MODX311 Lecture: Biosafety and Waste Management PDF

BIOSAFETY AND WASTE MANAGEMENT LEC 1 TRANS 1 MOLECULAR BIOLOGY AND DIAGNOSTICS Instructor: Prof. Justin Kim Vergara, RMT, MPH Date: February 15, 2022 Outline At the end of the session, the student must be able to learn: I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. ▪ Laboratory Qualit...

BIOSAFETY AND WASTE MANAGEMENT LEC 1 TRANS 1 MOLECULAR BIOLOGY AND DIAGNOSTICS Instructor: Prof. Justin Kim Vergara, RMT, MPH Date: February 15, 2022 Outline At the end of the session, the student must be able to learn: I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. ▪ Laboratory Quality System Safety Decontamination/Disinfection A. Different Forms of Decontamination Standard Precaution Decontamination Solutions A. Chemical Agents B. Types of Chemical Disinfectants Spill Control Inactivation of Prions Inactivation of Microorganisms Biosafety Cabinet A. Types of Biosafety Cabinet Protection Filter Biosafety Management A. Health and Medical Surveillance B. Safety Practices C. Develop Personal Safe Work Habits D. Maintain Clean and Orderly Work Space E. Proper Disposal of Biowastes Laboratory Waste Management Chemical Hazards in Genotyping Laboratory A. Ethidium Bromide B. Carcinogens C. Dyes, Probes, Labels Action Plan for Implementing Safety Practices Definitions ⚫ ⚫ side notes LABORATORY QUALITY SYSTEM Biosafety and waste management- necessary part of molecular biology laboratory Provide us knowledge needed to solve key problems in human health One of major components of laboratory quality system The application of the knowledge and techniques is very important to prevent the Laboratory and personnel exposure to potentially infectious agents or biohazard In any laboratory, safety is very important. We must break the chain of infection and break the transmission of diseases through proper hand hygiene. ⚫ Primary goal of LQS: ensure that lab personnel is safe from potentially harmful of infectious agents ⚫ ⚫ ⚫ ⚫ ⚫ ⚫ Page 1 of 7 2021 – 2022 2nd Semester MODX311 LEC SAFETY Why is safety important? Coming in contact with human blood or blood products (plasma, serum, etc.), or with certain chemicals used in the laboratory, is potentially hazardous. Fundamental objective of any biosafety in molecular biology lab is to contain potentially harmful agents Safety involves taking precautions to protect you and coworkers against infection, injury or poisoning. Hand Hygiene Persons must wash their hands after working potentially hazardous materials and before leaving the Laboratory. Lather and Scrub: 20 seconds Rinse: 10 seconds Do not use Hand Dryers! Performance of entire Handwashing: 40-60 seconds Alcohol & Sanitizers - alternatives Alcohol-based cleansers are not recommended after contact with sporeforming bacteria, including Clostridium difficile and Bacillus sp. ▪ Hands should be washed regularly -before and after touching the specimens -before and after putting your gloves A number of infectious diseases can be spread through contaminated hands (Ex: respiratory infection, influenza, coronavirus, G.I.T infection, salmonella) Handwash with soap & water if hands are visibly soiled ◼ Liquid soap is better than bar soaps to prevent higher chances of transmission If hands are not visibly soiled, use alcohol-based sanitizers Hand dryers- not used in the lab; produce aerosols (some bacteria are transmitted through aerosols) DECONTAMINATION/DISINFECTION Decontamination – Process of removing or neutralizing chemical or Biological Agents so that they no longer pose a hazard. (https://ncbi.nlm.nih.gov) ◼ Reduce level of microbial contamination ◼ Free from the risk of infection transmission SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER [MODX311] BIOSAFETY AND WASTE MANAGEMENT | Prof. Justin Kim Vergara, RMT, MPH 1. Sterilization Removal or destruction of all forms of life, including bacterial spores. Killing all forms of microorganisms from vegetative to spore forming organisms ◼ Autoclave- most common sterilization in the laboratory ◼ Gamma radiation- deep penetrating destroying DNA and RNA of microorganism - Short wavelength but high energy - Used only for HEAT SENSITIVE materials (ex: syringe) 2. Disinfection Removal, inhibition, or killing of microorganisms including potential pathogens by using chemical agents usually on inanimate objects; does not remove all bacterial spores. ▪ Eliminate most pathogen but not all microbes ▪ Reduce level of microbial contamination ▪ Chemical disinfectants- don’t kill spores ▪ Most commonly used chemical disinfectant: Sodium Hypochlorite (NaClO) Ethyl Alcohol (ETOH) ▪ Used in surfaces, inanimate objects 3. Antisepsis Antimicrobial substances typically applied in the skin to reduce possibility of sepsis formation or putrefaction. ◼ Similar with disinfectant (can’t kill bacterial spores) ◼ Solutions used for cleaning wounds, can be applied directly to skin (humans and animals) ◼ Most commonly used antiseptic: Iodine Solution Hydrogen Peroxide 10% NaOCl 70% ETOH Benzalkonium Chloride (found in Lysol but it cannot be used directly in the skin since it has other components) ◼ DIFFERENT FORMS OF DECONTAMINATION 70% Ethyl Alcohol 10% Sodium Hypochlorite: Spills (1:10) 1% Sodium Hypochlorite: General Surface Decontamination (1:100) Contact Time of Bleach: 10-15 minutes More spill requires higher contact time All surfaces and equipment must be decontaminated before and after usage. CHEMICAL AGENTS Efficacy is based on different factors: ▪ ⚫ ⚫ ⚫ ⚫ ⚫ Organic Load ▪ Composition of chemical agents Microbial Load ▪ Number of bacteria or microorganism present in the specimen ▪ Higher viral load= higher conc. of reagents used Type of Organism ▪ Spore forming, capsulated, enveloped, vegetative (bacteria) enveloped, or non-enveloped(virus) Condition of surface to be decontaminated Disinfectant concentration, pH, temperature, contact time, environmental humidity RESISTANCE OF MICROORGANISMS TO DISINFECTANTS More Resistant Bacterial Spores Mycobacteria Hydrophilic Viruses (non-lipid, nonenveloped) Fungi - STANDRAD PRECAUTION Minimum infection prevention practices that apply to all patient care, regardless of suspected or confirmed infection status of the patient, in any setting where health care is delivered (CDC) ▪ Wear proper PPE, Avoid physical hazards a. Avoid touching eyes, nose, or mouth with gloved or unwashed hands b. ALL SPECIMENS shall be treated as infectious (even if specimen is free from disease; regardless if blood is present) ◼ all body fluids EXCEPT SWEAT c. Avoid wearing jewelry d. Refrain from using mobile electronic devices inside the Laboratory e. Keep work area tidy, clean, and free of cluster and materials not necessary for the work being done DECONTAMINATION SOLUTIONS Use of decontamination solutions with proven activity against enveloped RNA viruses Page 2 of 7 TYPE OF MICROBE Prions Less Resistant ▪ ▪ Vegetative Bacteria Lipophilic Bacteria (lipid containing, enveloped) EXAMPLES Bovine spongiform encephalopathy (Mad cow) Creutzfeldt- Jakob disease Bacillus subtilis; Clostridium sporogenes Mycobacterium tuberculosis; Mycobacterium bovis Rhinovirus; Adenovirus Cryptococcus sp. Candida sp. Streptococcus pneumoniae; Staphylococcus aureus Herpes Simplex; Cytomegalovirus; HIV Prions- most resistant to disinfection also known as Infective proteins. They don't have nucleic acids. The patient who has CJD undergoes brain surgery, all the equipment and instrument must be disinfected. CJD / Creutzfeldt-Jakob disease is an example of human prion. Disinfectants- destroys nucleic acids (DNA and RNA) Bacterial spores can survive in higher temperature. SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER [MODX311] BIOSAFETY AND WASTE MANAGEMENT | Prof. Justin Kim Vergara, RMT, MPH Mycobacteria has different cell membrane so it is harder for the disinfectants to penetrate. Hydrophilic Viruses are the least resistant due to their lipid in the membrane. The disinfectant can easily melt the lipid. 1. TYPES OF CHEMICAL DISINFECTANTS Sodium Hypochlorite Contact Time: 10-15 Minutes Bought in concentrations of 5-8.25%, most common: 5.25% Preparation: 1:10 or 1:100 – At least 5000 ppm, no more than 10,000 ppm Chlorine for decontamination CORROSIVE (highly reactive): Needs to be washed after (Steel) ▪ DO NOT USE CONCENTRATED SOLUTION (produces toxic gas) DO NOT AUTOCLAVE BLEACH SOLUTIONS! Effect: Oxidative effect of Hypochlorous acid Avoid the following: – Exposure to air (↓ free Chlorine concentration due to Chlorine evaporation) – Storage indirect sunlight and varying temperatures (NaOCl solutions can be stored up to 6 months if this is followed!) – Bleach loses 20-50% of its concentration/efficacy after 6mos ▪ Household bleach active ingredient (Ex: Zonrox) Different Cleaning Jobs Require Different Bleach Solutions General lab use - Hypochlorite Solutions SPILLS GENERAL DISINFECTION [Chemical/Blood/Specimen spills] 10% 1% (1PART + 9PARTS H2O) (1PART + 99PARTS H2O) *WHO Laboratory Biosafety Manual 2. Alcohol Best preparation: 60-90% Alcohol (↑ Water conc while maintaining high Alcohol concentrations ↑ contact time of Alcohol to microorganism) ▪ Disinfection: Ethyl Alcohol ▪ Antisepsis: Ethyl or Isopropyl ▪ 70% Alcohol- best concentration to achieve contact time requirement ▪ 40% is also available in the market, cheaper since it has a lower concentration ▪ 70% is more effective than 90% since it achieved the contact time requirement. ▪ While 90% is more volatile – easily evaporates so it does not meet the contact time requirement If used as a disinfecting agent: – Without Bleach:2-5minutes – With Bleach: No need for contact time Bactericidal, Fungicidal, Virucidal, non-Sporicidal Allowed to evaporate from the surface to which they Were applied to achieve maximum effectivity. Effect: Cause denaturation of protein and dissolution Page 3 of 7 of lipid membrane Should not be used as a lone decontaminant for MOLECULAR TESTING – causes aggregation of Nucleic Acids not eradicating them 70% Ethyl Alcohol: Disinfectant & antiseptic at the same time; it can be consumed by human since it is found in liquors Isopropyl Alcohol: also known as rubbing alcohol which is commonly used as antiseptic only 3. Ultraviolet Light ▪ Room disinfectant A type of non-ionizing radiation that causes damage to cellular DNA by producing Thymine dimers – Most lethal wavelength: 260nm Longer wavelength (>1 nm) and low energy Microorganisms are destroyed when water is passed under the UV Lamps Quality Control Indicator: Bacillus pumilus Disadvantage: Can only destroy those under its direct contact. ▪ Eye and skin irritation, skin cancer: Basal cell carcinoma, squamous cell carcinoma, malignant melanoma Wears out overtime. Has little to no effect on RNA. 4. Lysol Active Ingredients: Benzalkonium Chloride or Hydrogen Peroxide ▪ Capable of oxidative burst ▪ Formerly has PHENOL (Carcinogenic) ▪ Phenolic Compounds- Earliest germicide KNOWN EFFECTIVE AGAINST MANY MICROORGANISMS Effects: – Benzalkonium Chloride: Disruption of cell membrane, resulting in leakage of cell contents. – Hydrogen Peroxide: Degrades Organic compounds by the highly reactive Hydroxyl radical (-OH). SPILL CONTROL General Steps: 1. Add concentration bleach (>5.25% available Chlorine) to a final concentration of 10% Bleach 2. Let the solution sit for 30-60 minutes. Larger volumes may require longer contact times. 3. Dispose of the solution down the sanitary sewer. ⚫ INACTIVATION OF PROINS Instrument: SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER [MODX311] BIOSAFETY AND WASTE MANAGEMENT | Prof. Justin Kim Vergara, RMT, MPH – Immerse in 1N NaOH or 2.5% NaOCl for 1 hour; remove and rinse in water, and then transfer to open pan and treat in a gravity displacement (121C) or porous load (134 C) autoclave for 1 hour. ⚫ Surfaces: – Spray or pour 1N NaOH or 2.5% on surface and let sit on 1 hour. Ensure surfaces stay wet for entire period. Surfaces should be clean of any gross contamination as organic material can reduce the effectiveness of the solutions. ▪ Incineration- used for disposable lab equipment 800-1000°C INACTIVATION OF MICROORGANISMS ⚫ AUTOCLAVE Principle: Steam under pressure ▪ Boiling point of water increases in high pressure Fastest and simplest method of sterilization – all organisms (except Prions) and spores are killed within 15 minutes. Biological Indicator: Geobacillus stearothermophilus ▪ ▪ ▪ ▪ Some HEPA filter have air purifier These are exhaust system of the biosafety cabinet that effective traps all of the infectious agents. Hence, the air that will come out is free of microbes. 