Nucleic Acid Extraction Techniques

Objectives:

• Understand the structural, functional, and physicochemical characteristics of DNA and RNA related to techniques used in molecular genetics labs.
• Master different DNA and RNA extraction techniques from various sample types.
• Determine parameters for assessing nucleic acid sample quality.

Introduction:

• Gene analysis using molecular genetics enables rapid and precise diagnosis of hereditary diseases.
• Mutation detection assays need high-quality, pure DNA samples.
• Nucleic acids located inside cells, isolated by membranes/cell walls.
• Obtaining nucleic acids involves pre-treatment, extraction, & purification.

Pretreatment of Biological Samples:

• Nucleic acids can be obtained from any biological sample.
• Specific pretreatment often needed due to sample diversity (blood, tissues, biopsies, etc.).
• Aim is to obtain cell suspensions (cells or aggregates in liquid) for nucleic acid extraction.
• Pretreatments vary depending on sample type.

Blood and Bone Marrow:

• DNA typically extracted from peripheral blood leukocytes suitable for genetic disease diagnoses.
• Steps:
• Obtain anticoagulated whole blood (EDTA, heparin, or sodium citrate).
• Perform extraction within 24 hrs (or refrigerate at 4°C for max. 5 days).
• Don't prolong storage for techniques requiring intact DNA (Southern blot).
• Lyse red blood cells with a 1:3 solution/buffer (blood:buffer).
• Commercial solutions use osmotic phenomena or mild detergents.
• Standard erythrocyte lysis solution uses osmosis.
• Erythrocyte lysis solution using detergent is also possible.
• Centrifuge sample, discard supernatant, & save cells.
• Washed cells proceed to nucleic acid extraction.
• Dried blood from Guthrie cards can be used for metabolic disease diagnosis in newborns.
• FTA paper lyses cells, extracts components, & fixes DNA.

Serum or Plasma:

• DNA obtained may not be high quality, requiring expertise.

Cell Cultures:

• Pretreatment involves cell collection by centrifugation (suspension cultures) or trypsinization (monolayer).

Fresh or Frozen Animal/Plant Tissues:

• Cells are trapped, forming 3D structures with interactions and extracellular matrix in tissue and cell cultures.
• Homogenization is required to purify the DNA.
• Homogenization mechanically or chemically releases cell contents.
• Processed quantity depends on tissue type & extraction method.
• 10-25mg animal tissue yields 10-40µg DNA; plant tissues need 100mg-1g for equal yield.

Mechanical Homogenization:

• Subjecting tissue to trituration for disruption, can be manual or automated.

Chemical Homogenization:

• Chemical liberation of cells, which can include proteases (collagenase, trypsin, pepsin), detergents (TX-100, Tween-20), & calcium chelators (EDTA).
• Chelators disrupt cell-to-cell and cell-to-matrix.

Methods for Cell Lysis:

• Physical methods include mechanical homogenization, manual grinding (mortar), glass beads, sonication, & temperature.
• Chemical methods use detergents (ionic detergents like sodium dodecyl sulfate) to solubilize proteins, tissues, and membranes via varying pH, salt concentration, temperature.
• Enzymatic uses enzymes like lysozyme (bacteria) and litiase (yeast); proteases break bonds in the plasma membrane and cell-cell interactions.

Formalin-Fixed Paraffin-Embedded Tissues:

• Useful in retrospective studies, FFPE tissues yield lower nucleic acid quality than fresh tissues.
• Quality loss due to DNA fragmentation during fixation and potential RNA degradation if storage is prolonged.
• Samples need deparaffinization via xylene or graded alcohols & distilled water.
• Finally, the deparaffinized and rehydrated tissue for chemical homogenization.

Other Samples:

• Other samples can include bacterial/yeast cultures, oral mucosa cells, and sputum.
• Pretreatments vary.
• Bacteria/yeast placed in suspension buffer (GTE).
• Commercial solutions used in the market obtain and preserve cells from the oral mucosa.
• Cellular sample obtained by mouth scraping using swab, swill, or saliva.

Other Acceptable Samples:

• Semen, hair, urine, or saliva are acceptable.
• Chorionic villi and amniotic fluid often used for prenatal diagnosis of a disease or genetic syndrome.

Nucleic Acid Extraction:

• After pretreatment extract DNA via manual techniques involving a series of processes.

Cell Lysis:

• Cell membrane rupture releases biological material.
• Accomplished through chemical, physical, and enzymatic methods.
• Chemical detergents solubilize proteins & plasma membrane components, release nuclear DNA.
• Physical process such as temperature, sonication, glass beads, or mechanical disruption releases nuclear DNA.
• Enzymatic involves specific proteases that sever all membranous links.
• Achieved by incubating cell suspension with lysis solution.

Lysis Solution:

• Components depend on cell type.
• Detergents disrupt lipid bilayers & denature integral proteins, release cellular & nuclear contents.
• SDS is commonly used for human and animal cell lysis.
• EDTA chelates divalent cations.
• Effective because it is a cofactor of DNAases.
• Proteases digest membrane & cytoplasm proteins (nucleases).
• Inactivating nucleases is important for obtaining high quantity and quality nucleic acids.
• Proteinase K is commonly employed to inactivate the nucleases.
• Chaotropic agents denature macromolecules.
  • Useful for inactivating nucleases such as guanidinium isothiocyanate, which halts ARNasas; also recommended for RNA purification.

Protein Digestion:

• Breaks/eliminates enzymes that coat/protect DNA with proteinase enzyme and it also frees the DNA.

