Week 3 Nucleic Acid Extraction PDF

NUCLEIC ACID 3 EXTRACTION ISOLATION OF DNA PREPARATION OF SAMPLE BACTERIA AND FUNGI VIRUSES BLOOD & BONE MARROW ASPIRATES PLASMA TISSUE SAMPLES DNA ISOLATION TECHNIQUES ORGANIC ISOLATION METHODS INORGANIC ISOLATION METHODS SOLID-PHASE ISOLATION PROTEOLYTIC LYSIS OF FIXED MATERIAL RAPID EXTRACTION ME...

NUCLEIC ACID 3 EXTRACTION ISOLATION OF DNA PREPARATION OF SAMPLE BACTERIA AND FUNGI VIRUSES BLOOD & BONE MARROW ASPIRATES PLASMA TISSUE SAMPLES DNA ISOLATION TECHNIQUES ORGANIC ISOLATION METHODS INORGANIC ISOLATION METHODS SOLID-PHASE ISOLATION PROTEOLYTIC LYSIS OF FIXED MATERIAL RAPID EXTRACTION METHODS ISOLATION OF MITOCHONDRIAL DNA ISOLATION OF RNA TOTAL RNA SPECIMEN COLLECTION RNA ISOLATION CHEMISTRIES ORGANIC ISOLATION SOLID-PHASE ISOLATION PROTEOLYTIC LYSIS OF FIXED MATERIAL ISOLATION OF MRNA NUCLEIC ACID EXTRACTION The purpose of nucleic acid extraction is to release the nucleic acid from the cell for use in subsequent procedures. the target nucleic acid should be free of contamination PROTEINS, CARBOHYDRATES, LIPIDS DNA: free of RNA RNA: free of DNA CELL LYSIS PURIFIFICATION CONCENTRATION ISOLATION OF DNA Miescher first isolated DNA from human cells in 1869 by precipitation Meselson and Stahl - density-gradient centrifugation 1. Preparing the Sample Nucleic acid is routinely isolated from human, fungal, bacterial, and viral sources in the clinical laboratory Some test systems include sample-collection vessels and reagents that begin the nucleic isolation in transport to the laboratory. Use of an improper collection tube or method will compromise the test results. Test validation should include the sample-collection methods. Specimens Adequate for Analysis Without DNA Amplification specimen Expected Yield Blood (1 mL, 3.5-10 x 10° WBCs/mL) Buffy Coat (1mL whole blood) 50-200 ug Bone marrow (1 mL) 100-500 ug Cultured cells (10' cells 30-70 ug Solid tissue (1mg) 1-10 ug Lavage fluids (10mL) 2-250 ug Mitochondria (10-mg tissue, 10' cells) 1-10 ug Plasmid DNA, bacterial culture (100-mL overnight culture) 350 ug - 1 mg 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 specimen Expected Yield 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 (1 mg) 1-10 ug Bone, teeth (500 mg) 30-50 ug Hair follicles 0.1-0.2 ug Fixed tissue (5-10x10 micron sections;10 mm2) 6-50 u Feces (animal cells, 1mg) 2-100 pg BACTERIA & FUNGI early recombinant DNA experiments were performed with gram-negative bacteria Bacteria and fungi have tough cell walls that must be broken to allow the release of nucleic acid 1. Mechanical Disruption not usually use since it may also destroy the nucleic acid does not give assurance that nucleic acid will be preserved well METHODS: Grinding: useD if the sample is solid Glass Beads: vigorous shaking 2. Enzymatic digestion less likely to damage chromosomal DNA and thus are preferred Proteinase K: digests proteins Lysozyme: digests other cell organelles 2. Alkaline Extraction most common method DETERGENT: 1% Sodium Dodecyl Sulfate STRONG BASE: 0.2 M NaOH: to lyse the cell EDTA: chelates DNAse Glucose: help to destroy the cell wall 4. Boiling Extraction method used if the sample is treated with lysozyme Diluted Sucrose Triton X-100 detergent Tris Buffer EDTA viruses Some procedures use cell-free specimens, such as plasma, for viral detection. used for intracellular parasite if its 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 blood and bone marrow Nucleic acid in human blood or bone marrow comes mostly from white blood cells (WBCs) methods: density-gradient centrifugation differential lysis A. 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 2nd layer in between buffy coat & RBC, used to prevent contamination from RBC b. Differential osmotic fragility of RBCs and WBCs Incubation in hypotonic buffer or water will result in the lysis of the RBCs PLASMA EXTRACTION OF DnA FROM PLASMA CAN BE USED FOR LIQUID BIOPSY REquires procedures to concentrate the target nucleic acid before isolation. Liquid biopsies may preclude surgical biopsies and allow serial biopsy testing A. 