99.97% efficiency at 0.3 microns and up. Made from Pleated borosilicate glass, arranged in random fibers ⚫ ULPA Filter – 99.999% efficiency at 0.12 microns Ultra-low Penetration Air ⚫ SULPA Filter – 99.9999% efficiency at 0.12 microns Super Ultra-low Penetration Air To achieve maximum cleanliness Used in reference laboratories & BSL4 ▪ ▪ ⚫ ⚫ ⚫ “Curtain of Air” at the opening- protection of product Laminar flow of filtered air within the BSC- Protection of the personnel Filtration of exhausted air- Protection of the environment Properties: – 121C, 15 psi, for 15 minutes: Media, Liquids, Utensils, Glass Pipettes, and Instruments for Assay – 132C, 15 psi, 30-60 minutes: Decontaminating Medical Wastes *PSI- pounds per square inch QUALITY CONTROL FOR AUTOCLAVE ⚫ Biological Indicator: ▪ Incubate for 24hours at 37ºC – No color change IF sterilization is complete – Turns color YELLOW if growth Happens (ineffective) ⚫ ⚫ Autoclave Tape: – Turns black if Sterilization is complete – The tape has white diagonal lines with Geobacillus stearothermophilus ⚫ ⚫ ⚫ ⚫ BIOSAFETY CABINET Calibrated and certified before use (by supplier) Practice proper usage, placement, and decontamination The Biosafety Cabinet is a requirement to process infectious specimens. ▪ Enclosed container minimizing our exposure to hazardous materials TYPES OF BIOSAFETY CABINET PROTECTION FILTER ⚫ HEPA Filter – Standard for Biosafety High Efficiency Particulate Air – 99.97% efficiency at 0.3 microns (pore size) – Made from Pleated borosilicate glass, arranged in random fibers ▪ Exhaust system filtering air that will circulate inside Biosafety cabinet ▪ Standard for Biosafety; most commonly used Page 4 of 7 ⚫ ⚫ ⚫ ⚫ BIOSAFETY MANAGEMENT Ensure the development and adoption of a biosafety management plan and a safety or operations manual. Ensure that regular training in laboratory safety is provided. Personnel should be advised of special hazards, and required to read the safety or operations manual and follow standard practices and procedures There should be an arthropod and rodent control program. ▪ Most infectious diseases are transmitted through these animals Appropriate medical evaluation, surveillance and treatment should be provided for all personnel in case of need, and adequate medical records should be maintained. HEALTH AND MEDICAL SURVEILLANCE Provision of active or passive immunization were indicated ▪ Ex: Passive- Hepa B Ig vaccine; Anti- tetanus Active- Ab produced Facilitation of the early detection of laboratoryacquired infections Exclusion of highly susceptible individuals (e.g., pregnant women or immuno-compromised individuals) from highly hazardous laboratory work Provision of effective personal protective equipment and procedures. SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER [MODX311] BIOSAFETY AND WASTE MANAGEMENT | Prof. Justin Kim Vergara, RMT, MPH SAFETY PRACTICES 1. Before Testing (Pre-analytical) -Specimen collection: use antiseptic technique -Specimen preparation -Specimen transport: use appropriate containers that properly sealed, with labels, and biohazard symbol 2. Testing (Analytical) -Testing: apply the safety rules 3. After Testing (Post-analytical) -Disposal DEVELOP PERSONAL SAFE WORK HABITS Wash hands before and after entering the lab Change gloves frequently to prevent cross contamination Wear lab coat or apron Dispose of contaminated sharps and waste immediately after testing Pipetting by mouth is strictly forbidden Never eat, drink or smoke at the test site Keep food out of the laboratory/testing site refrigerator MAINTAIN CLEAN AND ORDERLY WORK SPACE Keep work areas uncluttered and clean Disinfect work surfaces daily Restrict or limit access when working Keep supplies locked in a safe and secure area Keep emergency eye wash units in working order and within expiry date PROPER DISPOSAL OF BIOWASTES Good Safety Practices: – Identify Hazards – Implement safety strategies to contain hazards – Audit existing practices to determine whether new ones are needed LABORATORY WASTE MANAGEMENT BIOLOGICAL WASTE ⚫ Exposure: Ingestion, Inoculation, Tactile Contamination, Aerosolization, Inhalation of Infectious materials ⚫ Blood – HBV, HCV, HIV – Blood-borne pathogens – Inactivated and Autoclaved ⚫ Swabs – Respiratory infections/ – Inactivated (Heat, Chemical) and autoclaved before disposal ⚫ Tissues – Should be fixed with a fixative – Unfixed tissues must be inactivated and autoclaved ⚫ Other body fluids – Sputum: Tuberculosis, other respiratory infections – Inactivated and autoclaved PREVENTION ⚫ Gloves – Primary barrier protection – Not reused or washed for reusing ⚫ Masks/Respirators, Protective Eyewear, Face Shield – for exposure from splashes to the mouth, eyes,nose ⚫ Decontaminate work area regularly CHEMICAL HAZARDS Stored in certain sections of the Laboratory – Not in use, packaging with leakage or corrosion: Replaced – Water reactive chemicals: No contact with water MATERIAL SAFETY DATA SHEET – Document that gives detailed information about a material and about any hazards associated with the material HANDLING CHEMICALS Ignitable, Corrosive, Toxic, Reactive Check label for substance verification ▪ CMRA (Carcinogenic, Mutagenic, Respiratory Harmful, Allergenic) Chemical-resistant gloves Containers held away from the body during transferring Do not heat with direct flame ACID TO WATER Do not touch, taste, or smell chemicals Use a Laboratory Chemical Hood. Clean spills properly and promptly, dispose accordingly. CHEMICAL HAZARDS IN THE GENOTYPING LABORATORY 1. Page 5 of 7 Guanidinium thiocyanate commonly used in lysis buffers ▪ denaturing agent for RNA extraction ▪ Carcinogenic SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER [MODX311] BIOSAFETY AND WASTE MANAGEMENT | Prof. Justin Kim Vergara, RMT, MPH ▪ 2. DO NOT MIX WITH BLEACH Ethidium Bromide (mutagen) DNA staining in agarose gels, buffers For Agarose Gel Electrophoresis Intercalating agent, Nucleic Acid stain Fluorescence: Orange Not directly mutagenic: metabolites are mutagenic ▪ For visualization of result for PCR or hybridization techniques ▪ Mutagenic- causes mutation ▪ Used for research purposes only 6. ❖ Large spill - Cover with paper towels and soak with 10% household bleach and allow to stand for 10-30 minutes Small spill - Wipe with paper towel soaked in 10% bleach Dispose all contaminated towels in biohazard bag and dispose it properly In Case of an Accident What types of accidents can happen? ❑ Potential Injury, i.e., needlesticks, falls ❑ Environmental, i.e., splashes or spills ❑ Equipment damage What should you do? ❑ Report to supervisor immediately ❑ Assess & take action ❑ Record using form ❑ Monitor situation 3. Carcinogens Any substance that can cause cancer. -Changes the metabolic processes by damaging the genomes Different types – Decontaminating Agents – Dyes – Probes and Labels DYES, PROBES, LABELS SYBR Green Replacement for Ethidium Bromide Can still bind to DNA with high affinity = potential Carcinogen ⚫ Acrylamide (neurotoxin) Used in PAGE (Polyacrylamide Gel Electrophoresis) Cross-linking agent for gel chromatography and electrophoresis Can cause peripheral neuropathy, possible carcinogen ⚫ Phenol (organic solvent) Used in Phenol-Acetic Acid-Urea Polyacrylamide Gel Electrophoresis (PAU-PAGE) ⚫ Chloroform (organic solvent) Extraction of Proteins Eye damage (sclera), fainting, life-threatening ❖ Action Plan for Implementing Safety Practices ⚫ Identify hazards Establish and implement safety policies and procedures Conduct safety specific training ❑ Must be a priority ❑ Communication is key Perform regular audits or assessments ❖ Policies and Procedures Safety reminders (biohazard, chemical, physical) should be included in lab SOPs (from DOH) Refer to general lab or institutional procedures and policies for safe handling and waste disposal ⚫ ACTION PLAN FOR IMPLEMENTING SAFETY PRACTICES ❖ 1. 2. 3. 4. 5. In Case of a Spill or Splash Evacuate Room and notify others to leave the room and post a warning sign for No entry Remove all contaminated clothing and /or lab coat and place in a biohazard bag Wash all exposed skin with antiseptic soap and water Inform supervisor Decontaminate the area: Assemble clean-up materials Page 6 of 7 DEFINITIONS Many different terms are used for disinfection and sterilization. The following are among the more common in biosafety: Antimicrobial – An agent that kills microorganisms or suppresses their growth and multiplication. Antiseptic – A substance that inhibits the growth and development of microorganisms without necessarily killing them. Antiseptics are usually applied to body surfaces. Biocide – A general term for any agent that kills organisms. Chemical germicide – A chemical or a mixture of chemicals used to kill micro-organisms. Decontamination – Any process for removing and/or killing microorganisms. The same term is also used for removing or neutralizing hazardous chemicals and radioactive materials. Disinfectant – A chemical or mixture of chemicals used to kill microorganisms, but not necessarily spores. Disinfectants are usually applied to inanimate surfaces or objects. Disinfection – A physical or chemical means of killing microorganisms, but not necessarily spores. Microbicide – A chemical or mixture of chemicals that kills microorganisms. The term is often used in place of “biocide”, “chemical germicide” or “antimicrobial”. Sporocide – A chemical or mixture of chemicals used to SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER [MODX311] BIOSAFETY AND WASTE MANAGEMENT | Prof. Justin Kim Vergara, RMT, MPH kill microorganisms and spores. Sterilization – A process that kills and/or removes all classes of microorganisms and spores. References: ◼ Page 7 of 7 Justin Kim Vergara, RMT, MPH. MODX311 Lecture. Our Lady of Fatima University, Valenzuela City. SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER NUCLEIC ACID AND PROTEINS LEC 2 TRANS 2 MOLECULAR BIOLOGY AND DIAGNOSTICS Instructor: JUSTIN KIM C. VERGARA, RMT, MPH Date: February 22, 2022 2021 – 2022 2nd Semester MODX311 LEC Outline At the end of the session, the student must be able to learn: I. II. III. IV. V. VI. History of Molecular Diagnostics Nucleic Acid A. Deoxyribonucleic acid (DNA) i. DNA structure ii. DNA Double Helix iii. DNA Replication iv. Restriction Enzymes v. Recombination (sexual reproduction) vi. Recombination (asexual reproduction) vii. Plasmids B. Ribonucleic acid (RNA) i. Types of RNA ii. Transcription iii. RNA Polymerases Proteins Amino acids Chromosomes Definition of terms ▪ ▪ HISTORY OF MOLECULAR DIAGNOSTICS YEAR 1865 1866 1949 KEY EVENT MENDEL’S LAW OF HEREDITY JOHANN MIESCHER, PURIFICATION OF DNA SICKLE CELL ANEMIA MUTATION WAS FIRST STUDIED WATSON AND CRICK’S DNA STRUCTURE RECOMBINANT DNA TECHNOLOGY DNA SEQUENCING IN VITRO AMPLIFICATION OF DNA (PCR) THE HUMAN GENOME PROJECT 1953 1970 1977 1985 2001 ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ 1865 – Gregor Mendel discovered that the traits of parents could be passed through to the children. 