DNA Isolation Methods PDF

Postgraduate Program MSc Molecular Medicine Prof. Dr. Mahmoud Zaki El-Readi Dr. Safaa Yehia Menesi Eid  2 Intended Learning Outcomes By the end of this lecture , the...

Postgraduate Program MSc Molecular Medicine Prof. Dr. Mahmoud Zaki El-Readi Dr. Safaa Yehia Menesi Eid  2 Intended Learning Outcomes By the end of this lecture , the student will be able to: Understand the sources of DNA in different organisms. Understand the cellular structure that needs to be disrupted to extract DNA. Understand the process of DNA and RNA isolation and the different methods employed. Understand the reasons for using specific chemicals in DNA extraction and what each does. 2  3 Cell Structures Animal and Plant Cell Organelles MCAS Biology (thinglink.com) 3 MSc Clinical Biochemistry-Biomedical Research & Biostatistics  Where Do we get the DNA from? 4 Cells and DNA In a plant cell, DNA is In bacteria, DNA is In an animal cell, DNA found in the nucleus, found in plasmids and is found in the nucleus mitochondria, and bacterial and mitochondria. chloroplast. chromosome. Knowing the DNA source that you are targeting is essential.  5 In humans, where can we get DNA from? All living cells contain hereditary information coded in DNA (some exceptions! What are they?). DNA in human cells is found in the nucleus and mitochondria. The DNA in the nucleus is packaged into 22 pairs of autosomal chromosomes and one pair of sex chromosomes (XX or XY – some exceptions).  6 In humans, where can we get DNA from?  7 Humans DNA Sources Blood is a connective tissue that is rich in proteins Plasma Erythrocytes (RBC) Leukocytes (WBC) No nucleus No DNA Nucleus → DNA → No DNA Birds and reptiles? Mammals RBC Birds RBC  8 Human DNA sources Saliva samples We can get DNA Can we get DNA Can we get DNA contain cells (mostly epithelial) only from living from trimmed from teeth What about bone? and can be a cells. hair? enamels? good source of DNA Easy to collect and DNA is easy to extract.  9 Human DNA sources Sources for DNA isolation are very diverse. Basically, it can be isolated from any living or dead organism. Common sources for DNA isolation include whole blood , hair, sperm , bones, nails, tissues, blood stains, saliva , buccal (cheek) swabs, epithelial cells, urine, paper cards used for sample collection, bacteria, animal tissues, or plants. Stored samples can come from archived tissue samples, frozen blood or tissue, exhumed bones or tissues, and ancient human, animal, or plant samples.  10 What are the essential components of a DNA extraction Procedure? Maximize DNA Remove or inhibit Maximize the Remove inhibitors quality of DNA recovery nucleases  11 How Much DNA Can We Recover? A Diploid Cell contains approximately 6 pg of DNA The average WBC of an adult is 5 - 10 X106 cells per ml of blood Therefore, the theoretical recovery of DNA per ul of blood is 30 - 60 ng. The PCR reactions call for on average 1 ng of DNA (single or double stranded). Many of the commercially available kits are sensitive below 1 ng of DNA (100-250 pg)  12 How to get DNA out of the cell? DNA isolation Lysis of cell membrane Separating Cell and tissue DNA from the disruption rest of the cell content.  DNA isolation 13 1- Cell and Tissue Disruption Homogenization Enzymatic Bead method pulverization or Grinding cryosection (Liquid N2)  14 DNA isolation 2. Lysis of cell membrane What is the cell membrane? What does the cell membrane contain?  15 Purposes of the Extraction Buffer Dissolve cellular Inactivation of Assist in the removal membranes DNase and Rnase of contaminants Detergents CTAB Detergents CTAB Chaotropic salts Reducing agents PVP Metal chelators Salts Detergent such as cetyl trimethyl ammonium bromide(CTAB) or SDS which disrupts the membranes Reducing agent such as B mercaptoethanol or DTT which helps in denaturing proteins which would facilitate their removal from the DNA by breaking the disulfide bonds between the cysteine residues Chelating agent such as Polyvinylpyrrolidone (PVP) and EDTA is used to remove negatively charged polysaccharides and chelates Mg2+ and Ca2+ ions required for DNase activity , a buffer which is almost always Tris at pH 8 Salt such as sodium chloride which aids in precipitation by neutralizing the negative charges on the DNA so that the molecules can come together.  