Genetic Engineering Class 3 Notes PDF

Genetic Engineering Class 3: Basic methods and tools 16/10/2024 - Oskar Schnappauf Outline for today Structure of our rst class 1. Quiz - Session 2 2. Advanced organizer 3. Basic methods and tools 1. Extraction of nucleic acids 2. Enzymes working on nucleic acids 3. Polymerase chain reac...

Genetic Engineering Class 3: Basic methods and tools 16/10/2024 - Oskar Schnappauf Outline for today Structure of our rst class 1. Quiz - Session 2 2. Advanced organizer 3. Basic methods and tools 1. Extraction of nucleic acids 2. Enzymes working on nucleic acids 3. Polymerase chain reaction (PCR) 1. Quiz - Session 2 fi  2. Advanced Organizer Transgenic plants and animals Generation of Generation of transgenic plants transgenic animals Golden rice GM salmon Medicine CAR T-cell therapy CRISPR/Cas in gene Basic methods and tools therapy Cloning strategies Inherited defects Isolation of nucleic acids Cloning vectors and plasmids Separation of nucleic acids Research Replacement gene PCR therapy Getting DNA into cells Ampli cation and quanti cation Host cells Analysis of gene DNA fragments into Enzymatic modi cations structure and function vectors Selection and screening Synthesis of cDNA Bread and butter Analysis of Genomes Bioinformatics Databases Alignment More advanced methods and tools RNA interference Others Methods Genome editing Applications Next generation sequencing 2. Advanced Organizer Transgenic plants and animals Generation of Generation of transgenic plants transgenic animals Golden rice GM salmon Medicine CAR T-cell therapy CRISPR/Cas in gene Basic methods and tools therapy Cloning strategies Inherited defects Isolation of nucleic acids Cloning vectors and plasmids Separation of nucleic acids Research Replacement gene PCR therapy Getting DNA into cells Ampli cation and quanti cation Host cells Analysis of gene DNA fragments into Enzymatic modi cations structure and function vectors Selection and screening Synthesis of cDNA Bread and butter Analysis of Genomes Bioinformatics Databases Alignment More advanced methods and tools RNA interference Others Methods Genome editing Applications Next generation sequencing 3. Basic methods and tools fi fi fi fi fi fi  Cell disruption or lysis: 3.1 Extraction of Nucleic Acids The lysis bu er and/or heat are used to General destroy structures that contain genetic 3 basic steps: material, such as the cell membrane and Cell disruption or lysis Separation and puri cation nuclear envelope. The lysate usually Concentration and precipitation contains SDS, as well as enzymes (e.g., Many kits available Plasmid Proteinase K), a chelating agent (EDTA), DNA RNA or Tris bu er, which can be added based Genomic DNA larger than plasmid DNA on the characteristics of the target —> can be separated sample. Separation and puri cation: The impurities, such as lipids and proteins, are removed from the cell lysates. Concentration and precipitation: After the preliminary puri cation, there will still be some impurities. Therefore, precipitation methods, such as alcohol precipitation, can be applied to further purify and concentrate nucleic acid molecules, resulting in higher concentrated nucleic acid molecules. Lysozyme: digests peptidoglycan 3.1 Extraction of Nucleic Acids Opening up the cells (main component of bacterial cell Lysozyme wall) Detergents (sodium dodecyl sulfate) Detergents (sodium dodecyl Physical force (ground) Proteinase K sulfate): bursts cell membranes by disrupting lipid bilayer Physical force (ground): tissue samples from animals and plants Proteinase K: degrades the tissue (i.e. tail tip) Cultured cells without cell walls: detergent alone releases the intracellular components ff ff fi fi fi  3.1 Extraction of Nucleic Acids Separate proteins from DNA - Phenol-chloroform Separation of cellular components using properties of phenol Removal of unwanted proteins from DNA Not soluble in water —> mixed with aqueous sample of DNA and protein —> protein dissolves in phenol layer and nucleic acids in aqueous layer Two phases are separated by centrifugation https://www.aatbio.com/ Aqueous DNA layer is removed from the phenol-layer 3.1 Extraction of Nucleic Acids Removal of RNA RNA also nucleic acid —> not soluble in phenol —> sample still contains RNA Ribonuclease Digests RNA into