1953 – It is one of the most important biological discovery of the 20th century when James Watson and Francis Crick described the structure of DNA. 1985 – Kary Mullis discovered the in vitro amplification of DNA. He won a noble prize because of the discovery of the Polymerase Chain Reaction (PCR). NUCLEIC ACID One of the Macromolecules in our body together with the Carbohydrates, lipids, and Proteins The building blocks of DNA and RNA are made up of nucleic acids. Macromolecules constructed out of long chains (strands) of monomers called nucleotides. Each nucleotides have three functional groups: (1) Nitrogenous base (2) Pentose Sugar (3) Phosphate groups Storage and transmission of genetic information. The main function of the Nucleic acid is to store and transmit the genetic information from the DNA to become Protein. Genotypic and Phenotypic characteristics of the organism. DNA is very important for the storage of genetic information of the living organism. TWO TYPES: Deoxyribonucleic acid (DNA) Ribonucleic acid (RNA) Page 1 of 6 ❖ ❖ ❖ ❖ ❖ ❖ Nitrogenous bases have two types: Purine and Pyrimidine bases. o Purine – made up of two or double ring structure Guanine Adenine o Pyrimidine – Cytosine Thymine – specific for DNA Uracil – for RNA Sugar building block of the Nucleic acid – Pentose sugar. o These sugars have five carbon and are positioned from the first carbon up to the fifth carbon. Each carbon has their own function. o Example: 1st carbon holds the nitrogenous bases 2nd carbon determines whether it is Deoxyribose or Ribose -OH (Hydroxyl group) – ribose; Oxygenated hydrogen -H (Hydrogen group) – Deoxyribose; Deoxygenated rd 3 carbon of the sugar, you can find the -OH group or Hydroxyl that attaches to the succeeding nucleotides forming a sequence of nucleotides. - Holds the phosphate group of the succeeding nucleotides through Phosphodiester bond. It also Supports the sugar phosphate backbone, that is made up of phosphate and a sugar. Remember: Whether it is Deoxyribose or Ribose, check if it is phosphorylated. If it is phosphorylated, it contains the Phosphate group making a monomer of Nucleotide. Nucleotide = Phosphorylated sugar + Base Nucleoside = Sugar + Base w/o the phosphate group We need to differentiated them through their structure. Whether they are single stranded or double stranded. Check the composition as well. Not all DNA are Double stranded, and not all RNA is single stranded. We also need to check where is the 3’ end and 5’ end of the DNA. o 5’ – It always end with the free phosphate group o 3’ – It is the free sugar A. DEOXYRIBONUCLEIC ACID (DNA) ▪ Primary function is to store genetic information. ▪ Usually found in Nucleus, and some (small amount) are found in the Mitochondria. Macromolecule of carbon, nitrogen, oxygen, phosphorous, and hydrogen atoms. Assembled in units of nucleotides that are composed of a phosphorylated ribose sugar and a nitrogen base. Nitrogen bases are attached to the SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER [MODX311] NUCLEIC ACID AND PROTEINS | Prof. JUSTIN KIM C. VERGARA, RMT, MPH Deoxyribose sugar which forms a polymer with the deoxyribose sugars of the other nucleotide through a phosphodiester bond. Adenine Cytosine Guanine Thymine COMPREHENSION CHECK!!! ✓ Anti-parallel, double stranded molecule ✓ Sugar Phosphate backbone ✓ Complementary base pairs joined by Hydrogen bond in the middle ✓ Each strand has the potential to deliver and code for information ✓ Length of DNA given in Base pairs ✓ ALWAYS READ THE STRAND FROM 5’ TO 3’ ❖ DNA STRUCTURE Double helical structure First Described by James Watson and Francis Crick Their molecular model of DNA was founded on previous observation of the chemical nature of DNA including the Diffraction analysis performed by Rosalind Franklin. Helical structure of DNA results from specific sequence (order) of nucleotides in the strand, as well as the surrounding chemical microenvironment. Two DNA chains form hydrogen bonds with each other in a specific weight, because these hydrogen bonds between the nucleotide are the key to the specificity of most nucleic acid base test in Molecular Laboratory. DNA REPLICATION ▪ ▪ DNA polymerase Enzyme responsible for polymerizing the nucleotide chains Template to determine which nucleotides to add to the chain Reads the template in the 3ʹ to 5ʹ direction It is a semi conservative replication means that the DNA replication results in two stranded DNA of which one parent strand and one daughter strand. The Parent strand will be divided into two that is happening during cell division. Before cell division, the DNA will copy itself forming two double stranded DNA. So that the Parent cell will have 2 daughter cells with the same genetic information. DNA DOUBLE HELIX ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ The sequences of the two strands that form the double helix are complementary Base pairing which follows Chargaff’s rule o The Adenine should bine with Thymine, and Cytosine should bine with Guanine. o Example: The Adenine binds with Guanine, our system will detect it and will remove the binding between the two non-complementary bases. o The nitrogenous bases are highly specific and they held together in a certain pattern. The complementary strands are Antiparallel orientation, meaning the 5’ end of one strand binds with the 3’ end of the other strand. Because of how the DNA is being replicated. The formation of hydrogen bonds between two complementary strands of DNA is called hybridization. In between the Adenine and Thymine, there is a hydrogen bond. But there’s only two hydrogen bond. Unlike in the Guanine and Cytosine, there are three hydrogen bonds. Between the two, the Guanine and Cytosine are more stable compared to the Adenine and Thymine. There is no two double ring structure that binds together. It is not possible because these bonded pairs will sort of fit in the diameter of the double helix. If you will bind two purines, the diameter of the DNA will be increased. It should be 1 Purine/1 double ring structure and 1 single ring structure. Once the base pair interaction forms the ladder. DNA ladder/Structure will twisted/coiled. It forms as a double helix because of nitrogenous bases. We need to protect the nitrogenous bases since they are Hydrophobic (waterfearing). Unlike the Sugar phosphate backbone, they are Hydrophilic (water-loving) in nature. In order to protect the nitrogenous bases, it will twist itself to prevent the water to come in contact with the nitrogenous bases. Page 2 of 6 ❖ ▪ ▪ ▪ ▪ Other key players for the DNA Replication: HELICASE – This will unzip the DNA strand. The site where the strand are being unwound by the helicase enzyme is called replication fork. SINGLE STRANDED BINDING PROTEINS – This will bind to the template/ parent strand to prevent the re-binding of the complementary bases. TOPOISOMERASE – Placed in front of the replication fork. This enzyme will prevent the super coiling of the DNA during the unzipping of the complementary strand. PRIMASE – Very important to produce RNA primer. An RNA primer is a short strand of nucleotide that are specific/ complementary to the parent strand. They will dictate where to start the replication. The primer will activate the DNA Polymerase 3. The DNA Polymerase 3 will start the adding of nucleotide bases to the daughter strand. It is only possible if it is toward the replication fork. POLYMERASE 3 DNA POLYMERASE 1 LIGASE ENZYME There is a possibility of re-binding of strands. SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER [MODX311] NUCLEIC ACID AND PROTEINS | Prof. JUSTIN KIM C. VERGARA, RMT, MPH ▪ ▪ ▪ ▪ Blue strand – Daughter strand; Yellow strand – Parent strand The DNA and RNA being synthesized starting from the 5’ by copying the complementary strand of the 3’. DNA polymerase will help for the addition or for knowing what nucleotides should be added to the parent strand. Leading strand – Top strand; This strand can build from one continuous strand towards the replication fork. Lagging strand – Bottom strand; cannot build one continuous strand because there is lagging. There are gaps present. It is building away from the replication fork. o The primer with the help of DNA Polymerase 3 will synthesize new strand of DNA. o RNAse H (Polymerase 1 enzyme) will enter to remove the primer. o The primer will jump into a new site. Those gaps are from the RNA primer and removed by the RNAse H. There is a possibility for these fragments will bind. o DNA Ligase will help to connect the two fragments forming a one continuous strands. o Okazaki fragment – is a short sequence that was formed in the lagging strand that will be connected together by the DNA ligase. RESTRICTION ENZYMES ▪ ▪ ▪ A type of Enzyme used for the degragation of the DNA. Nucleases/DNAses 1. Exonucleases – it degrades the DNA from its end. Either in the 5’ or 3’. - very useful if you want to proofread the newly synthesized DNA. Or to remove the non-complementary base by breaking the phosphodiester bond and will replaced it by the correct one. 2. Endonucleases – In the middle. It is used if you want to insert a new sequence or cut them in the middle part. And to identify a recognition site of endonuclease. o Restriction enzymes Attacks specific sequence of DNA Also called as Molecular scissors. Very useful for Molecular testing specifically the Type 2 restriction endonucleases. o Three types of Restriction Endonucleases - The Restriction Endonucleases are mostly found in bacteria. And later on, it was discovered that all nucleated cells contain restriction enzyme. TYPE 1 – Random cut TYPE 2 – most important one because it makes specific cut. It will find a recognition site. TYPE 3 – Non specific cut Before, the purpose of the Restriction enzyme serves as the Bacteria’s immune system. Example, a bacteriophage (virus that attacks a bacteria) will attack the bacteria releasing its nucleotide sequence inside. The bacteria will protect itself from the invading bacteriophage. The bacteria containing restriction enzyme will cut the nucleic acid of the Bacteriophage for it to stop its replication. To ensure that the restriction enzyme will not cut it self nucleic acid. Methyltransferase enzyme will add methyl group to the self nucleic acid. Kailangan methylated na yung nucleic acid, they are resistant to degragation so that when the restriction endonuclease attacks, it will only cut the invading nucleic acid and not your own nucleic acid. Most of these enzymes are isolated from bacteria. For each recognition site, it will make a specific cut depending on what enzyme you will use. Deoxyriboendonucleases or Endonucleases Break the sugar-phosphate backbone of DNA. Restriction enzymes Endonucleases that recognize specific base sequences and break or restrict the DNA polymer at the sugarphosphate backbone DNA Ligase Catalyzes the formation of a phosphodiester bond between adjacent 3ʹ-hydroxyl and 5ʹ-phosphoryl nucleotide ends Other DNA Metabolizing Enzymes : Nucleases- degrade DNA from free 3ʹ-hydroxyl or 5ʹphosphate ends. Methyltransferases- catalyze the addition of methyl groups to nitrogen bases Helicases- separation of the sugar-phosphate backbones in both strands RECOMBINATION (SEXUAL REPRODUCTION) ▪ ▪ ▪ Mixture and assembly of new genetic combinations Common for Humans Example is the Mendel’s law, each generation of sexually reproducing organism is a new combination of parental genomes. Recombinant chromosomes are found in the children. Half of the traits are from the mother, and half are from the father. (Diploid) ▪ ▪ RECOMBINATION (ASEXUAL REPRODUCTION) Genetic information in asexually reproducing organisms can be recombined in three ways: o Conjugation – The Transfer of genetic material from one organism to one organism through direct contact. ▪ 1 Bacteria (donor) and 1 Bacteria (Recipient) o Transduction – Also passes the genetic material from the donor to the recipient through the use of carrier. ▪ Example: Viruses, Bacteriophages o Transformation – very useful in modern day recombination techniques. - The target sequence from the donor cell could be cut using the restriction enzyme (specific sequence), and that specific sequence will be inserted inside the chromosome of the recipient cell. - Genetic Engineering / Genetic Loaning of the Nucleic acid. Page 3 of 6 SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER [MODX311] NUCLEIC ACID AND PROTEINS | Prof. JUSTIN KIM C. VERGARA, RMT, MPH TYPES OF RNA 1. Ribosomal RNA (rRNA) The largest component of cellular RNA 80% to 90% of the total cellular RNA Important structural and functional part of the ribosomes, cellular organelles where proteins are synthesized Various types of ribosomal RNAs are named for their sedimentation coefficient (S) Three rRNA species in prokaryotes 16S, 23S, 5S rRNA species in prokaryotes Single 45S precursor RNA (pre- ribosomal RNA) ▪ Most abundant; and it has catalytic roles and structural roles 2. Messenger RNA (mRNA) In prokaryotes, mRNA is synthesized and simultaneously translated into protein. Prokaryotic mRNA is sometimes polycistronic Eukaryotic mRNA is monocistronic, In eukaryotes, copying of RNA from DNA and protein synthesis