16 DNA isolation 2. Lysis of cell membrane Detergent (SDS) Buffer (Tris-HCl) Lysis buffer: Salt Reducing agent (mercaptoethanol) Chelating agent (EDTA)  17 3- Separating DNA from the rest Organic extraction Enzymatic proteinase k (Phenol-chloroform isoamyl Column extraction alcohol extraction) (DNA selective binding)  18 Principles of Phenol-chloroform isoamyl alcohol DNA extraction Sample (blood, bacteria, tissue, etc) Soup- add C:I (24:1) Mix and Spin Soup- add ethanol and sodium acetate Add lysis buffer (Tris-Hcl, EDTA, Nacl, Mgcl2, SDS, pH 8.0) Mix and Spin Mix and Spin Pellt- add 70% ethanol Mix and Spin and repeat 3x Pellet- add saturated phenol (In Tris-HCl) DNA Dry Pellet Mix and Spin Soup- add P:C:I (25:24:1) Dissolve in TE buffer or DNAase protein free H2O and store  19 Role of each Chemical and Reagents Tris-HCl Maintains the pH of the solution EDTA Inhibits DNase Enzyme NaCl Prevents denaturation of DNA MgCl2 Prevents mixing of DNA with other cell organelles SDS Break cell envelope and denatures protein Saturated Phenol Non-polar solvent, repulse DNA and denatures proteins Chloroform Sharpens fuzziness between organic and aqueous layers Isoamyl alcohol Prevent foaming of phenol Sodium acetate Precipitate DNA (Na+ interacts with PO3-) Ethanol Precipitate DNA (lowers dielectric constant of water) TE buffer Dissolves DNA or can used DNase H2O Procedure: Phenol-chloroform isoamyl alcohol 20 DNA extraction from tissue Procedure: Phenol-chloroform isoamyl alcohol 21 DNA extraction from blood  22 Proteinase K methods of DNA extraction Sample (blood, bacteria, tissue, etc) Add ethanol and sodium acetate Mix and Spin Pellet- add 70% ethanol Add TE buffer Mix and Spin and repeat 3x Mix and Spin Dry Pellet Add lysis buffer (Tris-Hcl, EDTA, Nacl, SDS, pH 8.0) and Proteinase K Mix and Spin Dissolve in TE buffer or DNAase Incubate at 56 ̊C for 1 hour free H2O and store  23 Role of each Chemical and Reagents Tris-HCl Maintains the pH of the solution EDTA Inhibits DNase Enzyme NaCl Prevents denaturation of DNA SDS Break cell envelope and denatures protein Proteinase K Degrades proteins at temp 56 ̊C and pH 8 Sodium acetate Precipitate DNA (Na+ interacts with PO3-) Ethanol Precipitate DNA (lowers dielectric constant of water) TE buffer Dissolves DNA or can used DNase H2O  24 DNA Isolation using kits The PureLink® Genomic DNA Kits allow rapid and efficient purification of genomic DNA. The kit is designed to efficiently isolate genomic DNA from mammalian cells and tissues, mouse/rat tail, blood samples, buccal swabs, bacteria, yeast, FFPE (formalin-fixed paraffin-embedded) tissue, and Oragene preserved saliva. After preparing the lysates, the DNA is rapidly purified from lysates using a spin column-based centrifugation procedure. The isolated DNA is 20–50 kb in size and is suitable for PCR, restriction enzyme digestion, and Southern blotting.  25 DNA Isolation using kits System Overview The PureLink® Genomic DNA Kits are based on the selective binding of DNA to silica-based membrane in the presence of chaotropic salts. The lysate is prepared from a variety of starting materials such as tissues, cells, or blood. The cells or tissues are digested with Proteinase K at 55°C using an optimized digestion buffer formulation that aids in protein denaturation and enhances Proteinase K activity. Any residual RNA is removed by digestion with RNase A prior to binding samples to the silica membrane. The lysate is mixed with ethanol and PureLink® Genomic Binding Buffer that allows high DNA binding PureLink® Spin Column (Mini Kit) or Binding Plate (96 Kit). The DNA binds to the silica-based membrane in the column or plate and impurities are removed by thorough washing with Wash Buffers. The genomic DNA is then eluted in low salt Elution Buffer.  26 Experimental Overview  RNA extraction: Handling RNA – The key factors 27 Working quickly but carefully is key! General handling Always wear latex or vinyl gloves to prevent Rnase contamination Change gloves frequently and keep tubes closed whenever possible Disposable plasticware The use of sterile, disposable polypropylene tubes is recommended These tubes are generally RNase-free and do not require pretreatment to inactivate RNases Non-disposable plasticware Non-disposable plasticware should be treated before use to ensure that it is RNase-free Rinse thoroughly with 0.1 M NaOH, 1 mM EDTA followed by RNase-free water Alternatively, chloroform-resistant plasticware can be rinsed with chloroform to inactivate Rnases Glassware Glassware should be cleaned with a