Molecular Diagnostic Practical Notebook PDF

Practical notebook FOR MOLECULAR DIAGNOSTIC 1 Page Table of Contents 1. Introduction to Molecular Diagnostic Laboratory................................. 3 1.1 General Lab Safety in Molecular Biology........................................... 5 1.2 Preparation...

Practical notebook FOR MOLECULAR DIAGNOSTIC 1 Page Table of Contents 1. Introduction to Molecular Diagnostic Laboratory................................. 3 1.1 General Lab Safety in Molecular Biology........................................... 5 1.2 Preparation of Solutions....................................................................... 5 Calculations to prepare Molar, % and X Solutions................. 5 Percent Solution...................................................... 5 X Solution............................................................... 5 2 Preparation of Genomic DNA from Blood................................................. 7 2.1 Introduction.......................................................................................... 7 2.2 principles.............................................................................................. 7 2.3 Materials................................................................................................ 7 Chemicals.......................................................................... 7 Equipments.........................................................................8 2.4 Experimental Protocol.......................................................................... 9 Principles............................................................................ 9 Blood Collection............................................................... 10 DNA Extraction................................................................. 10 Measurement of concentration… ………………..………11 Measure the purity………………………………...………..11 Storage……………………………………………..……….12 Application…………………………………………..………12 2.5 Results................................................................................................... 13 2.6 Discussion............................................................................................. 13 2.7 Questions............................................................................................... 13 2.8 References.............................................................................................. 14 2 Page  1. INTRODUCTION TO MOLECULAR DIAGNOSTIC "Molecular Biology" is the study of biology at the molecular level and is concerned with the understanding of the genetic material. Molecular diagnostics is a dynamic and transformative area of diagnostics, leading to insights in research and treatment in many disease states that are revolutionizing health care. Molecular diagnostics detect and measure the presence of genetic material or proteins associated with a specific health condition or disease, helping to uncover the underlying mechanisms of disease and enabling clinicians to tailor care at an individual level facilitating the practice of “personalized medicine.” Molecular diagnostics are the tools that are driving the continuing discovery of biomarkers at the research level, which in turn leads to treatments designed around these biomarkers. Then molecular diagnostics play an additional critical role by ensuring that these new therapies are delivered to the right patients through more accurate diagnosis of the exact nature of their individual disease. This has led to the emerging field of companion diagnostics, in which a molecular diagnostic test is used to identify whether a specific therapy (a companion to the diagnostic) is likely to be effective for an individual patient. Molecular diagnostic tests can help a woman understand the likelihood that her breast cancer will reoccur later in life, or tell a doctor what drug is the right treatment for a late-stage melanoma patient, they can make it possible for couples 3 Page considering a family to know if they are carriers of a cystic fibrosis gene mutation and therefore at risk of having a child affected by cystic fibrosis. Molecular diagnostics can identify multiple strains of respiratory viruses in a single test, or monitor the level of HiV virus in a patient’s blood to determine how well their treatment is working in these and many other ways, molecular diagnostics are transforming health care. Molecular diagnostics today are routinely used in hospitals, reference labs, and blood banks. In the latter, molecular tests are used to screen donated blood products for infectious diseases like hepatitis and HIV. In hospitals, testing is often performed to identify pathogens in patients with infections. In fact, infectious diseases are one of the strongest growing areas within the molecular diagnostics field. There is also increasing demand for such technologies at the point of care (testing that occurs at the point of treatment or patient interaction with a health care provider, such as a doctor’s office or a clinic). The techniques used for these studies are referred to as "Techniques of Molecular Biology". The first step is to isolate DNA or RNA, for these techniques to be carried out. The DNA or RNA can either be obtained from the cells (e.g. plasmid DNA, genomic DNA, mRNA) or can be prepared [complimentary DNA (cDNA). 