from the RNA are separated by the nuclear membrane barrier mRNA Transcription Constitutive Transcription Inducible or Regulatory Transcription ▪ Most important; it will transport information from DNA to Ribosome. ▪ mRNA has the same sequence that can be found from the DNA. ▪ mRNA will go to the cytoplasm specifically in the Ribosome, because Ribosome is for protein synthesis. ▪ It is a single stranded molecule only. It is just a copy of one strand of the DNA. 3. Transfer RNA (tRNA) Translation of information from nucleic acid to protein requires reading of the mRNA by ribosomes, using adaptor molecules or transfer RNA (tRNA) Relatively short, singlestranded polynucleotides of 73 to 93 bases in length MW 24,000 to 31,000 ▪ Responsible in carrying individual amino acid to the Ribosome wherein they will be joined together by peptide bonds to make that protein. ▪ It contains three letter nucleotides: UAG ▪ This UAG carries an amino acid molecule ▪ The mRNA should be complementary to the tRNA containing the specific amino acid PLASMIDS Most plasmids are double-stranded circles of 2,000 to 100,000 bp (2 to 100 kilobase pairs) in size. Plasmids can carry genetic information Plasmids were found to be a source of resistant phenotypes in multidrug- resistant bacteria. They carry the Antibiotic-resistant gene. Very short sequence Separated from the actual chromosome of the bacteria These are circular in form If an organism is an antibiotic resistance, it can passed the information from the plasmid from one bacteria to another through direct contact or Conjugation. Cloning is also possible through transformation. ▪ ▪ ▪ ▪ ▪ ▪ B. ▪ ▪ ▪ ▪ RIBONUCLEIC ACID (RNA) Polymer of nucleotides similar to DNA Synthesized as a single strand rather than as a double helix RNA strands do not have complementary partner strand Nitrogen bases Adenine Cytosine Guanine Uracil Single Stranded; smaller than the DNA Its function is to translate the DNA to become proteins (phenotypic characteristics) Most DNA are found in the Cytoplasm mRNA is found in the Nucleus 4. Small Nuclear RNA (snRNA) functions in splicing in eukaryotes RNAs sediment in a range of 6 to 8S ▪ Found in the Nucleus only ▪ Product of RNA splicing. Splicing is happening to remove the unnecessary sequences in the mRNA Other RNAs ▪ Mostly they are just structural and catalytical functions but they does not have the ability to code the genes. 5. sRNAs ▪ Untranslated RNA molecules 6. ncRNAs ▪ Non-coding RNA TRANSCRIPTION ▪ Page 4 of 6 This is the copying of information from the DNA to mRNA because DNA can only store information, in order for this SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER [MODX311] NUCLEIC ACID AND PROTEINS | Prof. JUSTIN KIM C. VERGARA, RMT, MPH ▪ information to be utilized it must be transcribed and translated into protein. Gene expression – is a process of transcription and translation Copying of one strand of DNA into RNA by a process similar to that of DNA replication. Catalyzed by RNA polymerase Three Types of RNA polymerase pol I – non-coding RNA pol II – most important; the one responsible for polymerizing mRNA pol III – non-coding RNA ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ ▪ The RNA polymerase uses one strand of the Double helix that will serve as a template for the synthesis of the RNA. About 10 base pairs of DNA would be unwind with the use of RNA Helicase to allow the DNA polymerase to work. The Primer will attach specifically where is the start of transcription. Specific part of the DNA only. It’s the same process with the DNA replication If you have abnormalities in the protein, maybe because it was written in your DNA or in the gene that was translated to become protein only. Abnormal proteins are product of abnormal genes as well. This amino acid that was carried by the tRNA to become a polypeptide chain. AMINO ACIDS Each amino acid has characteristic biochemical properties determined by the nature of its amino acid side chain Grouped according to their polarity Determine the shape and biochemical nature of the protein. The molecule of an amino acid is composed of four different groups: o Carboxyl group – COOH- ; they are Ionized. (at pH 7) o Amino group – Not Ionized. (at pH 7) o Side chain group o Hydrogen group All of them are connected with the Carbon on its middle. Zwitter ion – it can switch from positive or negative charge. But at physiologic pH, most of the amino acids are negatively charge. RNA POLYMERASES OTHER RNA-METABOLIZING ENZYMES ❖ ❖ Ribonucleases Degrade RNA in a manner similar to the degradation of DNA by deoxyribonucleases Classification: Endoribonucleases – to cut the RNA at the middle part Exoribonucleases – for the proofreading of noncomplementary of nucleotide sequence RNA Helicases Catalyze the unwinding of double-stranded DNA Used for Double stranded RNA ▪ ▪ PROTEINS Products of transcription and translation of the nucleic acids They manifest the phenotype directed by the nucleic acid information Polymers of amino acids Proteins are polypeptides that can reach sizes of more than a thousand amino acids in length Most abundant macromolecule in cells Proteome vs Genome o Proteome – Sometimes we are doing protein analysis. This is a collection of proteins encoded in all organism’s DNA. o Genome – Collection of all genes. Sometimes, we need to check or need to do protein analysis whether there is a problem with the transcription or translation. If there is something with the messages/ nucleotide sequence in our genes, definitely it will affect the product. Page 5 of 6 ▪ ▪ ▪ There are 20 naturally occurring amino acids. They are group according to their polarity, whether they are Nonpolar or Polar. Polar amino acids are subclassified into three: o Uncharged o Negatively charged o Positively charged The sequence of amino acids determines the nature and activity of that protein. Primary Structure ▪ ▪ ▪ ▪ The linear sequence of amino acids joined together by peptide bonds Once they are formed inside the Ribosome, they will form a long chain of amino acid called as the primary structure. The sequence of the primary structure is very important. We will read starting amino terminal end up to carboxyl terminal end, and they are being held together by peptide bond. Minor changes in primary structure will definitely have an effect (Disorder or Abnormality) Example: Hgb S (Sickle cell anemia) is a product of amino acid substitution. ON the 6th amino acid, Valine is seen instead of glutamine. It is a genetic problem. SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER [MODX311] NUCLEIC ACID AND PROTEINS | Prof. JUSTIN KIM C. VERGARA, RMT, MPH ▪ ▪ Secondary Structure ▪ ▪ ▪ ▪ The regular folding of regions of the polypeptide chain There is a folding or coiling because of the R-side chain. The R-side chain of each amino acid will interact with each other and formed hydrogen bond. Folded – β-pleated sheet Coiled – α helix CHROMOSOMES DNA double helix that carries genes Seen during cell division Creates identical copy of itself o Centromere The X shaped is rarely seen in a cell, they are usually in a threadlike only – Chromatin threads. But during cell division, they will formed the X shaped chromosome. But at normal times, the DNA are found as long chromatin thread. CHROMOSOME FORMATION Tertiary Structure ▪ ▪ ▪ ▪ ▪ The 3D arrangement of all the amino acids in the polypeptide chain Is also important for protein function. This folds are specific with each other. The folding of a protein is specific as well. If there’s any problem with the folding or changes with the Rside chain, magkakaroon na rin ng abnormality with the folding of the Protein. Denatured protein – loss of tertiary structure; non-functional. ▪ ▪ Quaternary Structure ▪ ▪ ▪ ▪ This is formed by the interaction of different polypeptide chains Groups of tertiary structure bound together to form either dimer, trimer, or tetramer, depending on how many tertiary structure are present. Oligomers – group of tertiary structure, they give complexity in an organism. Monomer - each component of protein; tertiary structure. ▪ During cell division, first, the DNA will coiled up and wrap itself around to the proteins called Histones. These Histones with wrapped DNA are called Nucleosome. And as the Nucleosome coils and twists up even more, it will produce the Chromatin fibre. Then this Chromatin fiber will later to be condensed and will coil and fold up even more until it will formed the Chromosome structure. This chromatin fibers begin to formed really tight loops and they coil up even more until they eventually formed the Chromatin structure. The Chromosomes are made up of DNA that is coiled up during cell division. Definition of terms KARYOTYPE Individual’s collection of chromosomes Used to check for abnormalities GENOTYPE Genetic DNA composition of organisms PHENOTYPE Physical appearance KARYOTYPING – the collection of 23 Pairs of Chromosomes. 22 pairs for the autosome and 1 pair for the sex chromosome. And check for any abnormalities. Trisomy 21 (Down syndrome) – There’s an extra chromosome present in the 21st chromosome of the patient. References: ◼ Page 6 of 6 Justin Kim Vergara, RMT, MPH. MODX311 Lecture. Our Lady of Fatima University, Valenzuela City. SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER NUCLEIC ACID ISOLATION LEC 3 TRANS 3 MOLECULAR BIOLOGY AND DIAGNOSTICS Instructor: Justin Kim C. Vergara, RMT, MPH Date: February 27, 2022 Outline At the end of the session, the student must be able to learn: I. II. III. IV.     Nucleic Acid Extraction Isolation of DNA A. Sample Preparation B. DNA Isolation Methods Isolation of RNA A. Sample Preparation B. RNA Isolation Methods Measurement of Nucleic Acid Quality & Quantity I. NUCLEIC ACID EXTRACTION Release of nucleic acid from the cell  Free of contamination with protein, carbohydrate, lipids, or other nucleic acids The initial release of the cellular material is achieved by breaking the cell wall (if present) and cell and nuclear membranes (cell lysis) Target material is purified Concentration and purity determination  In purity determination, we need to quantify & qualify the samples before testing  once the specimen is submitted in the laboratory, we need to prepare the materials  to determine success of result, we must have quality, efficient samples of DNA/RNA/Nucleic Acid II. ISOLATION OF DNA Miescher (1869)  First isolated DNA from human cells  First DNA human samples were isolated through Alkaline lysis method  if you will add Nucleic Acid to alkaline solution, it will precipitate and we can separate them through Density Gradient Centrifugation (based on the molecular weight of the nucleic acid) Meselson and Stahl (1958)  Demonstrate semi-conservative replication of DNA Alkaline Lysis  procedures were used extensively for extraction of 1- to 50-kb plasmid DNA from bacteria during the early days of recombinant DNA technology.  Purpose: DENATURE first the DNA and then, after alkaline treatment, we need to acidify it to RENATURE (to go back to its state)  Alkaline lysis before was effective only in plasmid DNA since it has shorter sequence  Large chromosomal DNA and protein cannot renature properly when neutralized at acetate at low pH after treatment, forming large aggregates, precipitate out of solution  1 Kilo base pair = 1000 nucleotides 1. 2.  A. SAMPLE PREPARATION identify what sample do you need choose the correct sample collection tube The initial steps in nucleic acid isolation depend on the nature of the starting material and the test method.  