detergent, thoroughly rinsed, and oven-baked at 240°C for at least 4 hours before use Autoclaving alone will not fully inactivate many RNases An alternative is Diethyl pyrocarbonate (DEPC) treatment: Fill glassware with 0.1% DEPC (0.1% in water), incubate overnight (12 hours) at 37°C, and then autoclave or heat to 100°C for 15 min Electrophoresis tanks Electrophoresis tanks should be cleaned with detergent solution (e.g., 0.5% SDS), thoroughly rinsed with RNase-free water, and then rinsed with ethanol and allowed to dry Plastics used for some electrophoresis tanks are not resistant to ethanol  28 RNA later RNA Stabilization Reagent Immediate RNA stabilization at room temperature RNA-protect Reagent RNAlater RNA Stabilization Reagent Cells Freshly harvested tissue Bacteria Saliva  RNA extraction: Precipitation Methods 29 Precipitation Method such as TRIzol Reagent: This method relies on phase separation by centrifugation of a mix of the aqueous sample and a solution containing water saturated phenol, chloroform and a denaturing solution (guanidinium thiocyanate) resulting in an upper aqueous phase and a lower organic phase (mainly chloroform). Other denaturing chemicals such as 2-mercaptoethanol may also be used.  30 RNA extraction: Spin Column Methods The PureLink® RNA Mini Kit provides a simple, reliable, and rapid method for isolating high-quality total RNA from a wide variety of sources, including cells and tissue from animal and plant samples, blood, bacteria, yeast, and liquid samples. The purified total RNA is suitable for use in a variety of downstream applications include:. Real-time-PCR (RT-PCR) Real-time quantitative–PCR (qRT–PCR) Northern blotting Nuclease protection assays RNA amplification for microarray analysis cDNA library preparation after poly(A)+ selection  31 RNA extraction: Spin Column Methods System Overview: Samples are lysed and homogenized in the presence of guanidinium isothiocyanate, a chaotropic salt capable of protecting the RNA from endogenous RNases. After homogenization, ethanol is added to the sample. The sample is then processed through a Spin Cartridge containing a clear silica- based membrane to which the RNA binds. Any impurities are effectively removed by subsequent washing. The purified total RNA is then eluted in RNase-Free Water (or Tris Buffer, pH 7.5).  32 Experimental Overview  33 RNA extraction: Precipitation X Spin Columns Precipitation Spin columns Cheap, no kits required, scalable Higher material cost Longer handling and incubation times Fast, easy to use Several rounds of precipitation are often High purity required for decent purity Risk to lose RNA pellet, especially with Special formats for different sample types small samples. and sizes, including very small samples  34 Using Nucleases to Remove Unwanted DNA or RNA Depending on when nuclease treatment is performed, it may be necessary to repeat purification steps for protein removal (e.g. phenol/chloroform extraction).  35 Assessing the Quality and Yield of Nucleic Acids Checking for DNA Running nucleic acid sample through an agarose gel is a common method for examining the extent of DNA degradation. Good quality DNA should migrate as a high molecular weight band, with little or no evidence of smearing.  36 Nucleic Acid Analysis via UV Spectrophotometry By measuring the amount of light absorbed by your sample at specific wavelengths, it is possible to estimate the concentration of DNA and RNA. Nucleic acids have an absorption peak of 1 OD at ~260nm. [dsDNA] ≈ A260 x (50 µg/mL) [ssDNA] ≈ A260 x (33 µg/mL) [ssRNA] ≈ A260 x (40 µg/mL)  37 Nucleic Acid Analysis via UV Spectrophotometry How pure is nucleic acid sample? Nucleic acids strongly absorb at 260 nm and less strongly at 280 nm while proteins do the opposite. The A 260/A280 ratio is ~1.8 for dsDNA, and ~2.0 for ssRNA. Ratios lower than 1.7 usually indicate significant protein contamination. The A 260/A230 ratio of DNA and RNA should be roughly equal to its A260/A280 ratio (and therefore ≥ 1.8). Lower ratios may indicate contamination by organic compounds (e.g. phenol, alcohol, or carbohydrates. 37 MSc Clinical Biochemistry-Biomedical Research & Biostatistics  38 DNA storage DNA is stored in TE buffer to ensure stability and inhibit DNase. Long term storage is done using -80 freezers. -20 and -80 freezers are used for frequent use of the DNA and RNA sample. Freezing and thawing a sample may cause damage to DNA.  39 THANK U