Basic Nucleic Acid Extraction Methods PDF

Basic Nucleic acid Extraction Methods Prelims: MLS 420  Overview on extraction of nucleic acids Learning DNA isolation techniques Points RNA isolation techniques Measurement of quality and quantity of nucleic acids Ex...

Basic Nucleic acid Extraction Methods Prelims: MLS 420  Overview on extraction of nucleic acids Learning DNA isolation techniques Points RNA isolation techniques Measurement of quality and quantity of nucleic acids Extraction overview  Release of nucleic acid from the cell Nucleic acid Should be free of contaminants (e.g. proteins, carbohydrates, lipids) Extraction Preliminary step in DNA and RNA studies Multiple methods and commercial kits available NA can be isolated from almost ANY intact cellular tissue Must be optimized depending on the sample source.  Efficiency of extraction and cost effectivity  Sufficiency of the quantity extracted for Choosing downstream applications a method  Purity of final nucleic acid extract  #1 #2 #3 Isolation PURIFICATION concentration  Scientific Research DNA/RNA studies in disease molecular biology research or in biotechnology Medical Identifying etiology of infections and possible USES of NA resistance to drugs Predicting disease progression EXTRACTION Forensic Paternity studies and identification of suspects DNA Extraction 1 pretreatment & weighing Some samples require pretreatment before isolation methods (e.g. tissues in paraffin) mRNA Weighing allows final % DNA yield to be calculated later on 2 Cell lysis The outer boundaries are disrupted for release of intracellular components mRNA Can be physical or solution-based 2 Cell lysis physical 1. Grinding 2. Shearing mRNA 3. Bead beating 4. Freeze-thaw 2 Cell lysis physical mRNA 2 Cell lysis Solution-based 1. Chemical Alkali (NaOH) Detergents (SDS, CTAB) mRNA Chaotropic agents (EDTA) 2. Enzymatic Proteinase K, Lysozyme, Lipase 2 Cell lysis Solution-based mRNA 3 REMOVAL OF CONTAMINANTS LIPIDS + detergent Formation of micelles surrounding hydrophobic lipid molecules e.g. SDS, CTAB 3 REMOVAL OF CONTAMINANTS proteins + proteases/detergents Proteinase K broad-spectrum serine protease that can also degrade nucleases SDS makes proteins anionic mRNA 3 REMOVAL OF CONTAMINANTS rna + RNAse For an RNA-free DNA extract mRNA 4 Dna precipitation Separation of freed DNA from cellular debris 1. ORGANIC DNA is separated from the other components in solution for retrieval 2. INORGANIC Materials depend on extraction method used 3. Solid-phase 4 Dna precipitation ORGANIC Makes use of high salt concentrations in low pH, and with an organic mixture (1:1 phenol-choloroform) Formation of a biphasic emulsion that is centrifuged 4 Dna precipitation ORGANIC The supernatant with DNA is placed into a new tube for ethanol precipitation using ice-cold reagents and in a low-temperature environment 4 Dna precipitation ORGANIC Solvation shell – cluster of solvent molecules surrounding and attaching to solute molecules in solution 4 Dna precipitation Advantages Disadvantages Efficient Time-consuming One of the most reliable Uses hazardous reagents methods Multiple tube transfers ORGANIC Can be used for a wide range of samples 4 Dna precipitation Also called “salting out” Uses low pH, high salt conditions, to selectively precipitate proteins and leave DNA in solution inORGANI DNA is then precipitated as in the organic methods C using isopropanol 4 Dna precipitation inORGANI C 4 Dna precipitation Advantages Disadvantages Faster and easier Inadequate removal of Non-toxic chemicals Lesser steps that can salts can affect mobility of DNA inORGANI introduce contaminants during electrophoresis (Band Shifting) C 4 Dna precipitation Solid matrices are used to bind and wash DNA and common in DNA extraction kits Solid These tubes with the solid phases are called columns phase and come in various sizes depending on the amount of DNA to be isolated E.g.: Silica-based, Magnetic, Anion exchange 4 Dna precipitation Solid phase 4 Dna precipitation Advantages Disadvantages Quick, reliable Expensive  Solid