In blood and bone marrow specimens, PREFERRED choice of collection is in yellow tubes with ACD (Acid Citrate Dextrose) for molecular studies  Other tubes that can be use: Tripotassium / K3 (Purple) Sodium Heparin (Brown) & Lithium Heparin (Green): for cytogenetic studies Non-additive Tubes (Red): if we want CELL FREE specimen, use serum Page 1 of 5  2021 – 2022 nd 2 Semester MODX311 LEC Sample types and nucleic acid sources  Viruses  Bacteria  Nucleated cells YIELD OF DNA FROM DIFFERENT SPECIMEN SOURCES Specimen Expected Yield* Specimens Adequate for Analysis Without DNA Amplification 6 Blood (1mL, 3.5-10 x 10 WBCs/mL) 50-200 ug Buffy Coat (1mL whole blood) Bone marrow (1mL) 100-500 ug 7 Cultured cells (10 cells) 30-70 ug Solid tissue (1mg) 1-10 ug Lavage fluids (10mL) 2-250 ug 7 Mitochondria (10-mg tissue, 10 cells) 1-10 ug Plasmid DNA, bacterial culture (100-mL 350 ug – 1 mg overnight culture) Bacterial culture (0.5mL, 0.7 absorbance units) 10-35 ug Feces (1mg; bacteria, fungi) 2-228 ug Specimens Adequate for Analysis With DNA Amplification Serum, plasma, CSF (0.5mL) 0.3-3 ug Dried blood (0.5 – 1 cm diameter spot) 0.04-0.7 ug Saliva (1mL) 5-15 ug Buccal cells (1mg) 1-10 ug Bone, teeth (500 mg) 30-50 ug Hair follicles 0.1-0.2 ug 2 Fixed tissue (5-10x10 micron sections;10 mm ) 6-50 u Feces (animal cells, 1mg) 2-100 pg  Example of Analysis WITH DNA amplification is polymerase reaction. Few amount of samples is already enough here (compared to specimens for analysis WITHOUT DNA amplification). 1. BACTERIA & FUNGI Bacteria and fungi have tough cell walls that must be broken to allow the release of nucleic acid  cell walls protect the DNA from degradation so we must disrupt it first  Enzymatic digestion: gently procedure  Proteinase K: digests proteins  Lysozyme: digests other cell organelles  Alkaline Extraction: most common method  1% Sodium Dodecyl Sulfate & 0.2 M NaOH: to lyse the cell  EDTA: chelating reagent; chelates DNAse; DNAse enzyme will degrade the DNA so we must chelate it.  Glucose: help to destroy the cell wall  Mechanical Disruption: not usually use since it may also destroy the nucleic acid; does not give assurance that nucleic acid will be preserved well  Grinding: use grind method if the sample is solid  Glass Beads: vigorous shaking  Boiling Extraction: method used if the sample is treated with lysozyme  Diluted Sucrose  Triton X-100 detergent  Tris Buffer  EDTA 2. VIRUSES Some procedures use cell-free specimens, such as plasma, for viral detection.  intracellular parasite  if active, it can be found in the host cell.  It is harder to extract once they are already in cell genome  Hence, we use plasma since we can look for FREE viruses SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBERS [MODX311] NUCLEIC ACID ISOLATION | Prof. Justin Kim C. Vergara, RMT, MPH  a. b. 3. NUCLEATED CELLS IN SUSPENSION Free WBCs carrying nucleic acids and cell-free nucleic acids are available from plasma.  We need to separate nucleated from non-nucleated cells  To achieve that, get the buffy coat since this is where the mononucleated cells are found Differential density-gradient centrifugation  whole blood or bone marrow mixed with isotonic saline is overlaid with Ficoll - a highly branched sucrose polymer  preferred method because it does not penetrate the biological membrane = no effect on structure of WBC nd  2 layer in between buffy coat & RBC, used to prevent contamination from RBC Differential osmotic fragility of RBCs and WBCs  Incubation in hypotonic buffer or water will result in the lysis of the RBCs 1. 2. 3. 4. 1. DNA ISOLATION CHEMISTRIES 1.1. Organic Isolation Method    4. PLASMA  In studying for molecular diagnosis, plasma can be of aid  Cell-free nucleic acid is a characteristic of tumors and transplanted organs Exosomes  Small vesicles form by invagination and budding from the inside of cellular endosome vesicles and are secreted by living cells.  Exosomes are released from solid tumors and transplanted organs  30-100 nm, formed by budding of endosome vesicles, can be collected by centrifugation Liquid Biopsy  sources of circulating nucleic acids  Isolation of cell-free nucleic acid requires procedures to concentrate the target nucleic acid before isolation  use of plasma for the purpose of diagnostic and prognostic analysis  we can also extract Nucleic Acid in liquid biopsy especially in viral infections & cancer cells cancer cells have the ability to separate from the tumor, and be found in body fluids. Hence, we can also study the molecular characteristics of cancer cells  Sources: plasma, CSF, ascites, pleural fluid Used to detect presence of extracellular viruses 5. TISSUE SAMPLES  Fresh or frozen tissue samples are dissociated before DNA isolation Frozen Tissue  Grinding: Grinding in liquid nitrogen and homogenizing tissue or  Mincing: Mince the tissue using scalpel, but mincing disrupts the whole tissue sample Fixed Tissue  Fixed tissue is harder to extract the cell, so we need to tease (teasing) it so it will shred into smaller pieces  to make extraction of nucleic acid easier  if still fixed in a paraffin wax & dehydrated, use the process of deparaffinization  get the tissue from the wax, add xylene or xylol to remove excess wax  the tissue is still dehydrated so rehydrate it with 70% ethanol (on decreasing concentrations)  you can now place it in a buffer solution  NBF (Neutral Buffered Formalin) is the least damaging among tested fixatives  Mercury based fixatives such as Bouin’s and B5 are the worst for DNA recovery  100 base pairs can be obtained from a fixed tissue Page 2 of 5 B. DNA ISOLATION METHODS DNA Isolation chemistries a. Organic Isolation Methods b. Inorganic Isolation Methods c. Solid-Phase Isolation Proteolytic Lysis of Fixed Material Rapid Extraction Methods Isolation of Mitochondrial DNA  Combination of high salt, low pH, and an organic mixture of phenol and chloroform  Phenol & chloroform are not used today since they are carcinogenic & irritants; used for research purposes only  We use low pH = acid for renaturation of denatured DNA after alkaline lysis Isolation of small amounts of DNA from challenging samples such as fungi can be facilitated by pre-treatment with Cetyltrimethylammonium bromide (CTAB)  CTAB is a detergent that will separate polysaccharide (chitin) from the DNA; chitin (present in the cell wall of fungi) interferes with the test procedure RNAse: Enzyme that degrades RNA  RNAse can be added in first step or last step of the procedure st Purpose in the 1 step: to remove the presence of RNA Purpose in the last step: to remove residual RNA Phenol (a caustic agent) and chloroform dissolves hydrophobic contaminants such as lipids and lipoproteins Procedure: a. Lysis: from the cell suspension, add NaOH, SDS to obtain lysate (suspension of lysed cells). b. Acidification: add acetic acid & salt for renaturation  Most preferred salts: sodium acetate, sodium chloride  Alternative salts: potassium acetate, lithium chloride c. Centrifuge: centrifuge the sample to separate supernatant from the cell debris. Transfer the supernatant into another tube d. Extraction: add equal amount of phenol & chloroform. In this step, you will form 3 layer:  Aqueous phase: hydrophilic components; nucleic acid is hydrophilic (due to its sugar-phosphate backbone) so you can find it in this layer; hence, you must obtain this layer.  Ampiphilic phase: both hydrophobic & hydrophilic components  Organic phase: lipids & hydrophobic organic elements e. Precipitation: add ethanol to precipitate DNA. You can follow either of the ratio below:  1:1 = 1 part aqueous solution, 1 part isopropanol  2:1 = 2 parts aqueous solution, 1 part ethanol - Higher amount since ethanol is volatile = easily evaporates f. The DNA at the bottom still has residual salts so we need to add another 70% ethanol  centrifuge again and you will now obtain PURE DNA. g. Resuspend them in buffer, Tris-EDTA or distilled water it is now ready for molecular testing. SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBERS [MODX311] NUCLEIC ACID ISOLATION | Prof. Justin Kim C. Vergara, RMT, MPH 1.2. Inorganic Isolation Method  This method was developed due to harmful effect of chloroform & phenol  high salt solution is used here (Na acetate, NaCl, potassium acetate, lithium chloride)  Problem: salt can precipitate the protein only but not the other contaminants cannot precipitate PURE DNA does not produce high quality DNA it is not as clean as in organic isolation method  Also called as “SALTING OUT”  Uses low-pH and high salt conditions to selectively precipitate proteins to isolate DNA  DNA can then be precipitated using isopropanol  Resuspended in TE buffer or water 1.3. Solid Phase Isolation Method     More rapid and comparably effective DNA extraction Uses Silica-based products - effectively bind DNA in high-salt conditions.  Diatomaceous earth – source of silica Solid matrices in the form of columns or beads  Commonly used to isolate viral and bacterial DNA from serum, plasma, or cerebrospinal fluid.  They are also used routinely for isolation of cellular DNA in genetics and oncology  Columns vary in sizes, spin and column. There is a carrier that has a capacity of >200 nucleotides in order to bind to the silica membrane  Column: test tube-like that must fit inside the microcentrifuge tube; solid matrix (carrier) is present inside Preparation of Solid-Phase Isolation  Cell lysis and release of nucleic acids  Solid-phase separation Column in High Salt Buffer  Adsorption Solid Matrix  Washing Buffer solution  Elution Water, TE, or another low salt buffer Procedure: st a. 1 Washing - Adsorption: After Lysis & Acidification, transfer the cell lysate (DNA in aqueous solution) to a column together with the high salt buffer  the DNA in aqueous solution will bind to the solid matrix  While the DNA is binding to the matrix, the hole below the column will be the way for other contaminants & debris to be washed out of the column  only the DNA will be left inside the column Page 3 of 5  The washing solution & the eluant can be drawn through the column by gravity, vacuum or centrifugal force. nd b. 2 Washing - Elution: instead of high salt buffer, we will now use low salt buffer to dissociate the DNA from the solid matrix  This whole procedure will take few minutes; centrifugation time is around 1-2 minutes only. 2. PROTEOLYTIC LYSIS OF FIXED MATERIAL Isolation of DNA from limited amounts of starting material  Fixed Tissues  Paraffin-embedded Tissues  we can use tissue sample if not yet stained stained samples are not allowed since the stain binds to nucleic acid Procedure: a. use xylene to remove the wax b. use 70% ethanol to rehydrate c. place the sample in a Tris-EDTA buffer d. add proteinase K to lyse the proteins in the sample (that is why it is called proteolytic procedure)  330 Particulate matter  Phenol & protein have wavelength near to the absorbance of 260 nm, so you may still read them; hence, they are common contaminants.        multiple bands = sample is degraded single & whole band = DNA is still intact 3. FLUOROMETRY Fluorometry, or fluorescent spectroscopy, measures fluorescence related to DNA concentration in association with DNA-specific fluorescent dyes 3,5-diaminobenzoic acid 2HCl (DABA): binds to all deoxyribose such as dsDNA & ssDNA Hoechst 33258: binds to adenine & thymine base pairs  Combines with adenine-thymine base pairs  Specific for intact double-stranded DNA  can detect 250 ug/mL of DNA  most PREFERRED than DABA 4. MICROFLUIDICS Lab-on-a-chip technology Sample is applied to a multi-well chip The sample then moves through microchannels across a detector  When it reaches the detector, we can now check the concentration of target material The instrument software generates images in electropherogram (peak) or gel (band) configurations.  small chip, automated, connected to a computer  we can use small amount here, even 1 uL  highly sensitive = so we can also use it in small RNA  mostly used in US (not common in the Philippines) in RNA: 2 bands are acceptable     2. SPECTROPHOTOMETRY Nucleic acids absorb light at 260 nm Follows Beer-Lambert Law  Absorptivity constants 50 = dsDNA 40 = RNA The absorbance at this wavelength is thus directly proportional to the concentration of the nucleic acid in the sample. One optical density unit or absorbance unit at 260 nm is equivalent to 50 mg/L (or 50 μg/mL) of double-stranded DNA and 40 μg/mL of RNA. References:   Justin Kim C. Vergara, RMT, MPH. MODX311 Lecture. Our Lady of Fatima University, Valenzuela City. Buckingham, L. Flaws ML. (2007). Molecular Diagnostics: Fundamentals, Method & Clinical Applications Example 1: A DNA preparation diluted 1/100 yields an absorbance reading of 0.200 at 260 nm. To obtain the concentration in μg/mL, multiply: 0.200 absorbance units x 50μg/mL per absorbance unit x 100 = 1,000μg /mL Page 5 of 5 SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBERS DNA HYBRIDIZATION TECHNIQUES AND PROBES MOLECULAR BIOLOGY AND DIAGNOSTICS Instructor: Prof. Justin Kim C. Vergara, RMT, MPH Date: March 8, 2022 I. Hybridization Technologies A. Southern Blot B. Northern Blot C. Prehybridization II. Probes A. Other Nucleic Acid Probe Types B. Probe Labelling C. Factors Affecting Hybridization III. Detection System IV. Interpretation of Result V. Array-Based Hybridization A. Genomic Array Technology B. In Situ Hybridization     Details About the details above  Side notes I. HYBRIDIZATION TECHNOLOGIES HYBRIDIZATION - It is a process of establishing noncovalent and sequence specific interaction between two or more complementary strands of nucleic acid into a single hybrid. The principle behind the DNA hybridization is a single stranded DNA molecule will recognized and specifically bind to a complementary dna strand by the use of DNA Probe or also known as Gene Probe.    