Molecular Biology: RNA, DNA & Nucleic Acids PDF

Objectives Molecular Biology in Lab Medicine Molecular biology is the study of biological processes at the molecular Most are a review of your BIOL1005 Not...

Objectives Molecular Biology in Lab Medicine Molecular biology is the study of biological processes at the molecular Most are a review of your BIOL1005 Notes level, focusing on DNA, RNA, proteins RNA/DNA Microbiology Central Dogma of Molecular Biology TB testing Purity Identification of microbes Lab Pathology Tumor markers Transfusion Medicine Opportunity for Test Review, if you have any questions please write at the bottom of the test paper Blood products Tissue typing Hematology Diagnosis and monitoring of disease RNA & DNA Nucleic Acids Long chains of nucleotides which consist of: Phosphate group Nucleotide sugar (pentose) Nitrogenous base DNA is a double-stranded, stable molecule with deoxyribose sugar, primarily used for long-term genetic storage RNA is a single-stranded, more transient molecule with ribose sugar, involved in protein synthesis and gene regulation. Nucleotide Sugars DNA sugar vs. RNA sugar Carbon 1: where the nitrogenous base is attached. RNA: ribose sugar Carbon 2: tells you if it is a has a hydroxyl group ribose or deoxyribose Carbon 3: the point of (-OH) at 2’ attachment for more DNA: deoxyribose sugar nucleotides Carbon 4: completes the ring has only a hydrogen via an oxygen (O) which bridges atom (-H) at 2’ to the carbon 1 (C1) Ribose Deoxyribose Carbon 5: hangs away from the ring and is the point of attachment for its phosphate(s) DNA – Deoxyribonucleic acid Two strands of the sugar phosphate forms a double helix RNA – Ribonucleic Acid structure Four different Bases Adenine Adenine Cytosine Cytosine Guanine Guanine Uracil Thymine Nucleotides in complementary base pairs adenine pairs with uracil Thymine – Adenine (T-A) guanine pairs with cytosine Guanine – Cytosine (G-C) Uracil replaces Thymine in DNA Transcription Held together with hydrogen bonds Apples in the Tree Cars in the Garage RNA Nucleotide Bases RNA exists in three forms Messenger RNA (mRNA) Pyrimidines: single Translates DNA coding into functional proteins hexagonal ring of carbon and The Blueprint nitrogen Transfer RNA (tRNA) Transports various amino acids to manufacture proteins The Delivery Truck (of Raw Materials) Purines: double rings made of a pyrimidine with a Ribosomal RNA (rRNA) the site of protein synthesis directed by mRNA pentagon added A large component of the ribosome http://www.synapses.co.uk/genetics/ The Factory Purines & Pyrimidines Central Dogma of Molecular Biology DNA RNA PROTEIN PURE AS GOLD The central dogma of molecular biology states that genetic C.U.T. THE PIE (Pie shape is like a pyramid?) information flows from DNA to RNA to protein, outlining the process of transcription and translation.  DNA Replication Process DNA Replication Helicase: separates the two DNA strands that make up the helix The process of DNA making a copy of itself during cell division Primase: puts down small Any mistake in the DNA code is pieces of RNA (primers) as called a mutation a starting point of DNA synthesis Chromatin: the DNA is loose within the nucleus Chromosome: when a cell is about DNA polymerase III: to divide, the DNA is tightly reads the code and condensed attaches DNA nucleotides to the ends of the primers DNA Replication Process Transcription Process in which DNA is copied into RNA DNA ligase: seals up breaks in the DNA RNA polymerase: backbone by forming Binds to a promoter region of the DNA (initiation sequence) covalent bonds between opens the DNA double helix; uses one strand as a template to make nucleotides mRNA following the base pair rules Stops at the terminator region (stop sequence) Semiconservative Synthesis of RNA molecule in the 5' to 3' direction using one of the DNA replication: each old half strands as a template. of the DNA double helix gets a new partner: resulting in two DNA molecules from one RNA Processing Translation Process in which mRNA directs the amino acid Pre-mRNA are edited before leaving the nucleus production during protein synthesis Introns are removed by spliceosomes The mature mRNA leaves the nucleus and enters Non-coding regions that do not code for the cytoplasm protein Takes place on ribosomes Remaining exons are spiced together Coding regions that code for protein The processed form is called Mature RNA Translation Nucleic Acid Quantification/Quality The information contained in the nucleotide sequence of the mRNA is read as three letters, called codons It is important to know how much DNA/RNA is present in your sample Each tRNA has an amino acid as well as an anticodon Many downstream reactions (i.e. that pairs with the mRNA codon, ensuring that the correct PCR) require a specific amount of amino acid is added DNA in order to be successful The final product is a new polypeptide Quality of DNA/RNA is assessed in two ways: The purity of the sample is measured by spectrophotometry The physical structure is characterized by electrophoretic techniques Spectrophotometry Beer’s Law Calculation Molecule (ug/ml)cm-1 Double stranded 0.020 DNA (dsDNA) DNA and RNA absorb UV light due to their nucleotide Single stranded RNA bases A= axbxc (ssRNA) 0.025 Double bonds of purines and pyrimidines absorb Single stranded DNA 0.027 260nm wavelength (ssDNA) A = absorbance of the solution Absorb UV light very efficiently making it possible to a = molar absorptivity coefficient detect and quantify at very low concentrations (how strongly the substance absorbs light) The best wavelength for DNA/RNA is 260nm (A260 or OD260) b = path length (usually 1cm cuvette) c = concentration Beer’s Law Absorbance is directly proportional to concentration Molar absorptivity coefficients have been calculated at If you plot absorbance vs. concentration the resulting A260: graph yields a straight line Example Example 2 You get an A260 of 1.787. What is You get an OD260 of 0.934. What is the concentration of your dsDNA sample? the concentration