Nucleic Acid Extraction Study Guide

Nucleic Acid Extraction Methods

Purpose of Nucleic Acid Extraction

• The purpose of extraction is to release nucleic acid from the cell for use in subsequent procedures.

• Ideal conditions for target nucleic acid:

  • Free from contamination with protein, carbohydrate, lipids, or other nucleic acids.

  • Specifically, DNA should be free of RNA, and RNA free of DNA.

Overview of Nucleic Acid Extraction (NAE) Steps

• NAE can be divided into four modifiable steps based on sample and downstream applications:

  1. Cell Lysis

  2. Removal of Membrane Lipids, Proteins, and Other Nucleic Acids

  3. Nucleic Acid Purification/Binding from Bulk

  4. Nucleic Acid Concentration

Detailed Steps in Nucleic Acid Extraction

1. Cell Lysis

• Cell lysis involves breaking the cell and nuclear membranes.

• Conditions for lysis should avoid damaging nucleic acid.

• After lysis, the target material is purified and its concentration and purity can be assessed.

2. Methods of Cell Lysis

Mechanical Methods

• High pressure homogenizer

• Bead mill

Non-mechanical Methods

• Physical Methods: Heating, osmotic shock, cavitation.

• Chemical Methods: Alkaline or detergents such as sodium dodecyl sulfate (SDS).

• Biological Methods: Enzymatic lysis (e.g., lysozyme).

3. Alkaline Lysis Procedure

• Modern procedures leverage differences in solubility of chromosomal DNA, plasmids, and proteins in alkaline buffers.

• Significant facts about alkaline lysis:

  • Large (50 kbp) chromosomal DNA and proteins aggregate and precipitate at low pH after neutralization, while plasmids remain in solution due to proper renaturation.

  • These procedures were pivotal in early recombinant DNA technology for extracting 1–50 kb plasmid DNA from bacteria.

4. Sample Preparation for Nucleic Acid Isolation

• 1. Nucleated Cells in Suspension:

  • White blood cells (WBCs) can be isolated via:

  • Differential density gradient centrifugation (using Ficoll).

  • Differential lysis in hypotonic buffer leading to WBC pelleting.

  • Ficoll is a branched sucrose polymer facilitating the separation of mononuclear WBCs from plasma and other cells based on density.

• 2. Tissue Samples:

  • Fresh/frozen tissues must be dissociated through methods like grinding in liquid nitrogen or homogenization.

• 3. Microorganisms:

  • Tough cell walls of bacteria and fungi necessitate breaking via:

  • Enzymes (e.g., lysozyme, zymolyase).

  • Mechanical methods (grinding or vigorous mixing with glass beads).

  • Treatment options include:

  • Detergent (1% SDS) and strong base (0.2 M NaOH) for bacterial cell walls.

  • Boiling in a sucrose and detergent mixture or direct NaOH boiling for rapid extraction.

5. Organic Isolation Methods

• Removal of contaminants (proteins, lipids, carbohydrates) accomplished with phenol and chloroform in high salt and low pH environments.

  • This method collects cell debris and strips away proteins, ensuring DNA purity.

  • To minimize RNA contamination, adding RNase at this stage is recommended.

  • After extraction, DNA is collected from the upper aqueous phase and precipitated using ethanol or isopropanol.

• DNA Precipitation Process:

  • Collect DNA precipitate via centrifugation, rinse with 70% ethanol to remove excess salt, and dissolve DNA pellet in rehydration buffer (e.g., 10 mM Tris, 1 mM EDTA).

6. Inorganic Isolation Methods (Salting Out)

• Utilizes low pH and high salt environments to precipitate proteins while allowing DNA to remain in solution.

• Similar to organic extraction, DNA can be precipitated subsequently using isopropanol.

7. Solid-Phase Isolation Method

• Uses solid matrices (e.g., silica-based products) for effective DNA extraction.

• Spin columns are common in clinical laboratories for isolating viral and bacterial DNA from bodily fluids.

  • Sample preparation follows similar steps as organic and inorganic methods, adjusted with specific buffers.

RNA Extraction

• Total RNA types in cells:

  • Ribosomal RNA (rRNA): 80-90% of total RNA.

  • Messenger RNA (mRNA): 2.5-5%; can be faintly visualized under agarose gel electrophoresis.

  • Transfer RNA (tRNA) and small nuclear RNAs also present.

RNA Isolation Process

• Cell lysis for RNA involves:

  • Detergents or phenol with high salt (0.2-0.5 M NaCl) or RNase inhibitors.

  • Guanidine thiocyanate may also be utilized as a strong denaturant.

• RNA Extraction:

  • Use acid phenol:chloroform:isoamyl alcohol solution for effective RNA extraction.

  • The upper aqueous phase containing RNA is isolated and precipitated using ethanol or isopropanol.

Measurement of Nucleic Acid Quality and Quantity

• Accurate quality and quantity measurement methods include:

  1. Electrophoresis:

  • Visual analysis of samples using agarose gel, with fluorescence dyes (ethidium bromide, SybrGreen I/II) for DNA or RNA.

  1. Spectrophotometry:

  • Nucleic acids absorb light at 260 nm wavelength, correlating with concentration.

  • Use of Beer-Lambert Law, with specific absorptivity constants:

    • 50 for DNA

    • 40 for RNA

  • Concentrations correlate with absorbance readings:

    • $(1 ext{ OD unit} = 50 ext{ µg/mL of DNA})$

    • $(1 ext{ OD unit} = 40 ext{ µg/mL of RNA})$

  • Avoid phenol contamination at 260 nm measurements.

Example Calculations

• Example 1 (DNA): Diluted 1:100 with absorbance reading of 0.200:

  • $0.200 ext{ absorbance} imes 50 ext{ µg/mL/OD unit} imes 100 = 1000 ext{ µg/mL}$

  • If resuspended in 0.5 mL: $1000 ext{ µg/mL} imes 0.5 ext{ mL} = 500 ext{ µg}$

• Example 2 (RNA): Diluted 1:10 with absorbance reading of 0.500:

  • $0.500 ext{ absorbance} imes 40 ext{ µg/mL/OD unit} imes 10 = 200 ext{ µg/mL}$

  • If resuspended in 0.2 mL: $200 ext{ µg/mL} imes 0.2 ext{ mL} = 40 ext{ µg}$

Quality Assessment of Nucleic Acids

• Protein Absorbance at 280 nm Comparison:

  • The 260 nm/280 nm ratio:

    • Should be 1.6-2.00 times.

    • Ratios below 1.6 indicate protein contamination.

  • Recommended to perform re-precipitation to enhance sample purity.

  • Alkaline buffers (pH 7.5) suggested for accurate assessments.

  • RNA typically shows a higher ratio (2.0-2.3).

  • Unexpectedly high ratios for DNA can indicate RNA contamination.