DNA PROBE / GENE PROBE – are synthetic single stranded DNA molecule that can recognize and specifically bind to a target DNA by complementary base pairing. 1.     And procedure performed in the clinical molecular laboratory are in at specifically target in geometry DNA and this requires visualization or detection of a particular gene or region of DNA. A. SOUTHERN BLOT First method for molecular analysis of specific DNA sites within complex background. Named after Edwin Southern  Goal of southern blot procedure is to analyze specific region of sample DNA.  For the southern blot we are using nucleic acid as the probe to target know the DNA or a this is our target material and we have also modification of the southern blot that are applied to analysis of RNA ,protein or lipids and this is important to study about gene expression regulation and protein modification.  Genomic dna is isolated and cut restriction enzymes and the fragments are separated by Gel Electrophoresis  The double stranded DNA will be cut by Endonucleases to separate or turn them into multiple pieces or fragments, and Page 1 of 7 2021 – 2022 2nd Semester MODX311 LEC the fragments that was formed will be separated using Gel electrophoresis  After the separation we need to denature the fragments.  DENATURATION – It is a separation of the double stranded DNA. Outline At the end of the session, the student must be able to learn:  LEC 4 TRANS 4     Disrupt the hydrogen bond in between the strand of DNA.  After denaturation, we need to transfer the denatured DNA to a solid support such as Nitrocellulose  Final step, the DNA fragments will be exposed to a labeled probe. Labeled probe is either complementary DNA or RNA that is highly specific to our target region of the DNA.  The labeled probe target is its complementary strand found in fragments or denatured fragments of DNA  And the signal is coming from the labeled probe is detected to indicate the presence or absence of sequence  Adding labeled probe it will have signal, meaning positive for particular sequence. But if there’s no signal meaning we don’t have the particular sequence.  It is a qualitative method wherein we detect the presence or absence of a particular sequence Can analyze any gene or gene region in prokaryotes or eukaryotes at the molecular level. Uses labeled PROBE Procedure:  Restriction Enzyme Cutting and Resolution  Preparation of Resolved DNA for Blotting (Transfer)  Blotting (Transfer)  The original method entailed hybridization is the use of a radioactive label of probe.  Before we’re using radioactive materials to detect or to give signals but now we already have Radioactive or signal.  Probe identity this procedure can analyze energy for Gene region in prokaryotes are even in eukaryotes at molecular level Restriction Enzyme Cutting and Resolution Digestion of DNA using Restriction Enzymes for three hours or more  We need to allowed complete cutting of all sites. since we are using the type 2 restriction enzymes which are highly specific to cut DNA at the specific site.  If incomplete cutting happens it will result in anomalous pattern so it will complicate on interpretation of southern blot data.  If more than one enzyme is to be used each sample must be digested separately for each enzyme and buffer.  Example i will use ECOR1 and i also use the bamH1 or the PST1. So for the digestion of DNA it should be separated we cannot mix this enzymes and then to specifically cut the dna on specific sequence we need to separately digest DNA by the use of different enzymes. the fragments that will form or result from the restriction digestion we will resolve it to gel electrophoresis. For example your ECOr1 cut your DNA into four fragments. so that we can identify or we can separate them from each other. 10-50 μg of high-quality (intact) genomic DNA is used for each restriction enzyme digestion for Southern analysis DNA is mixed with the appropriate restriction enzyme and Buffer  Diluted 1/10 into the final reaction volume The fragments resulting from the restriction digestions are resolved by Gel Electrophoresis  Can identify or separate them from each other. Observe the cut DNA after electrophoresis  Form after the enzyme digestion and all electrophoresis need to use a molecular weight standard. for the molecular with standard those are your known  standards (molecular weight ) should be run with the test samples. SATURDAY, NEMO, DORY, KEMERUT– TRANSCRIBER [MODX311] DNA HYBRIDIZATION TECHNIQUES AND PROBES | Prof. Justin Kim C. Vergara, RMT, MPH    Large fragments require longer runs at low voltage to get the best resolution  WHY Longer time?  Kasi hindi pwedeng biglain because there's a tendency that your Gel will be destroyed because of the source of this large fragments Genomic DNA cut with restriction enzymes should produce a smear Large aggregate on top indicates:  Restriction enzyme activity was incomplete 3.  Large fragments (>500 BP) are more efficiently denatured if they are depurinated before denaturation.  Before moving DNA fragments from the gel to the membrane for blotting.  The double stranded fragments are denatured as the DNA remains in place of the gel. Although those short fragments can be denatured directly as describe for the following section DEPURINATION - removing of purine          Denaturation  DNA is denatured by exposing the gel to a strong base (NaOH)  Promotes breakage of the hydrogen bonds holding the DNA strands to one another. You will observe a smear represents the billions of fragments of all sizes released after enzyme digestion. For example your chromosomal DNA it is a very long fragment and it will cut your DNA into multiple, the fragment even millions or billions of fragment that's why those fragments that will form is have available sizes and it looks have smear. If you will separate a large fragments you can use those low concentration of Gels. But if you want to separate small fragments does the time you have to use higher concentration of your gel because higher concentration will impede migration. both uncut DNA and degraded DNA it will prevent accurate analysis. Preparation of Resolved DNA for Blotting (Transfer)  Denaturation of dsDNA fragments in the gel  Transfer of separated ssDNA Page 2 of 7  The resulting single strand will break hydrogen bonds in between nitrogen bases.  Ex. You have single-stranded DNA after denaturation the single stranded is now ready know to bind with a complementary probe (are short sequence of either DNA or RNA that will specifically bind to the target sequence)  into Nitrocellulose Membrane  The act of transferring the resulting fragments to a solid support medium meaning know from gel to a solid support medium, we need to transfer this band from the gel to our solid-faced medium.  usually uses NYLON or you can also use the NITROCELLULOSE MEMBRANE because if membrane filters are the medium of choice connected and to achieve optimal hydrogen bonding between the probe and its complementary sequence later so in the results sample DNA to the doublestranded DNA fragments in the gel must be separated into single strand before you will transfer into nitrocellulose membrane. Fragments are still double-stranded during the separation of  them in the gel electrophoresis; they are still double-stranded and we need to make them single-stranded before we transfer it. before transfer we should convert your double-stranded fragments into single stranded fragments and that's what you called as DENATURATION. Uses HCl solution which loosen up the larger fragments by removing purine bases from the S-P (SUGAR- PHOSPHATE) backbone. Because of this removal of purine bases it will loosen up the larger fragments for more complete denaturation. 4. Smear in the lower region indicates that it is degraded DNA is degraded 2. Depurination  The target sequence is ATCG so the probe that will used should be TAGC (has a label help for production of signals).You will know if you have a particular sequence for not and remember those probe they are complementary to the target sequence that we are looking for.  Single strands of DNA they are now ready to hydrogen bonding with the single strand probe. Further the single-stranded DNA will bind more tightly than double-stranded DNA to the nitrocellulose membrane upon transfer Ex. You have a gel and your bonds before you transfer to our nitrocellulose membrane you should be single stranded. So after transferring it to single-stranded DNA then that’s a time we can add the probe. Then the probe will bind to the specific regions fragments wherein complementary and resulting single-strand are available for hydrogen bonding with single-stranded probe. Then the single-stranded DNA will bind to the nitrocellulose membrane after transfer.  Forming ssDNA  ssDNA > dsDNA in membrane transfer DENATURED IN GEL → TRANSFER TO NITROCELLULOSE MEMBRANE/NYLON (SINGLE STRANDED) 5.  Blotting (Transfer) Transferring or Blotting the denatured DNA to a solid substrate that will facilitate probe binding and signal detection  Nitrocellulose (MEDIUM OF CHOICE) SATURDAY, NEMO, DORY, KEMERUT– TRANSCRIBER [MODX311] DNA HYBRIDIZATION TECHNIQUES AND PROBES | Prof. Justin Kim C. Vergara, RMT, MPH  Nylon (MEDIUM OF CHOICE)  Modified Cellulose (Diethyl aminoethyl or Carboxymethyl)  Polyvinyl difluoride (PVDF)  Single-stranded DNA avidly binds to nitrocellulose membranes with a non covalent, but irreversible, connection  Nitrocellulose is the most versatile medium for molecular transfer applications A covalent attachment of nucleic acid to these membranes is achieved by exposure of the DNA on the membrane to UV-light cross-linking. Genomic fragments are permanently bound to the membrane. So we must first convert non-covalent to covalent attachment of the nucleic acid to those membrane and how you will convert non-covalent connection of the single-stranded DNA after transfer or blotting to become covalent attachment so you can expose our DNA on the membrane using UV light to convert noncovalent bond to covalent bond   Dry membrane will inhibit the binding of the sample (may be prevented by using Transfer buffer).  NOTE: Do not forget before you transfer your sample in the membrane make sure that your membrane are moisten by fluting them on the surface of the transfer buffer will be using to wet or to moist the membrane before transferred Ex. The nitrocellulose membrane should be moisten and using the transfer buffer because any area or dry spot area that does not properly hydrate it will remain white and while of the rest of the membrane darker with the buffer. Rinse the entire membrane using your transfer buffer because if the membrane does not hydrate evenly the dry spot will inhibit binding of your singlestranded DNA. So the color white will know that if it is hydrated already if the color is darker. 6.  