of RNA in your sample? A=axbxc 0.934 = 0.020 x 1 x c A=axbxc c = 0.934 / 0.020 1.787 = 0.025 x 1 x c c = 46.7 ug/mL 1.787 / 0.025 = c c = 71.5 ug/mL  Quality (Purity) by Spectrophotometry Qualification of Nucleic Acids A260 /A280 Pure DNA has an ideal purity ratio of ~1.80-1.85 Reliable measurement of DNA/RNA purity is also Pure RNA has an ideal purity ratio of ~2.0 important for many applications in molecular biology Impurities can lead to an inaccurate measurement of Acceptable value for DNA will be 1.75-1.95 DNA/RNA 1.95 indicates possible contamination with RNA (if you are looking for DNA) The ratio of the absorbance at 260nm vs. 280nm is a measure of the purity of DNA & RNA in the sample Look at your laboratory ranges for acceptable purity A260 /A280 Calculations Calculations Calculate the purity of your DNA sample given the Calculate the purity of your DNA sample given the following data: following data: A260 = 0.934 A260 = 0.934 A280 = 0.653 A280 = 0.519 0.934/0.653 = 1.43 BAD L 0.934/0.519 = 1.80 GOOD J May indicate protein contamination in your DNA sample  Qualification by Gel Electrophoresis Good Lab Practices The physical quality or presence of nucleic acids can be validated by agarose gel electrophoresis High-quality DNA should appear as crisp molecular weight bands on the agarose gel Small fragments of nucleic acid can As the DNA quality gets worse, smears leading synthesize more than 10 million copies of more towards the low molecular weight will RNA or DNA molecules occur Contamination can result in the wrong substance being amplified, i.e. false positive results Minimize Contamination Gloves & Equipment Laboratory Construction and Environmental Control Dedicated Consumables and Equipment At minimum, two areas designated for pre-amplification and post- Each room should have its own centrifuge, vortex, pipettes amplification Dedicated lab coats, gloves Separate independent environmental controls with separate duct work Pipette tips DNAse & RNAse free tips Unidirectional Workflow Sterile May use barrier tips to prevent aerosolization One direction only: Pre-amplification à Post-amplification Some labs have a “Reagent preparation or Clean room” room to prepare their reagents. This would be before Pre-amplification Frequently changing gloves room Should always wear fresh gloves and change often especially if We never go backwards and take stuff back you suspect they have become contaminated Consumables and products should not be reintroduced to the Avoid touching hair, face, etc. pre-amplification room Change gloves & wash hands when going from one room to another.  Cleaning Techniques Contamination & Pipetting Aseptic Cleaning Techniques All work surfaces including bench tops, pipettes, Environmental monitoring fridge/freezer handles, centrifuge buttons & biosafety for the absence of contamination in reagents, consumables, cabinets (if required) work surfaces You need to wipe down your pipettes Swab of benchtop or heavily used items and test for recommend wiping them before usage as well contamination Example Cleaning procedure Pipetting Techniques Wipe down with 10% bleach Critical to quality of results After 15 min, use DI water-dampened paper towel to remove Minimize contamination between samples leading to false bleach residue positive results Then 70% alcohol-dampened paper towel Ensures accurate volume is aspirated and dispensed May include Commercial RNAase Away in the procedure If you contaminate your tip, just dispose and get a new one UV Use new tip for each different reagent/sample You may use the same tip if you are pipetting out the same Procedure differ depending on place of work reagent into multiple (empty) wells/tubes First molecular lab Extraction Lab 2 Sample types 1. Whole blood and we will separate out the buffy coat There are 6 basic steps to nucleic acid extractions: 2. Cheek swabs to get buccal cells 1. Lyse Cells - Disrupt/break open cell membranes to release nucleic acid. Methods: mechanical, chemical, enzymatic Extract out the DNA 2. Lyse Nuclei - In eukaryotes, the nucleus in each cell must be Quality your samples using a Nanodrop Spectrophotometer broken open in conjunction with the cell membrane. 3. Digest/Denature Cellular Debris - Breakdown and remove Some Terms unwanted cellular components including proteins, lipids and Supernatant vs Precipitate unwanted nucleic acid. Supernatant – clear liquid above the solid material 4. Precipitate Nucleic Acid - DNA & RNA are soluble in water. Precipitate – the solid mass that settles at the bottom of the tube Solutions are used to precipitate (clump) nucleic acid so that it comes out of solution (e.g. ethanol). 5. Purify Nucleic Acid - Wash steps to separate and remove Eluate vs Waste eluate/Flow-Through contaminants. Eluate – Final purified DNA/RNA/Protein that was intentionally released 6. Resuspend and Evaluate - Precipitated nucleic acids are dissolved from the binding matrix back into a liquid. Quality, quantity and purity of the nucleic acid are assessed. Waste Eluate – unwanted materials such as contaminated that were not bound to the binding matrix  Reagents used Kit Reagents Phosphate buffered Saline PBS is a balanced salt solution that maintains pH & osmotic Digestive Enzymes & detergents balance Proteinase K – enzyme that More buffering stability in comparison to just 0.9% saline will digest proteins (release DNA) as well as nucleases Can be used for washing cells, tissues & other contaminants A nuclease is an enzyme that digests DNA Histopaque SDS – sodium dodecyl sulfate A specifically densed medium that will help isolate the A detergent that breaks cell membranes open, lymphocytes and other mononuclear cells from whole blood releasing DNA EtOH: precipitates DNA and enhances attachment to silica Wash Buffers – remove contaminants Elution Buffer – Remove DNA that is attached to silica Spin column Silica Based NANODROP Spectrophotometer High affinity for nucleic acids Do not poke the bottom with your tip! Apply extracted DNA sample to measure quality