Peptide Removal:

• Removes remaining peptides using organic extraction.

Nucleic Acid Recovery:

• Nucleic acids are precipitated using ethanol with/without sodium chloride.

Extraction Considerations:

• Verify lysis homogeneity; with clumps or leftover cells, prolong protocol, add lysis solution, & incubate at 37-65°C.
• Pure DNA requires ARNase A to remove RNA after lysis and before purification, incubating at 37°C for 15-45 min.

Manual DNA Extraction Techniques:

• Salting-out and phenol-chloroform-isoamyl alcohol extraction.
• Commercial kits pre-mixed reagents at optimal extraction concentrations.

Phenol-Chloroform-Isoamyl Alcohol DNA Extraction:

• Based on differential solubility in organic solvents.
• Four phases: Remove non-nucleic acid compounds that are soluble in organic solvents, precipitate the aqueous phase (with salts and carbohydrates lipids), perform more rounds, and wash the DNA.
• Nucleic acids, unlike other molecules, are not dissolved in phenol.
• Mixing phenol, chloroform, and isoamyl alcohol at a 25:24:1 ratio is common.
• Combine equal volumes of lysate and reagent, vortex vigorously. Isomers are dissolved.
• The chlorophorms will dissolve the isomers.
• Phenol denatures & dissolves lysate proteins and integrates in the organic phase. Isoamyl alcohol prevents foaming.
• Centrifugation separates into two phases: aqueous (top) with nucleic acids, salts, other components.

DNA Precipitation:

• Transfer aqueous phase to a clean tube, precipitate DNA using sodium acetate.
• Add alcohol to remove hydration layer, which causes clumping, and it can be visible to the naked eye.

DNA Washing:

• Remove the supernatant, wash DNA pellet using 70-95% ethanol, and centrifuge again.
• Removes the supernatant, wash DNA pellet using 70-95% ethanol, and centrifuge again.

DNA Resuspension:

• Eliminate all ethanol, because some will interfere with molecular biology.
• Re-suspend the DNA in a solution of distilled water.

Salting-Out DNA Extraction:

• Protein precipitation with high salt concentrations occurs.
• High salt concentration intensifies interaction between the protein and other agents which decreases solubility and precipitates it.
• The method is use for blood.
• There are five stages:
• Wash cells with saline.
• Lyse erythrocytes using lysis buffer.
• Lyse leukocytes and digest proteins.
• Perform saline extraction.
• Store the product with specific agents.

Automated Techniques:

• Cytogenetic labs with many samples use automated DNA extraction.
• Automated tools use silica-coated magnetic particles for DNA binding, obtaining high-quality DNA.
• Advantages: highly purified nucleic acids, reduced protein contamination, speed.

Nucleic Acid Purification:

• After lysis, extract nucleic acids from cell extract with proteins, salts, soluble components, & extraction reagents.
• Process isolates nucleic acids.

Magnetic Bead Purification:

• Binds nucleic acids to magnetic micro-spheres that are retained.
• Magnetic micro sphere generally consist of magnetita, the binding is due to adsorption.
• Adsorption employs silica/functional groups for nucleic acid affinity on micro spheres.

Ultrafiltration:

• Retains appropriately sized nucleic acids by using membranes with determined pore sizes in collection tubes.
• PCR products >150bp purified this way.

RNA Extraction Techniques:

• Objectives include:

• Eliminate unusable ARNr and ARNt for gene quantification analysis.

• Maintain structure integrity with non-rupture reagents.

• Obtain sufficient quality and quantity of ARNm.

• RNA is highly prone to ARNasas (present everyw), esterilization is ineffective.

• Avoid contamination, use guanidine isothiocyanate in lysis solution, use RNase-free materials/reagents during ARNm extraction and be careful of the RNasas.

• ARN techniques involving phenol and chloroform like TRIzol allow extraction of high quality ARN.

• ARN r is the most abundance ADN followed by ARNt and finally ARNm.

• Commercial kits are available for quicker separation.

• Magnetic particles are used for NA isolation purposes such as purification and extraction, they possess an overall positive charge when pH is low.

• OligodT, small magnetic beads can be used for specific purpose like ARNm purposes.

ARNm Purification Steps:

• Efficient cell lysis & nucleoprotein complex denaturation from cell extracts.
• Ribonuclease activity inactivation within the cell.
• Protein elimination (especially ribonucleases, RNasas).
• Contaminant DNA elimination.
• Elution (separation) of ARNm.

Sample Collection and Storage:

• Nucleic acids must be well-preserved after extraction.
• DNA may be dissolved in the buffer at 4°C, DNA and proteins often require water which is often frozen to further usage.
• DNA can be stored in a fridge.
• ARN samples need cold temperature for appropriate storing and often benefit from specific agents.

Nucleic Acid Quality:

• Determined by parameters:
• Integrity.
• Concentration.
• Purity.
• Determine if acids have maintain the original size if that were processed correctly.
• Electrophoresis determines if degradation has happen.
• Intact ADN samples create a unique band, degradation creates smear.

Concentration

• A important point with biology tecniques such as molecular
• Can precisely determine concentration with UV absorbtion if not contamination is present.
• ADN 1 unit equals 50 ug/ml.
• ADN 1 unit equals 33 ug/ml.
• RNA, 1 unit equals 40 ug/ml.
• Account for dilution before measuring AND has be correct at 320 nm.

Purity

• Determine the absence.
• ADN/ RNA 1.8 ADN pure RNA 2 is considered correct if there id no purity.
• Distortions due to substances such as proteins at 280 nm.
• Solved by using absorption coeficients.