4 Page Molecular diagnostics examine the molecules in the cell, i.e. the DNA, RNA or proteins, and how their role in human biology and disease. 5 Page  Laboratory safety 1.1. Safety rules and regulations in Laboratory of molecular biology In molecular biology lab, a number of chemicals are used that are hazardous and can cause severe burn and long term sickness requiring immediate medical attention. The following chemicals are especially noteworthy: Phenol: cause severe burns. Ethidium bromide: a strong carcinogen In order to assure the safe handling of the chemicals, always follow the following safety precautions: 1- It is forbidden to eat, drink and smoke in the laboratory. 2- Wear gloves while handling hazardous chemicals, safety glasses or goggles, and lab coat. 3- Always use fresh tips or pipette for each solution and samples to avoid contamination of the samples and the solutions. 4- Always keep the work area clean of any unwanted tubes, beakers and dirty dishes. 5- All reagents should be marked clearly with reagent name and concentration. 6- All samples should be numbered and labeled correctly with the names and dates. 7- Make sure that after use the reagents and chemical are placed in the fridge or freezer as required 8- Do not collect samples if you are ill or from anyone who might have a serious communicable disease. 9- Always discard the waste, Supernatants and samples in appropriate waste disposal as instructed by the instructor. 6 Page 10- If any chemical is accidently spilt on the skin, immediately rinse with a lot of water and inform the instructor. 11- Wash your hands thoroughly at the end of the lab period 12- If you have questions about procedures, ask your laboratory instructor. 1.2. Preparation of Solutions Calculations to prepare molar, percentage and X solutions A Molar Solution is one in which 1 liter of solution contains the number of grams equal to its molecular weight M.Wt x M x volume needed 1000 e.g. to make up 100 ml of a 5M NaCl solution =58.456 (mw of NaCl) g/mol x 5 moles x 0.1 liter/ 1000 = 29.29 g in 100 ml of solution. Percent Solution: w/v weight (gms) in 100ml. e.g. to make a 0.7% solution of agarose in TBE buffer, weigh 0.7 of agarose and bring up volume to 100 ml with TBE buffer. Percentage v/v is = volume (ml) in 100ml. X Solution: Many enzyme buffers are prepared as concentrated solutions. e.g. 5X, 10X (five or ten times the concentrated of the working solution). These concentrated solutions are then diluted accordingly to give the final concentration of 1X of working buffer. The following is useful for calculating amounts of stock solution needed: ci x vi = cf x vf , where ci = initial concentration, or conc of stock solution; vi = initial vol, or amount of stock solution needed cf = final concentration, or conc of desired solution; vf = final vol, or volume of desired solution. 7 Page e.g. to set up a restriction digestion in 25 μl of IX buffer, add 2.5 μl of a 10X buffer, the other reaction components, and water to a final volume of 25 μl. Before Class Students will receive “Background Reading” to read for homework the night before starting the lab. They will write 2-3 questions they have about the information Students will also highlight any unfamiliar terms and write the meaning of one of the terms that they have highlighted according to the context in which the word is used in the“Background Reading.” 8 Page  2. PREPARATION OF GENOMIC DNA FROM BLOOD 2.1 Introduction There are different protocols and several commercially available kits that can be used for the extraction of DNA from whole blood. This procedure is one routinely used both in research and clinical service provision and is cheap and robust. It can also be applied to cell pellets from dispersed tissues or cell cultures (omitting the red blood lysis step). 2.2 Principles: There are four basic and one optional step in a DNA extraction:  Cells, which are to be studied, need to be collected.  Breaking the cell membranes open to expose the DNA along with the cytoplasm within (cell lysis).  Lipids from the cell membrane and the nucleus are broken down with detergents and surfactants.  Breaking proteins by adding a protease.  Breaking RNA by adding an RNase (optional).  The solution is treated with concentrated salt solution to make debris such as broken proteins, lipids and RNA to clump together.  Centrifugation of the solution, which separates the clumped cellular debris from the DNA.  DNA purification from detergents, proteins, salts and reagents used during cell lysis step.  