Transfer Method Move the DNA from the gel to a membrane substrate for probing  Three types:  Capillary Transfer  Electrophoretic Transfer  Vacuum Transfer         EX. example you have your Agarose gel is placed on the top of a reservoir of buffer. So we have your buffer here and on top of your buffer you have a plastic support in the middle or we will place our gel. so we have your buffer show it should not be overflowing on the plastic support. So we should have a reservoir of buffer which can be a shallow container or have filter paper and soaked on high salt buffer. The purpose of your filter paper is to absorb your buffer going to your gel EX. 10x saline or sodium citrate as a buffer. Commercially available transfer buffer. The nitrocellulose or the nylon membrane is placed directly on the gel. Agarose gel on the top we have your nylon membrane or your nitrocellulose membrane. The nitrocellulose membrane directly place under gel and the dry absorbent filter paper. The dry absorbent filter paper or paper towels are stuck on the top of the membrane So what will happen there so the buffer on your reservoir will move by capillary action so it will be absorbed by this filter paper breach, it will be absorbed in the filter paper going to your A.Gel and the bonds in your A.Gel will be trapped in the nylon membrane. The other buffer solution will be absorbed of the paper. B. Electrophoretic Transfer   Simple and inexpensive Uses electric current to move the DNA from the gel to the membrane  utilizes electrodes attached to the membrane above shows ANODES and below the membrane is CATODES. The current carries the DNA transversely from the gel to the membrane  Tank Transfer  The electrons transfer current to the gel and membrane to electrophoresis buffer.  Preferred for large proteins.  No instruments are required  A. Capillary Transfer  Advantages:  can be less than optimal especially whenever you're using large gel and the presence of bubbles salt crystals or other particles between the membrane and the gel can cause loss of information or staining artifacts because of the presence of these bubbles or even salt crystals between the membrane and the gel so it can cause staining artifacts once the single-stranded DNA was blotted to nitrocellulose membrane it is a reversible.   Prone to staining artifacts  Use for immobilizing protein - Antibody - usually used for western blot test.    Disadvantages:  Slow  Page 3 of 7  Semidry Transfer  The electrodes contact the gel membrane sandwich directly. It require small amount of buffer just to soaked gel and membrane.  Small proteins it can take for few hours even overnight specially for large fragments. SATURDAY, NEMO, DORY, KEMERUT– TRANSCRIBER [MODX311] DNA HYBRIDIZATION TECHNIQUES AND PROBES | Prof. Justin Kim C. Vergara, RMT, MPH      C. Vacuum Transfer  Uses suction to move the DNA from the gel to the membrane in a recirculating buffer it avoids discontinuous transfer due to air trapped between the membrane and the gel         On this method transfer the dna more rapidly as compared with your a capillary transfer 2 -3 hours is done for vacuum transfer. DISADVANTAGE: Expense and maintenance of the electrophoresis and vacuum equipment.  Analysis of RNA structure and quantity indirectly reveals mutations in the regulatory or splicing signals in DNA Perform RNA Isolation and quantification Uses approximately 30 μg total RNA or 0.5 to 3.0 μg polyA RNA are applied to 0.8% to 1.5% agarose gel/polyacrylamide gels  Polyacrylamide - Used for short or smaller transcript Complete denaturation is also required for efficient transfer of the RNA from the gel to the membrane Denaturant must be removed from the gel by rinsing the gel in deionized water before transfer because it inhibits binding of RNA to nitrocellulose C. PREHYBRIDIZATION Prevent the probe from binding to nonspecific sites on the membrane surface Involves incubation of membrane in the prehybridization buffer solution (blocking agents)  Denhardt solution (Ficoll, polyvinyl pyrrolidine, bovine serum albumin) - prevents sites that possibly have non specific bindings.  Salmon sperm DNA  0.01% SDS with formamide (for RNA) Incubate for 30 mins to several hours The membrane is exposed to the prehybridization buffer at the optimal hybridization temperature for 30 minutes to several hours, depending on the specific protocol. At this stage the sample is ready for hybridization with the probe, which will allow visualization of the specific gene or region of interest. II. 7.   Blotting (Transfer) cont. After transfer, the DNA can be permanently immobilized to the membrane through: BAKING  Vacuum Oven (80°C, 30 to 60 mins)  UV Cross linking (covalent attach) Prevents washing away the bound DNA (during extended procedures)    PROBES Single-stranded fragment of nucleic acid attached to a signalproducing moiety Hybridize specifically with the target DNA or RNA Contain normal nitrogen bases that can hybridize with complementary DNA or RNA and resistant to nuclease degradation due to :  Reduced negative charged on their backbone. The purpose of your probe is to identify one or more sequences of interest with large amount of nucleic acid.  The probe that using are: 1. RNA- will directly bind to the target sequence.   Denatured DNA - as probe  Modified nucleic acid Probe length: range from tens to thousands of base pairs. Peptide nucleic acids (PNAs) and locked nucleic acids have also been used as probes. These structures contain normal nitrogen bases that can  hybridize with complementary DNA or RNA, but the bases are connected by backbones different from the natural phosphodiester backbone of DNA and RNA.  These modified backbones are resistant to nuclease degradation and, because of a reduced negative charge on their backbone, can hybridize more readily to target DNA or RNA.  Probes for Western blots are specific binding proteins or antibodies. A labeled radioactive substance or even ezymes to have signal.         B. NORTHERN BLOT Modification of the Southern blot technique Designed to investigate RNA structure and quantity  Level of gene expression  Structural Abnormalities RNA structural abnormalities resulting from aberrations in synthesis or processing, such as alternate splicing. Splicing abnormalities are responsible for a number of diseases, such as beta-thalassemias and familial isolated growth hormone deficiency. Page 4 of 7 SATURDAY, NEMO, DORY, KEMERUT– TRANSCRIBER [MODX311] DNA HYBRIDIZATION TECHNIQUES AND PROBES | Prof. Justin Kim C. Vergara, RMT, MPH A. OTHER NUCLEIC ACID PROBE TYPES  Synthesized using chemical methods  Resistant to nucleases that degrade DNA and RNA  to prevent the degradation or breaking of the phosphodiester backbone be modified it The negative charge phosphodiester of DNA and RNA counter act hydrogen bonding between the bases of the probe and the target sequence  Types:       1. Peptide nucleic acid (PNA) - doesn’t have negative charges.   Ex. dT and dU - resistant to nucleases  2. Locked nucleic acid / Bridged - refer as inaccessible RNA (2nd & 4th carbon )  3. Unlocked nucleic acid probes - locks or doesn’t 2nd and 3rd of C2 and C3 → Flexibility → distabilize double helix B. PROBE LABELLING For visualization of bound probe to the target fragments in the membrane Classic method: Probe labeling with 32P and generate detectable signal  This labeling was achieved by introduction of nucleotides containing radioactive phosphorus to the probe. New methods uses non-radioactive label Based on indirect detection of a tagged nucleotide  incorporated in or added to the probe Commonly used nonradioactive tags : 1. BIOTIN   2. DIGOXIGENIN - bind to UTP  either of which can be attached covalently to your nucleotide triphosphate UTP (uridine triphosphate) or CTP (cytidine triphosphate) 2.   RNA Probe Labeling Transcribed from cloned DNA or amplified Labeled during their synthesis with radioactive, biotinylated, or digoxigen- in-tagged nucleotides. 3.   Nucleic Acid Probe Design Necessary to determine the specificity of the results Longer probes of 500 to 5000 BP offer greater specificity with decreased background noise  Long probes are less affected by point mutations or polymorphisms within the sequence targeted by the probe or within the probe itself  Disadvantage: difficult or expensive to synthesize Shorter probes Method widely used to detect protein and RNA as well as DNA structure in place in the cell NC2  The principle of FISH is the same as ISH FISH targets specific sequences of chromosomes with fluorescent probes Detect and localize the presence or absence of specific DNA sequences on chromosomes  + Denature DNA (using NaOH), Detection/localization of specific nucleic acid sequences within structurally intact cells or morphologically preserved tissues sections by “hybridizing” a complementary probe If nucleic acids are preserved in a histological specimen, then it can be detected by using a complementary probe  standards/ control 1. C. In Situ Hybridization INTERPRETATION OF RESULT(ISH) Solution Hybridization In solution hybridization, neither the probe nor the target is immobilized. Probes and targets bind in solution, followed by resolution of the bound products. Solution hybridization has been used to measure mRNA expression, especially when there are low amounts of target RNA. Page 7 of 7 SATURDAY, NEMO, DORY, KEMERUT– TRANSCRIBER NUCLEIC ACID AMPLIFICATION LEC 5 TRANS 5 MOLECULAR BIOLOGY AND DIAGNOSTICS Instructor: Prof. Justin Kim Vergara, RMT, MPH Date: March 22, 2022 Outline ▪ At the end of the session, the student must be able to learn: I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. Nucleic Acid Amplification Target Amplification 1. Polymerase Chain Reaction Amplification Program Elements of PCR Cycle 1. Denaturation 2. Annealing 3. Extension/Elongation Amplification By PCR 1. First Four Cycles 2. Completed Amplification Cycle Components of PCR 1. Short oligonucleotide primers 2. DNA template 3. Nucleotides 4. Polymerase 5. Buffers 6. Other Components Thermal cycler/ Thermocyclers The Reaction in PCR Controls for PCR Contamination of PCR Reactions Contamination Control Mispriming Hot Start Touchdown PCR PCR Product Clean-up ▪ Side notes - NUCLEIC ACID AMPLIFICATION Nucleic Acid Amplification make many copies of target DNA ▪ ▪ ▪ ▪ 2021 – 2022 2nd Semester MODX311 LEC Kary Mullis: He realized that he can perform in vitro DNA replication by using same enzymes used in vivo DNA replication (using the parent strand to form daughter strand) In vitro replication (used 4 different Nucleotide- ATCG) + enzymes → double test target makes multiple copies = 2n 2n= know how much DNA will be generated after the cycles of PCR N= number of cycles Example: 230 = 1B copies of DNA - The first successful amplification was a short fragment of the Escherichia coli plasmid (pBR322) - The first practical application is the amplification of amino acid sequence of beta- globin and analysis for diagnosis of patients with sickle cell anemia ▪ Formerly called PCCR- Polymerase Catalyzed Chain Reaction - All use enzyme-mediated processes, to synthesize copies of target nucleic acid ▪ Parent strand, daughter strand- copy the template to make amplicons (daughter stand formed, PCR products) ▪ 4 nucleotide bases- dNTP deoxynucleotide triphosphate dATP dTTP dGTP dCPP ▪ all are used to synthesize amplicons ▪ Polymerase enzyme- for addition of dNTP to growing strand ▪ In vivo= copy entire DNA because you replicate the entire DNA; ▪ PCR= copy specific sequence only which is the target sequence Three Categories: 1. Target amplification systems- target nucleic acid based (PCR) 2. Probe amplification system– probe specific for target sequence 3. Signal amplification system TARGET AMPLIFICATION - TARGET is a short region of double-stranded DNA - Involves making many copies of a specific DNA sequence ▪ analogous to the in vivo replication of DNA within cells ▪ cell culture- DNA replication for days to months (to make indetectable levels of DNA) → in culture media for months to make billions of copies of DNA - PCR is the first and prototypical method for amplifying target nucleic acid - Discovered by Kary Mullis in 1983 (Nobel Prize Awardee) BOOK: Buckingham & Flaws – Molecular Diagnostics Target Amplification Involves making copies of a target sequence to such a level (in the millions of copies) that they can be detected in vitro (be visualized on an agar plate). PCR is the first and prototypical method for amplifying target nucleic acid. ▪ ▪ ▪ Page 1 of 7 POLYMERSE CHAIN REACTION AKA Polymerase catalyzed chain reaction Parent strand = in vivo template strand = in vitro ▪ ▪ ▪ ▪ Target sequence- use specific primer Primer- useful for initiation of amplicon synthesis Forward & Reverse primers Primers bind adjacent or near to the target sequence - Amplification products detected