ribonucleotides Ethanol precipitation Add equal volume of alcohol —> large DNA falls out of aqueous phase Isolated by centrifugation Smaller ribonucleotides stay soluble Many based on silica: 3.1 Extraction of Nucleic Acids Di erent kits available Silica column based Beads based https://www.integra-biosciences.com/ ff  Guanidinium thiocyanate used in 3.1 Extraction of Nucleic Acids protein degradation Isolation of RNA - Guanidinium thiocyanate method Disrupts cells Denatures proteins Deactivates nucleases Stabilizing DNA and RNA Chloroform added: Centrifugation separates https://www.addgene.org/protocols/kit-free-rna-extraction/ solution Upper phase contains RNA Lower phase contains DNA and protein Top layer is extracted —> RNA is precipitated using isopropanol Guanidinium thiocyanate–phenol solution commercially available as TRIzol, TriFast, or TRI Reagent In the widely used guanidinium Isolation of RNA thiocyanate phenol-chloroform method, Guanidinium thiocyanate method the cell extract is extracted with phenol/ chloroform at low pH. Guanidinium thiocyanate is a chaotropic agent used in protein degradation. The principle of this single-step technique is that RNA is separated from DNA after extraction with acidic solution consisting guanidinium thiocyanate and phenol. The guanidinium thiocyanate–phenol solution, which is commercially available as TRIzol, TriFast, or TRI Reagent, disrupts the cells, denatures the proteins, and deactivates the nucleases, thereby stabilizing the DNA,  RNA, and protein. Chloroform is then added, and after centrifugation the solution separates into an upper aqueous phase containing the RNA and a lower organic phase containing the DNA and protein under acidic conditions (Fig. 6.10). Recovery of total RNA is then done by precipitation with isopropanol. Sample Lysis and Homogenization: 3.1 Extraction of Nucleic Acids Add lysis bu er to break down Isolation of RNA - Column methods membranes and release RNA RNA Binding and Column Loading: Binding bu er to create conditions for RNA binding to column Add mixture to column Allow low through column matrix by centrifugation On-Column DNase Treatment: Apply DNase-containing solution directly on column matrix Incubate for the DNase to digest DNA contaminants Washing: Wash steps remove proteins, salts, and cellular debris Elution: Add elution bu er to column to release puri ed RNA Collection of puri ed RNA in collection tube fi ff ff ff fi  3.2 Enzymes working on nucleic acids 4 classes of restriction enzymes: 3.2 Enzymes working on nucleic acids Restriction enzymes I: large, rst discovered, cut away Cut DNA at de ned from recognition sites sites Important for DNA II: most used, recognize inverted manipulation Protection system palindrome sites for bacteria III: large subunits, cut 20bp away from recognition site, often do not cleave completely IV: cleave modi ed sites (methylated) Type II most used in GE 3.2 Enzymes working on nucleic acids Restriction enzymes Cut blunt, 3’-overhang, 5’- overhang (‘sticky ends’) Expected frequency of cutting: 4n (4=A,T,C,G; n=length of recognition sequence) Tetranucleotide-seq cuts every 256bp Hexanucleotide-seq every 4096 Sometimes useful, i.e. NGS fi fi fi  Enzyme activity in units (1 unit is the 3.2 Enzymes working on nucleic acids Restriction enzymes amount of enzyme that cuts 1µg Often inverted palindromes DNA in 1h at 37C) Palindrome (‘step on no pets’) Inverted palindrome —> sequence reads the same on each of the two strands ≠ mirror palindromes, i.e. 5’- TAGGAT-3’ 3.2 Enzymes working on nucleic acids Restriction enzymes Isochizomeres vs. neoschizomers Isochizomeres: Recognize same recognition site and cut the same way SphI (CGTAC/G) and BbuI (CGTAC/G) Neoschizomers: Recognize same recognition site but cut di erently SmaI (CCC/GGG) and XmaI (C/ CCGGG) Often inverted palindromes 3.2 Enzymes working on nucleic acids Restriction enzymes Palindrome (‘step on no pets’) Inverted palindrome —> sequence reads the same on each of the two strands (≠ mirror palindromes, i.e. 5’-TAGGAT-3’) Enzyme activity in units (1 unit is the amount of enzyme that cuts 1µg DNA in 1h at 37C) ff  Often inverted palindromes 3.2 Enzymes working on nucleic acids Restriction enzymes Palindrome (‘step on no pets’) Inverted palindrome —> sequence reads the same on