The most commonly used procedures are: o Ethanol precipitation usually by ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. Precipitation of DNA is improved by increasing of ionic strength, usually by adding sodium acetate. 9 Page o Phenol–chloroform extraction in which phenol denatures proteins in the sample. After centrifugation of the sample, denaturated proteins stay in the organic phase while aqueous phase containing nucleic acid is mixed with the chloroform that removes phenol residues from solution. o Minicolumn purification that relies on the fact that the nucleic acids may bind (adsorption) to the solid phase (silica or other) depending on the pH and the salt concentration of the buffer. 2.3 materials: chemicals The common lysis solutions contain sodium chloride tris HCl which is a buffer to retain constant pH Cell Lysis Buffer - Non-ionic detergent (SDS), EDTA - designed to lyse outer cell membrane and nuclear membrane. Detergent: Breaks apart membranes by attaching to the lipids (fats) & proteins in the membranes EDTA (Ethylenediaminetetraacetic disodium salt) is a chelating agent of divalent cations such as Mg2+. Mg2+is a cofactor for Dnase nucleases. If the Mg2+is bound up by EDTA, nucleases are inactivated. Proteinase K: it is usual to remove most of the protein by digesting with proteolytic enzymes such as Pronase or proteinase K, which are active against a broad spectrum of native proteins, before extracting with organic solvents. Protienase K is approximately 10 fold more active on denatured protein. SDS: Proteins can be denatured by SDS or by heat. 10 P a g e Equipment's 1.Waterbath set at 65°C. 2. Centrifuge tubes (15 mL; Falcon). 3. Microfuge (1.5 mL) tubes 4. Nanodrop isolation of nucleic acids using silica-gel based membranes four basic steps in spin column silica based DNA extraction procedure: Lysis: Break down cells to access DNA in the nucleus Lysis buffer Chaotropic salts Destabilise hydrogen bonds, hydrophobic interactions, proteins (including nucleases) Disrupts the association of nucleic acids with water in preparation to bind to silica membrane Detergent (protein lysis and solubisation) Enzyme Heat Purification-bind Separate purified DNA from cell material and any PCR inhibitors Ethanol is added to enhance the binding of DNA to silica Load sample to column 11 P a g e  Centrifuge DNA binds to the membrane and remaining lysate discarded Purification - Wash Impurities and proteins should now have passed through the column and been discarded The membrane, however, is still dirty with residual proteins and salt, if using blood it may be tinted yellow or brown The column is washed with buffers to remove any residual impurities There are typically 2 washes with a centrifuge step after each Wash 1 will contain a low amount of chaotropic salt to remove any remaining proteins and coloured contaminants Wash 2 contains a high concentration of ethanol to remove the remaining salts. Salts MUST be removed for good DNA yields and purity. Wash 2 can be repeated to ensure this. All ethanol MUST be removed so that the DNA can be successfully removed/eluted from the silica membrane. Centrifuge the sample until the column is completely dry Elute When elution buffer is added, the nucleic acids become hydrated and will release from the membrane. If the column still has ethanol on it, the nucleic acids will not fully rehydrate Buffer AE in the QIAamp mini kit. DNA more stable at a slightly basic pH and will dissolve more easily in a basic buffer. Elution of DNA can be maximised by allowing the buffer to sit in the membrane for a few minutes before centrifugation. Can be repeated 12 P a g e 2.4 Experimental Protocol Blood Collection Draw blood in EDTA-containing Vacutainer tube by venipuncture. Store at room temperature or 40 C and extract within the same working day. DNA Extraction Thermo Scientific GeneJET Genomic DNA Purification Kit Mammalian Blood Genomic DNA Purification Protocol Procedure 1. Add 400 μl of Lysis Solution and 20 μl of Proteinase K Solution to 200 μl of whole blood, mix thoroughly by vortexing or pipetting to obtain a uniform suspension. 2. Incubate the sample at 56°C while vortexing occasionally or use a shaking water bath, rocking platform or thermomixer until the cells are completely lysed (10 min). 3. Add 200 μl of ethanol (96-100%) and mix by pipetting or vortexing. 4. Transfer the prepared lysate to a GeneJET Genomic DNA Purification Column inserted in a collection tube. Centrifuge the column for 1 min at 6000 x g. Discard the collection tube containing the flow-through solution. Place the GeneJET Genomic DNA Purification Column into a new 2 ml collection tube (included). 13 P a g e 5. Add 500 μl of Wash Buffer I (with ethanol added). Centrifuge for 1 min at 8000 x g.Discard the flow-through and place the purification column back into the collection tube. 6. Add 500 μl of Wash Buffer II (with ethanol added) to the GeneJET Genomic DNA Purification Column. Centrifuge for 3 min at maximum speed (≥12000 x g). Optional. If residual solution is seen in the purification column, empty the collection tube and re-spin the column for 1 min. at maximum speed. Discard the collection tube containing the flow-through solution and transfer the GeneJET Genomic DNA Purification Column to a sterile 1.5 ml microcentrifuge tube(not included). 