample SDS-PAGE Protein transfer to membrane Detection by antibody (primary and secondary) Detection methods: Detection Method Description Radioactive labeling Uses radioactive isotopes to label Fluorescent labeling Uses fluorescent dyes to label an Enzyme labeling Uses enzymes to catalyze a chemilumin Primary and Secondary Antibodies: Primary antibody: binds to the protein of interest Secondary antibody: binds to the primary antibody and is labeled for detection Southern, Northern, and Western Blotting Mnemonic 🤔 SDNRPW: Southern (DNA), Northern (RNA), Western (Protein) Enzyme-Linked Immunosorbent Assay (ELISA) 🌡 ELISA is a laboratory technique used to detect and quantify proteins in a sample. It is similar to Western blotting but uses an immobilized antibody on a dish instead of a gel. ELISA steps: Add protein sample to dish with immobilized antibody Add detection antibody to bind to protein Detect bound antibody using enzyme or fluorescent labeling Types of ELISA: https://www.turbolearn.ai/content/f2b8e2bc-f9ab-4a6e-a07e-277d516c98a4 3/5 9/3/24, 5:03 PM Laboratory Techniques in Genetics Overview Direct ELISA: uses conjugated secondary antibody Indirect ELISA: uses primary antibody and labeled secondary antibody Genomics and Miscellaneous Techniques 🔬 DNA Microarray 🔍 DNA Microarray: a technique used to detect specific DNA sequences in a sample. It involves hybridizing DNA to a chip with immobilized known DNA sequences and detecting bound DNA. DNA Microarray Output: Intensity graph for each probe Heat plot of bound DNA sequences Transgenic Animals 🐭 Transgenic animals: animals that have been genetically modified using gene editing techniques. Methods of generating transgenic animals: Injecting genes directly into a fertilized egg Using stem cells to modify genes and insert them into an embryo## Generating Chimeric Animals 🐭 Chimeric animals are organisms composed of cells from two or more different individuals. In genetic engineering, this technique is used to study the effect of a specific gene or mutation on an organism. To create a chimeric animal, scientists take cells from an embryo and modify them by adding genes of interest. These modified cells are then added to another embryo, resulting in an organism with two sets of cells: one from the original embryo and one from the modified cells. Advantages of Chimeras Allows for the study of lethal mutations in a controlled environment Enables the production of animals with specific genetic modifications Modification Gene of interest added to embryo Cells with the m Germ cells contain gene of interest Progeny with the Cloning 🐑 Cloning is a process that creates an exact genetic replica of an organism. This is achieved by taking an unfertilized egg and removing its nucleus, replacing it with the nucleus of an adult somatic cell (e.g., skin cell). Cloning Process Remove nucleus from unfertilized egg Remove nucleus from adult somatic cell Insert somatic cell nucleus into egg The resulting embryo is a clone, containing 100% of the DNA from the adult animal Epigenetic modifications: These are chemical modifications to DNA or histone proteins that can affect gene expression. In cloning, epigenetic modifications can cause clones to die prematurely. Scientists have developed techniques to reprogram adult nuclei to better recapitulate embryogenesis, improving cloning success rates. Analyzing Diffusion with FRAP and FLIM 🔍 https://www.turbolearn.ai/content/f2b8e2bc-f9ab-4a6e-a07e-277d516c98a4 4/5 9/3/24, 5:03 PM Laboratory Techniques in Genetics Overview Fluorescence Recovery After Photobleaching (FRAP) "FRAP is a way to analyze diffusion in a sample." How it works: Label molecules of interest with a fluorescent dye Bleach a specific area with a high-intensity light Measure the time it takes for fluorescence to recover in the bleached area This indicates the rate of diffusion of the labeled molecules Fluorescence Lifetime Imaging Microscopy (FLIM) "FLIM irradiates a cell with light and then measures the fluorescent lifetime in order to quantify the concentration of ions, molecules, and gases." How it works: Irradiate cells with light Measure fluorescent lifetime to quantify concentrations of ions, molecules, and gases https://www.turbolearn.ai/content/f2b8e2bc-f9ab-4a6e-a07e-277d516c98a4 5/5