each of the two strands (≠ mirror palindromes, i.e. 5’-TAGGAT-3’) Enzyme activity in units (1 unit is the https://www.neb.com/en/tools-and-resources/selection-charts/compatible-cohesive-ends-and-generation-of-new-restriction-sites amount of enzyme that cuts 1µg DNA in 1h at 37C) Can be used to generate 3.2 Enzymes working on nucleic acids Restriction enzymes recombinant DNA Cut out target gene Cut target molecule, i.e. plasmid Mix together and wait https://www.khanacademy.org/ https://www.snapgene.com/guides/restriction-enzyme-cloning 3.2 Enzymes working on nucleic acids Nucleases Degrade DNA by breaking down phosphodiester bonds RE are examples of endoucleases that cut within DNA strand Exonuclease degrade DNA from the end of the molecule https://international.neb.com/  3.2 Enzymes working on nucleic acids Nucleases 4 types of nucleases important in GE: Bal31 —> shortens dsDNA from both ends Exo III —> 3’-exonuclease, generates 5’ overhangs DNAse I —> cuts ssDNA and dsDNA S1 —> speci c for ssDNA https://international.neb.com/ 3.2 Enzymes working on nucleic acids Ribonucleases Cut RNA Used to get rid of RNA if DNA is wanted Sometimes not wanted, i.e. during RNA extraction Can be secreted with sweat RNase-inhibitors often used for RNA extraction https://international.neb.com/ Used to join DNA molecules 3.2 Enzymes working on nucleic acids DNA-Ligase In cell to repair phosphodiester bonds Often used: T4 DNA ligase (from E.coli) Most e ective when sealing gaps in sticky ends, blunt end much lower https://geneticeducation.co.in/ https://assets.thermo sher.com/ e ciency Works best at 37C, often used at 16C to prevent thermal denaturation of ssDNA ffi ff fi fi Polymerase Chain Reaction Polymerase Chain Reaction Overview Generate millions of copies of a speci c piece of DNA (usually 200 - 1000 bp) Not the complete genome is ampli ed Development by Kary Mullis in 1983 Two essential things: Two primers ( ank the target region) Thermostable DNA polymerase (i.e. Taq-polymerase) Polymerase Chain Reaction Overview fi fl Polymerase Chain Reaction Components Other ingredients include: Template DNA (i.e. DNA extracted from human blood) dNTPs as building blocks for DNA polymerase Bu er (containing co-factors for DNA polymerase) Tubes are placed into Thermocycler Changes temperature Polymerase Chain Reaction Cycles Polymerase Chain Reaction Thermocycler Typical sequencing program: Initial denaturation 30-35 cycles of: Denaturation Annealing Elongation Final Elongation Store at 4C or 16C ff Quantitative PCR Quantitative PCR Observe what is ampli ed Monitors PCR while it happens by detecting uorescence label after each cycle Cycle threshold is set to avoid background noise Number of PCR cycles to reach threshold depends on amount of DNA at start of reaction Quantitative PCR Two commonly used variations fl fi  Quantitative PCR Ct (cycle threshold) method Widely used technique in qPCR to determine amount of speci c DNA/ RNA Ct value de ned as cycle at which uorescence signal crosses a de ned threshold Threshold usually set at a level that is above background uorescence Ct value inversely proportional to amount of target DNA/RNA in sample The higher the Ct, the lesser DNA/RNA in the beginning Digital PCR Be or not to be Extension of quantitative PCR PCR reaction distributed into thousands of sub-nanoliter reactions Chamber digital PCR Droplet digital PCR Ideally 1 DNA molecule per reaction Each unit with either 0 or 1 or more target fragments Each will score either 0 or 1 The more target, the more 1s Digital PCR Be or not to be fl fi fl fi Gel electrophoresis Separate your DNA by size DNA with multiple negative charges —> migrate towards positive electrode Mobility depends on fragment length Two gel types are used: Agarose Polyacrylamide Evaluate using UV light Loading bu er DNA ladder https://www.aatbio.com/catalog/gel-electrophoresis Quantification of nucleic acids How much did I get DNA/RNA often in aqueous solution Measuring absorbance at 260nm using a spectrophotometer A260 of 1.0 equals 50µg/ml dsDNA and 40µg/ml of RNA The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA Ratio of ~1.8 accepted as “pure” for DNA Ratio of ~2.0 accepted as “pure” for RNA Lower ratio indicates presence of protein/ phenol/other contaminants that absorb strongly at 280nm ff