BM UD6. Extracción y Purificació de Acidos Neucleicos

• Nucleic acids can be obtained from virtually any biological material like blood, biopsies, or tissues
• A specific pre-treatment is needed to obtain cellular suspensions that contain free cells for extraction and purification

Blood as a Sample

• Blood is commonly used to study nucleic acids by purifying them through leukocytes
• Blood components can inhibit basic molecular biology techniques, such as hemoglobin interfering with PCR
• Pre-treatments aim to eliminate hemoglobin and plasma through erythrocyte lysis

Blood Sample Collection

• Ideal to use total anticoagulated blood with EDTA
• Heparin- or citrate-anticoagulated blood is usable
• Nucleic acid extraction should occur within 24 hours of sample collection
• Refrigerate samples for up to five days at 4°C

Erythrocyte Lysis Protocol

• Used to break erythrocytes, often done by diluting samples, often in a 1:3 ratio
• Common lysis reagents include:
  • Standard Solution: 150mM Ammonium Chloride, 1mM EDTA, 10mM Potassium Bicarbonate
  • Detergent Solution: 10mM Tris-HCl, 1% Triton X-100, 11% Sucrose
• Centrifuge lysed sample at 2000 rpm for 10 min, eliminate remaining plasma, hemoglobin, and collect cell pellet

Obtaining Mononuclear Cells

• Blood allows for nucleic acid isolation from retroviruses infecting lymphocytes, like HIV or HTLV
• Peripheral blood mononuclear cells (lymphocytes and monocytes) can be isolated before nucleic acid extraction through density gradient centrifugation using Ficoll
• Ficoll is a synthetic polysaccharide with sodium diatrizoate
• Centrifuge for 20-30 minutes at 1500 rpm
  • Erythrocytes are contained in the lower layer.
  • Granulocytes sediment and found immediately superior to the erythrocytes.
  • Mononuclear cells found between the separation medium and plasma interface.
• Aspirate the plasma and transfer to a clean tube for monolear cells
• Wash with saline solution to remove any plasma and/or platelets by centrifuging for 10 minutes at 1500 rpm

Cell Cultures

• In vitro cell obtaining methods are by controlled seeding in appropriate media for genomic and gene expression studies

Preparation for Nucleic Acid Extraction and Types

• These samples recquire recollection
  • Suspension cultures: Collect cells in tube and eliminate growth media using centrifugation, then wash suspended cells with physiological serum or buffer before nucleic acid extraction
  • Monolayer cultures and trypsinization: Cells in a monolayer adhered to flask wall undergo extraction directly or are detached mechanically using a scraper or enzymatic to collect for wash and nucleic acid extraction

Fresh Animal or Plant Tissues

• Obtaining nucleic acids from fresh tissue requires mechanical or chemical homogenization to release cells.
• For animal tissues 10-25 mg material is required; for plant tissues, 100mg-1g
  • Mechanical homogenization: cut the tissue, then triturate with abrasive agents like glass or ceramic spheres undergoing violent oscillation
    • Tissue breakage can release enzymes, therefore perform procedure in cold or utilize enzymatic inhibitors
    • A mortar may be used with liquid nitrogen to quickly congeal and prevent degradation, and pulverized to obtain fine powder
  • Chemical homogenization: Tissue is subjected to proteases and detergents, commonly used for small tissue and tissue cut from cryostat

Formalin-fixed Paraffin-embedded Tissues (FFPE)

• Tissues are ideal for retrospective studies.
• Recovered nucleic acids may be lower than those from tissues
• Formalin fixation requires more than 24 hours to degrade DNA
• Pre-treatment involves deparaffinization with xylol and rehydration with decreasing ethanol concentrations, followed by chemical homogenization like with fresh tissue

Uncommon Samples

• Bacterial and Yeast cultures: Suspend microorganisms in buffer, centrifuge liquid cultures to remove supernatant, resuspend isolated colonies from agar plates in buffer
• Mucosa Cells: Commercial kits are available for collecting and preserving mucosa samples by oral scraping, rinsing, or direct collection of saliva
• Sputum: Used for detecting respiratory infection-causing pathogenic microorganisms; pre-treat by liquefying with N-acetyl-cysteine and decontaminate with sodium hydroxide

Nucleic Acid Extraction

• Cellular suspensions (prokaryotic or eukaryotic) require nucleic acid extraction to obtain naked nucleic acids from cellular structures using lysis
• Inactivates cellular nuclease so that it prevents nucleic acid degradation using DNAase and RNAase
• Involves incubating suspension in lysis solution

Lysis Solution Components

• Composition varies by cell type (variations for tough lysis via cells with walls), and include detergents, EDTA, proteases, etc
  • Detergents: The main component to breaking and destabilizing membranes. SDS is often used in combination with EDTA
  • EDTA: Chelates Mg cations to inhibit ADNasas
  • Proteases: Like proteinase K, digest membrane and cytoplasmic proteins for dissolving membranes; also removes nucleases
  • Chaotropic Agents: Disable DNA and RNA nucleases. A typical agent is guanidinium isothiocyanate - denatures macromolecule-water interactions

Degrading the Wall

• Breaking down walls of bacteria, yeast, and vegetal cells
  • Enzymatic: Specific enzymes destroy bacterial (lysosyme) or yeast and vegetal cell walls.
  • Mechanical: Bacteria sonification, cycles of freeze-thaw
  • Use CTAB to eliminate polysaccharides

Lysing Verification

• Result should be solution homogeneity
• Grumes and cellular masses should be eliminated immediately
• To get rid of RNA, it can be eliminated via reaction media ARNase incubating at 37 °C for 15-45 minutes in blood samples