7. Add 200 μl of Elution Buffer to the center of the GeneJET Genomic DNA Purification Column membrane to elute genomic DNA. Incubate for 2 min at room temperature and centrifuge for 1 min at 8000 x g. Note  For maximum DNA yield, repeat the elution step with additional 200 μl of Elution Buffer.  If more concentrated DNA is required or DNA is isolated from a small amount of starting material (e.g., 50 μl) the volume of the Elution Buffer added to the column can be reduced to 50-100 μl. Please be aware that smaller volumes of Elution Buffer will result in smaller final quantity of eluted DNA. 8. Discard the purification column. Use the purified DNA immediately in downstream applications or store at -20°C. 14 P a g e DNA concentration It is estimated by measuring the absorbance at 260nm, adjusting the A260 measurement for turbidity (measured by absorbance at 320nm), multiplying by the dilution factor, and using the relationship that an A260 of 1.0 = 50µg/ml pure dsDNA. DNA purity: The A /A ratio is ~1.8 for dsDNA. Ratios lower than 1.7 usually indicate 260 280 significant protein contamination. The A /A ratio of DNA and RNA should be roughly equal to its A /A 260 230 260 280 ratio (and therefore ≥ 1.8). Lower ratios may indicate contamination by organic compounds (e.g. phenol, alcohol, or carbohydrates). Turbidity can lead to erroneous readings due to light interference. Nucleic acids do not absorb light at the 320 nm wavelength. Thus, one can correct for the effects of turbidity by subtracting the A from readings at A , A and A 320 230 260 280 15 P a g e Storage The following properties of reagents and conditions are important considerations in processing and storing DNA and RNA. Heavy metals promote phosphodiester breakage. EDTA is an excellent heavy metal chelator. Free radicals are formed from chemical breakdown and radiation and they cause phosphodiester breakage. 5°C is one of the best temperatures for storing DNA. –70°C is probably excellent for long-term storage. For long-term storage of DNA, it is best to store it in high salt (>1 M) in the presence of high EDTA (>10 mM) at pH 8.5. Storage of DNA in buoyant CsCl with ethidium bromide in the dark at 5°C is excellent. Applications: It is needed for genetic analysis which used for:  1- scientific: use DNA in number of Applications , such as introduction of DNA into cells & animals or plants for diagnostic purposes (gene cloning)  2- Medicine: is the most common. To identify point sources for hospital and community-based outbreaks and to predict virulence of microorganisms  3- forensic science: needs to recover DNA for identification of individuals ,( for example rapists, petty thieves, accident , or war victims) , and paternity determination. 16 P a g e 2.5 Results. …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …… 2.6 Discussion …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… …………………………………………………………………………………………………………………………………………………… ………………………………………………………… 17 P a g e 2.7 Questions 1- What do you think is the purpose of the cell lysis solution? 2- Isolated DNA should be free from contaminating protein, heme and other cellular macromolecule, what precautions did you take to solve this situation? 3- Heme, the non-protein iron component of hemoglobin, is a primary contaminant of DNA from blood preparations, how can you detect this type of contaminant in the isolated DNA? 4- Do you know for sure that your DNA is in the sample you have stored? 5- Is it possible prove that your DNA is in the sample? 6- How is DNA extraction important to forensic scientists? 18 P a g e 2.8 References Bartlett JMS and Stirling D, Methods in Molecular Biology, second edition (226):29-33 Helms C. Salting out Procedure for Human DNA extraction. In: The Donis-Keller Lab - Lab Manual Homepage [online]. 24 April 1990. [cited 19 Nov. 2002; 11:09 EST]. Available from: http://hdklab.wustl.edu/lab_manual/dna/dna2.html. Epplen JE and Lubjuhn T. DNA profiling and DNA fingerprinting. Birhkhauser Verlag, Berlin. 1999; p.55. Genome.gov: https://www.genome.gov/ Howard Hughes Medical Institute Biointeractive: http://www.hhmi.org/biointeractive NCI-Understanding Cancer Series, Genetic Variation: http://www.cancer.gov/cancertopics/understandingcancer/geneticvariation Scripps Genomics Primer: http://www.stsiweb.org/education_training/primer/ DNAInteractive: http://www.dnai.org/index.htm 123Genomics: http://www.123genomics.com/ Go Molecular: http://www.gomolecular.com/discover/what_is_molecular_diagnostics.html AACC Trainee Council Clinical Pearls: https://www.aacc.org/publications/clinchem/ClinChemTrainCouncil/pearls/P ages/default.aspx# 19 P a g e