by 2 oligonucleotide primers - Produce 108-109 copies of targeted sequences (AMPLICONS) - Amplicons= PCR products - Sensitive to contamination à false-positive reaction ▪ Primer vs Probe o Primer: binds beside the target sequence o Probe: binds directly to the target sequence BOOK: Buckingham & Flaws – Molecular Diagnostics Polymerase Chain Reaction When the cell replicates its DNA, it requires the existing double-stranded DNA that serves as the template to give the SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER [MODX311] NUCLEIC ACID AMPLIFICATION | Prof. Justin Kim Vergara, RMT, MPH order of the nucleotide bases, the deoxyribonucleotide bases themselves: adenine, thymine, cytosine, and guanine; DNA polymerase to catalyze the addition of nucleotides to the growing strand, and a primer to which DNA polymerase adds subsequent bases. Within one to two hours PCR can produce millions of copies called amplicons of DNA. The real advantage of the PCR is the ability to amplify specific targets. AMPLIFICATION PROGRAM Components: 1. DNA template- PARENT DNA ▪ Example: To know if the patient is positive to sars-COV2, look for the N-gene in his sample. ▪ N-gene is the sequence of RNA of the virus ▪ If N-gene is present = it will serve as a template. Use the template in amplification to produce amplicons = if you produced amplicons, you are (+) to sars-COV2 ▪ If N-gene is not present = no template = no amplicons = (-) result 2. Short oligonucleotide primers- FORWARD AND REVERSE 3. Nucleotides ▪ dNTP (deoxynucleotide triphosphate): most commonly used since there is a lesser chance of degradation ▪ Alternatives: dATP, dTTP, dcTP 4. Polymerase- useful in addition of dNTPs to growing strands 5. Buffers- maintain and stabilize condition→ pH (stabilize enzyme) and Mg, NaCl for temp ▪ ▪ DENATURATION separation of double stranded DNA First step in amplification program- dsDNA will become ssDNA Single stranded (ss) Double stranded (ds) Why perform denaturation: Primers will not bind to double stranded DNA, it will only bind to single stranded DNA Removal of hydrogen bonds in between nitrogenous bases by: Exposing it to high temperature (90-96’C) Hydrogen bond will be hydrolyzed (Hydrolysis) BOOK: Buckingham & Flaws – Molecular Diagnostics Denaturation The double-stranded DNA is denatured into two single strands in order to be replicated. This is accomplished by heating the sample at 94C–96C for several seconds to several minutes, depending on the template. The initial denaturation step is lengthened for genomic or other large DNA template fragments. Subsequent denaturations can be shorter. About the Temperature ▪ ▪ ▪ ▪ Thermal cycler or thermocycler is use to facilitate the change in the temperature The temperature is near the boling point of water so ssDNA or ssRNA must be converted first into dsDNA so it can take the temperature After maging dsDNA, gawin uli single stranded (singlestranded is the requirement) Very high ang temperature, kapag hindi ginawang dsDNA ang ssRNA, maha-hydrolyzed lang or masisira ▪ ▪ ▪ ▪ 1. 2. 3. ▪ ELEMENTS OF PCR CYCLE Denaturation Annealing Extension/Elongation ▪ In every cycle, the number of DNA will be doubled. ▪ Each step require different temp and time ▪ Page 2 of 7 PRIMER ANNEALING Attachment of primer to template Primers will not bind directly to, it will bind to the nearest Most critical for the specificity of the PCR based on primer placing, size of target region can be determined Important to initiate synthesis of DNA or amplicons Primer should be complementary to the template but not exactly on the target sequence, just adjacent. SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER [MODX311] NUCLEIC ACID AMPLIFICATION | Prof. Justin Kim Vergara, RMT, MPH Consider: -The primers dictate the part of the template that will be amplified ▪ Anneal the primer to the template, Primer must hybridize to the template TEMPERATURE – set the correct temp Annealing temperatures range from 50°C to 70°C AT- temp that primer will hybridize, attachment/bind to the template - 5°C lower than MT - A starting point can be determined using the Tm (MELTING TEMPERATURE) of the primer sequences MT- temp where there is separation of primer from the template -The Tm is a way to express the amount of energy required to separate the hybridized strands of a given sequence BOOK: Buckingham & Flaws – Molecular Diagnostics Primer Extension This is essentially when DNA synthesis occurs. In this step, the polymerase synthesizes a copy of the template DNA by adding nucleotides to the hybridized primers. DNA polymerase catalyzes the formation of the phosphodiester bond between an incoming dNTP determined by hydrogen bonding to the template (A:T or G:C) and the base at the 3′ end of the primer. In this way, DNA polymerase replicates the template DNA by simultaneously extending the primers on both strands of the template. It occurs at the optimal temperature of the enzyme, 68C–72C. Tm = 4°C × number of GC pairs + 2°C × number of AT pairs AMPLIFICATION BY PCR ▪ 20-30 bases (oligonucleotides- short sequences) - based on composition and length and primer, you will be able to, check GC and AT pairs Example: 60°C – MT 55°C- AT (not optimal temp, just starting temp → to find optimal temp, do series of temp for each cycle until optimal temp is found) BOOK: Buckingham & Flaws – Molecular Diagnostics Annealing It is important that the annealing temperature be optimized with the primers and reaction conditions. Annealing temperatures will range 50C–70C and are usually established empirically. A starting point can be determined using the Tm of the primer sequences. Reaction conditions, salt concentration, mismatches, template condition, and secondary structure will all affect the real Tm of the primers in the reaction. ▪ ▪ ▪ ▪ ▪ ▪ ▪ After 1st cycle → 2 dsDNA → 2nd cycle → 4 dsDNA 1. PCR: First Four Cycles 2. ▪ ▪ ▪ PCR: Completed Amplification Cycle Usually set to 30-50 cycles but depends on protocols Used for molecular testing Presence of amplicons = POSITIVE for disease COVID 19: Detection of N gene → amplified = POSITIVE (viral RNA) *PCR is very sensitive- can detect even little amounts of N gene PRIMER EXTENSION Last step of PCR cycle DNA synthesis occurs Forward and Reverse catalyze addition of DNA primer activates polymerase enzymes, adding dNTP forming amplicons or products In this step, the polymerase synthesizes a copy of the template DNA by adding nucleotides to the hybridized primers - DNA polymerase replicates the template DNA by simultaneously extending the primers on both strands of the template - This step occurs at the optimal temperature of the enzyme 68°C to 75°C (higher than annealing temp) ▪ primer will extend and form new strand by the use of polymerase enzyme Page 3 of 7 SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER [MODX311] NUCLEIC ACID AMPLIFICATION | Prof. Justin Kim Vergara, RMT, MPH COMPONENTS OF PCR 1. SHORT OLIGONUCLEOTIDE PRIMERS ▪ analogous to the probes used for hybridization techniques ▪ PRIMERS- does not bind directly to target sequence, used for amplification technique vs PROBES- detect specific sequences, they are oligonucleotide, used for hybridization technique ▪ GC is triple bond = harder to separate; unlike AT pairs which is double bond only ▪ as much as possible, reverse and forward must have the same temperature; if not, as long as it it is within 2C difference. if forward is 60C and reverse is 55C; mas mauunang maganneal si forward; kaya dapat, same temperature para sabay-sabay ang procedure ▪ - The primers are the critical component of the reaction because they determine the specificity of the PCR Primers are designed to contain sequences complementary to sites flanking the region to be analyzed ▪ Commercially purchased primers → gene and specific target sequence you want to amplify → they will determine what primer they need to synthesize ▪ Purchased in lyophilized form – reconstitute using a buffer *A paper included with composition, purification, amount of primer, modification and exact sequence of primer - Single-stranded DNA fragments, usually 20-30 bases in length ▪ exact sequence should be included ▪ - The placement of the primers will also dictate the size of the amplified product TWO TYPES OF PRIMER Mispriming ▪ attachment of primer away from the target sequence ▪ amplicons will become larger (due to unintended sequence) 1. Forward Primer - always bind to the minus strand or anti-sense strand (TEMPLATE STRAND) - formation of amplicons is Forward from 3’ to 5’ 2. Reverse Primer - binds to plus strand or sense strand (NON-TEMPLATE) - READ IN REVERSE! 5’ to 3’ Melting Temperature - Primer Tm then serves as a starting point for setting the optimal annealing temperature MT- equal amount of dsDNA and ssDNA, avoid annealing temp set with the same MT, AT should be lower Tm = 4°C × number of GC pairs + 2°C × number of AT pairs ▪ ▪ the primer must bind BESIDE the target sequence; kapag masyadong malayo, lalaki ang amplicon size because even the unintended sequences will be copied. larger copies will be formed; the size will be larger than the expected PCR product Primer Dimer ▪ attachment of forward and reverse primer, they are complementary especially on 3’ end (very critical) ▪ Example: TAG forward, CTA reverse → bind together→ catalyzed to form amplicons that are longer/larger - Binding of primers onto each other through short (2- to 3-base) homologies at their 3ʹ ends and the copying of each primer sequence - Forward primer Tm= Reverse primer Tm Difference= 2°C Melting Temperature is affected by 2 factors: 1. Length of primer ▪ ▪ the longer the primer, melting temperature will increase the longer the primer, the harder to separate them; hence, the higher the temperature required to denature them 2. Composition of primer ▪ ▪ ▪ Check for AT pairs & CG pairs the higher the AT pairs = the lower the temperature the higher the GC pairs = the higher the temperature Page 4 of 7 SATURDAY, NEMO, DORY, KEMERUT – TRANSCRIBER [MODX311] NUCLEIC ACID AMPLIFICATION | Prof. Justin Kim Vergara, RMT, MPH BOOK: Buckingham & Flaws – Molecular Diagnostics Primer Dimer These are PCR products that are just double the size of the primers. They result from the binding of primers onto each other through short (2–3 base) homologies at their 3′ ends and the copying of each primer sequence. The polymerase will not form a phosphodiester bond if the 3′ end of the primer is not hydrogen-bonded to the template. This characteristic of primer binding has been exploited to modify the PCR procedure for mutation analysis of the template. Non-Complementary Extensions ▪ location in primer not complementary to the template ▪ binding of 3’ end is the most critical, must be highly specific to the template ▪ 5’ hanging may put non- DNA molecules (ex. Fluorescent label, biotin) Other Purposes: Restriction enzyme site Visualization of non-DNA Binding site - Designed to add or alter sequences to one or both ends of the PCR product 4. DNA POLYMERASES 1. Taq polymerase - most known - Thermostable enzyme (stable even at high temp) - Isolated from the thermophilic bacterium Thermus aquaticus (bacteria from volcanic craters, can withstand very high temp and enzyme is still active) - DNA polymerase added once at the beginning of the procedure and would maintain its activity throughout the amplification cycles. 2. Tth polymerase - From Thermus thermophilus (also from volcanic crater) - Has reverse-transcriptase activity, so it can be used in reverse transcriptase PCR (RT-PCR) ▪ convert RNA → DNA using reverse-transcriptase enzyme ▪ always convert to double stranded so temp can separate the dsDNA to ssDNA 3.*Vent polymerase (Proofreading enzyme) - Allows Taq or Tth polymerase to generate large products over 30,000 bases in length. - exonuclease, removing non-complementary and unnecessary bases, exons especially for very long DNA bases Modified Polymerase Enzymes ▪ modification for Taq polymerase, removed some amino acids to make it more stable, higher half-life 2. DNA TEMPLATE - Genomic DNA will have only one or two copies per cell equivalent of single-copy genes to serve as amplification targets - For routine clinical analysis, 100 ng to 1 ug of DNA is usually used; DNA cloning- may use less - Goo