Purification

• Separates out the RNA and DNA from other components with physicochemical properties
  • Purification is done with solvents organiques, with sales, ionic chromatography, adsorption chromatography with filtration

Eliminating Solvents

• Compounds integrate lysed material in organic solvents
• Consists of four phases
  • Equal volumes are mixed with lysed material mixture of organic solvents (phenol, choloroform, isoamilic alcohol volume 25:24:1)
  • Mixture undergo centrifuge
    • Top Phase: nucleic acids and sales
    • Bottom Phase: proteases and lipids

DNA Preciptation

• Aqueous phase is removed to clean test tube and DNA is precipitated by adding cold sodium acetate
• The reaction acts as a desiccant while DNA loses covalently joined molecules, rendering a loss of macromolecule
• DNA is at the bottom
• Ethanol is used to wash remaining contaminants that coprecipitated
• DNA will be able to resuspend in whatever solution is used

Saline Preciptation

• Proteins are precipitated by saline solutions with high concentration
  • Elevation of ionic levels will diminish hydrophobic solubility
• Procedure consists of the follow
  • Sample is combined with saline
  • Mixture is centrifuged. Nucleic acids, salt, and water will be at top, along with lipid and proteins at bottom will form an impure solution
• Isopropanol will then be used to precipitate out DNA due to lack of solubility caused by diminished acids
• Washing with 70% ethanol will leave DNA ready to be resuspended water desalinized to low ionic buffer

Chromatic Exchange

• Uses ionized reversible electrostatic bond exchanging ionic charge to joined stationary phase functional groups
  • Anionic: when negative ions bind a solution
  • Cationic: when postiive ions bind a solution
• Depends on mobile and stationary phase of ionic charge
• Nucleic acids are purified through cellulous and silica matrices in polypropylene columns
• Nucleic aicds are covalently line to anioinic exchange, as they are negative by a posphate group
  • MAE - Methylaminoenthanol
  • DEAEC - Diethylaminoetyl - Column chromatography consists of - Charged chromatographic with lysed material - Nucleic acids uncharged (negative) bind functional groups - Protease, polysaccharides, contaminants are the columns elution - Eluting column must contain saline concentrate to elliminate any lingering charge - DNA is freed with saline mix and pH - Nucleic acids precipitate with isopropyl as needed, eluting TE

Absorbing Chromatogrpahy

• Based on absorbtion capacity of nucleic acids to glass and silica presence of a saline solution
• To bond silica and nucleci acids, they are coered with hydrate from phosphates, silicats
• Caps hydrate interfere as a buffer
• When saline is added, it removes it from the structures creating a hyrdrophobic environment for silicic-aicd to conjoin

Adsorption Steps

• Load material into a buffer column containing caotropic, and sentrifuge to retain nucleci, and collects into collection tube as it eliminates
• Columns cleaned with etanol 70% removing contaminants
• Rehydrate column with tris-EDTA to separate and extrude via centrifugation

Magnetic Sphere Purification

• Based on union of nucleic aids and microspheres of magnetite in a matriix
  • Absorbtion: Add siilica coated micropheres to lyze material
    • Put into magnet and separate lysing material from protein
    • 70% ethanol is used to wash contamination away
    • Micropheres and nucleic acids are sepearted and incubated in water
    • Affinity: Polymer-coated spheres for the desired acids - In other words add Olio-t micropheres to a sample to bind RNA for separation through magnets in the previous process. Eluir in Tris-Edta

Ultrafiltration

• Retention by size of nucleic acids and membranes of pore sizes
• Commonly used in PCR amplificiation of 150 PB base - PCR Is run with cellulose for filtration, and washed away with 70% ethenol

Plasmid Purificatiojn

• Vectors use cloned sequences for recombination - Small extra chromosomal molecules, often for resistance to antibiotics
  • Chromosomas are separate and made of RNA
    • Density gradient through centrifuge
    • Saline lysis will destroy desaturation of cellular molecuels - Desaturation- renaturization (PH SDS) - After being centrifuged, the sample is set to eliminate solubles from DNA plasmids
      • Material undergoes ARNase, chromatic adorption with isopropyl, and magnet extraction

RNA Purification

• The material is very sensative to arnas - Sterlized by heat but regained after - Conditions - Gloves - Commercial cleaning - Clear Material - Use lysis/Iso guanidium

Characteriztion for NA

      - Consists of  Integrity, Purity Concentration
		  - Integredity
			      - If a material has been fragmented
				         - Valued with lectropherisis gel
          - Gel: Sugar phosphate to distribute load
				          -DNA genome single in upper part 
					          - Will be a smear if disintegrated
									          - Plasmids: Band
						 RNA: smear or mix

Purity

   - Pure if they do not have containments as measure with spectrophotometry
		   - A:260 measures for bases

• .Azs0/Azso: measures levels with a range from 1.7 -2 - Under 1.2 there is a large contaiment. 1.8 RNA

• Az230 Measure for contamination Range: 1.50-2.2

  • UV - no need to subtract if there is solution - 260, 280 will be turbid if the range is there

Conc

• Measured by molecular parts in solute. Factors may matter in bio molecule use - .260 nm measure is best, then test against the beir lamert -Directly proporonal/ optical aspect - Flurescent double strand can be measured

Functionalioty

• DNA Amount in tech
• Checked without use quality control by PCR

Storage of Acids

       - Two goals: 1: no disintegrating. 2 can tract it
    - Tube
		             - Closed / Nuclease Free
    - Frozen samples
	           - DNA: 4 degrees is fine for 5 days, or long term freezer to prevent long
           -RNA 80
To

Traction

        - Direct coding with ink and temp
	             -Label
		                 - 2-d bidi code
               - Recorded to the tubing