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 b. Liquid Biopsy sources of circulating nucleic acids Isolation of cell-free nuclei acid requires procedures to concentrate the target nucleic acid before isolation can also extract nucleic acid from viral infections and cancer cells cancer cells have the ability to separate from the tumor, and can be found in body fluids. sources: plasma, csf, ascites, pleural fluid used to detect presence of extracellular viruses tissue samples fresh or frozen tissue samples are dissociated before dna isolation A. frozen tissue grinding: grinding in liquid nitrogen and homogenizing tissue or mincing: mince the tissue using scalpel, but mincing disrupts the whole tissue sample B. fixed tissue harder to extract the cell; teasing is required to shred to smaller pieces and make extraction of nucleic acid easier. 1. deparaffinization: xylene or xylol 2. rehydration: 70% alcohol 3. buffer solution: a. neutral buffer formalin: least damaging b. mercury based (bouin’s): worst dna isolation methods dna isolation chemistries 1. organic isolation method Combination of high salt, low ph, and organic mixture of phenol and chloroform to dissolve lipids and lipoproteins phenol and chloroform: carcinogenic and irritants low pH: for renaturation of dna after alkaline lysis cetylmethylammonium bromide (ctab) detergent that will separate polysaccharide (chitin) from the dna of fungi chitin: interferes with the test procedure rnase enzyme enzyme that degrades rna added to first step or last step of the procedure 1st step: to remove the presence of RNA 2ns step: to remove residual rna procedure: 1. lysis: from the cell suspension, add naoh, sds to obtain lysate (suspension of lysed cells) 2. acidification: add acetic acid and salt for renaturation most preferred salts: sodium acetate, sodium chloride alternative salts: potassium acetate, lithium chloride 3. centrifuge: centrifuge the sample to separate supernatant from the cell debris. transfer the supernatant into another tube 4. extraction add equal amount of phenol and chloroform. in this step, there is a formation of 3 layers: aqueous phase: hydrophilic components (nucleic acids) ampiphilic phase: both hydrophobic and hydrophilic components organic phase: lipids and hydrophobic organic elements 5. precipitation addition of ethanol to precipitate dna 1:1 = 1 part aqueous solution, 1 part isopropanol 2:1 = 2 parts aquesous solution, 1 part ethanol higher amt. since ethanol easily evaporates because of its volatility 6. ethanol addition 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. 7. resuspension Resuspend them in buffer, Tris-EDTA or distilled water it is now ready for molecular testing. 2. inorganic isolation method This method was developed due to harmful effect of chloroform & phenol high salt solution is used here (Na acetate, NaCI, 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 3. Solid Phase Isolation Method More rapid and comparably effective DNA extraction Uses Silica-based products (Diatomaceous earth) effectively bind DNA in high-salt conditions. 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. 1st 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 The washing solution & the eluant can be drawn through the column by gravity, vacuum or centrifugal force. 2nd 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. 4. 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: use xylene to remove the wax use 70% ethanol to rehydrate place the sample in a Tris-EDTA buffer 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. 3. fluorometry Fluorometry, or fluorescent spectroscopy, measures fluorescence related to DNA concentration in association with DNA-specific fluorescent dyes 3,5-diaminobenzoic acid 2HCI (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) quiz 2 questions: 1. what is the initial step in nucleic acid extraction? 2. what is the best specimen for dna extraction? 3. what is the least preferred method for extracting bacterial dna 4. development of this method preclude the need for surgical biopsy 5. why plasma is the preferred sample for viruses