method Produces high quality phase DNA Usually comes in a kit with its own prepacked reagents 5 DNA resuspension Resuspension in Tris-EDTA (TE buffer) is done so the extract can be stored and diluted for downstream uses EDTA chelates divalent ions that could potentially form salts with the phosphate groups of DNA & inhibits DNAses Kept in basic pH (Tris buffer) to reduce interactions between DNA, histones, and polycationic amines RNA Extraction  RNA is not as stable as DNA RNAses are ubiquitously present in the environment and hardy How is it different? While dissociating tissue, sample must be frozen in liquid nitrogen or immersed in buffer to inactivate intracellular RNAses  Organic extraction Cell lysis in detergent or phenol with high salt (0.2-0.5 M NaCl) or RNAse inhibitors; DNAse may also be added here  Organic extraction Option 1: Separation of proteins by acid phenol:chloroform:isoamyl alcohol  Organic extraction Option 2: Separation of phases by TRIzol reagent (acid-guanidinium-phenol)  Organic extraction RNA is precipitated by 2x ethanol or 1x isopropanol  Organic extraction RNA is resuspended in nuclease-free water to ensure that RNA will not be degraded while being stored or during use  Organic extraction Advantages Disadvantages Rapid elimination of nucleases Time-consuming Stabilization of RNA Laborious Can be used for smaller or larger Makes use of hazardous reagents samples Protocols are well-established and more routinely used  Solid-phase extraction Cell lysis is similar to organic extraction  Solid-phase extraction Lysate is added to the spin column (microfuge tubes) with high salt chaotropic buffers  Solid-phase extraction RNA is washed and eluted off of the membrane  Solid-phase extraction Advantages Disadvantages Simple and straightforward Large amounts of sample cannot be Used in kits, making them highly used in one go as it can clog the convenient membranes Usable in large-scale extractions Expensive  and automated methods Quantity and quality  To check the concentration and purity of yield Determines success of isolation why assess? Calculations used for downstream applications  Agarose gel SPECTROPHOTOMETR electrophoresis Y  spectrophotometry Makes use of Beer-Lambert’s Law to determine concentration of DNA spectrophotometry Nucleic acids have conjugated double bonds in their purine and pyrimidine rings Maximum absorbance at 260 nm  spectrophotometry 1 absorbance unit at 260 nm = 50 μg/mL of DNA 40 μg/mL of RNA DNA preparation diluted 1/100 yields an absorbance of 0.200 at 260 nm. What is Sample the concentration of your sample? problem  Sample 1000 μg/mL problem From the above sample, what is the final yield if it was resuspended in 0.5 mL of Sample Tris EDTA buffer? problem  Sample 500 μg problem What if the sample was RNA resuspended in nuclease-free water? What is now the Sample concentration and yield? problem Concentration: 800 μg/mL Sample Yield: 400 μg problem  Determined most commonly by UV spectrophotometry Purity of A260/A280 ratio often used to assess purity nucleic acid Absorbance at 260 nm for nucleic acids Absorbance at 280 nm for proteins Additional A230 nm for other contaminants and 270 nm for phenols  OPTIMAL RATIOS Purity of A260/A230 A260/A280 nucleic acid RNA 2.0-2.2 1.8-2.00 DNA 2.0-2.2 1.8-2.00* *DNA leans more towards an A260/A280 ratio of 1.8  NANODROP spectrophotometry Requires only 1-2 μL of sample instead of needing 50-75 μL Displays the entire absorbance spectrum in graphical form Can determine a wide range of sample concentrations without serial dilution (2 ng-15,000 ng/μL)  Agarose gel electrophoresis (AGE) Separation method based on size under the influence of an electric current (max: 15 V)  Agarose gel electrophoresis (AGE) A calibration curve is done after to plot the log of the observed molecular weight based on the marker against the distance traveled by each of the bands Agarose gel electrophoresis (AGE) After running, the bands of interest can be isolated out and reextracted for further studies Basic Nucleic acid Extraction Methods