NOTE: The study and thought of DNA extraction theory

QBIT Detection Method

This technique utilizes fluorescent dyes that emit fluorescence upon binding to specific target molecules. The instrument measures fluorescence values and converts them into concentration data. Compared to spectrophotometry, this method is more sensitive, specific, and precise in detection.

Considerations:

(1) Considering costs, methods 1.5.1 i 1.5.2 are currently more convenient during the development of extraction methods.

(2) To confirm the effectiveness of extraction, subsequent quality control measures are needed. When developing methods independently, choosing effective quality controls is essential.

Introduction to DNA Extraction and Purification Reagents Information

I’ve compiled summaries of various reagents that might be used for better understanding. This compilation is for reference purposes, not original, solely intended for educational exchange.

Proteinase K Proteinase K, isolated from white fungi, is a potent protein-digesting enzyme with high specific activity, crucial for DNA extraction. It remains active within a broad pH range (4-12.5) and at high temperatures (50-70 degrees Celsius). Chelating agents like EDTA or deionizing agents like SDS do not deactivate it.

Proteinaza K, a serine protease with broad cleavage activity, cuts peptide bonds at the carboxyl end of aliphatic and aromatic amino acids. It digests various membrane proteins and glycoproteins on cell and nuclear membranes and also digests histones bound to DNA.

Sodium Chloride (NaCl) NaCl aids in removing proteins bound to DNA. By neutralizing the negative charge on DNA, it enables molecules to aggregate and keeps proteins dissolved in the water layer, preventing their precipitation with DNA in alcohol.

Salt concentration plays a significant role when cells are exposed to hypotonic or hypertonic solutions, leading to osmosis.

EDTA EDTA chelates divalent cations like Mg2+ and Ca2+, which exist in enzymes, reducing the enzymatic activity of DNases and RNases. For instance, DNase enzymes require Mg2+ ions as cofactors for their activity. Chelating Mg2+ ions with EDTA renders DNase inactive, protecting DNA. Magnesium ions are also required for nucleic acid-protein aggregation, while calcium ions are essential for cell wall integrity and membrane stability.

Phenol, an organic solvent insoluble in water, is used with chloroform and isoamyl alcohol for DNA purification, removing protein and polysaccharide contaminants. When shaken with cellular extracts, nonpolar components of cells will fractionate in phenol, leaving polar components in water. DNA remains undissolved in phenol because it is a nonpolar solvent. On the other hand, proteins contain both polar and nonpolar groups due to different amino acids in their long chains. The folding of proteins into secondary, tertiary, and quaternary structures also depends on amino acid polarity.

Centrifugation with a mixture of phenol: and chloroform:

isoamyl alcohol in a 25:24:1 ratio yields three layers: aqueous, interphase, and organic. DNA, negatively charged and polar at neutral to alkaline pH, remains hydrophilic and is retained in the aqueous phase. Hydrophobic amino acids in proteins shield the DNA in the aqueous solution. Međutim, upon denaturation, nonpolar nucleic acids are exposed, causing proteins and certain polysaccharides to precipitate at the interphase.

The phenol-chloroform combination reduces the distribution of poly(A) and mRNA in the organic phase and minimizes the formation of insoluble RNA-protein complexes at the interphase. Phenol retains about 10–15% of the aqueous phase, resulting in similar RNA loss. Chloroform prevents water retention, thereby enhancing yield.

Only neutral phenol should be used as acidic phenol dissolves DNA, or phenol, via oxidative action, transforms into quinones, generating free radicals that degrade nucleic acids.

Chloroform

Chloroform is a nonpolar (hydrophobic) solvent in which nonpolar proteins and lipids dissolve. This encourages the distribution of lipids and cellular debris into the organic phase, protecting the separated DNA in the aqueous phase. With a relatively high density (1.47 g/cm³), chloroform ensures the separation of the two liquids, allowing the organic and aqueous phases to distinctly separate and aiding in removing the aqueous phase while minimizing cross-contamination with the organic phase. Since chloroform is inherently volatile, it doesn’t hinder subsequent processes.

Supplement: How to Use Phenol and Chloroform for DNA Extraction to Remove Proteins?

Phenol and chloroform are nonpolar molecules, while water is a polar molecule. When a protein-water solution mixes with phenol or chloroform, water molecules between protein molecules are displaced by phenol or chloroform, causing proteins to lose their hydrated state and denature. After centrifugation, denatured proteins, denser than water, separate from the aqueous phase and precipitate below it, separating from the DNA dissolved in the aqueous phase. Phenol and chloroform, being organic solvents, have their advantages and disadvantages in protein removal. Phenol has a more significant denaturing effect but partly dissolves in the aqueous phase, resulting in a loss of about 10% to 15% of the DNA in the aqueous phase. Chloroform’s denaturing effect is not as effective as phenol, but it doesn’t dissolve in water, hence doesn’t carry DNA. Thus, a combination of phenol and chloroform yields the best results during the extraction process.

Isopentanol (Isopropanol)

Chloroform exposed to air forms harmful gas, phosgene. If only chloroform is used, gas trapping leads to foaming or bubbling, making it difficult to purify DNA correctly between the two phases. At this point, combining chloroform with isopropanol or octanol prevents the emulsion of the solution during the extraction process.

RNaza A

RNaza A is an endoribonuclease that catalyzes the hydrolysis of the 3′,5′-phosphodiester bond of RNA at the 5′-ester bond in a two-step reaction. The first step is the transphosphorylation, resulting in the termination of an oligonucleotide ending in pyrimidine 2′,3′-cyclic phosphate. The second step is the hydrolysis of the cyclic phosphate to produce a terminal 3-phosphodiester. DNA lacks the crucial 2′-OH, making it resistant to digestion by RNase A.

Isopropanol/Ethanol

Ethanol precipitates DNA from the extraction solution. DNA, with a phosphodiester backbone, is essentially hydrophilic. Water molecules form a hydration shell around DNA through hydrogen bonding. Isopropanol/ethanol is used to precipitate DNA, disrupting the hydration shell. Isopropanol is a good choice for DNA precipitation. It requires a smaller volume (0.6–0.7 times the volume of the supernatant). RNA, with an additional 2’OH, tends to be more soluble in water and is selectively precipitated, leaving behind DNA. Isopropanol can dissolve nonpolar solvents like chloroform, thereby removing impurities from previous steps.

Typically, cold isopropanol is used, but researchers suggest room temperature to prevent polysaccharide precipitation. While low temperatures increase DNA yield, they might increase impurities.

Isopropanol (Prednosti: requires less volume, quick precipitation, suitable for low concentration, high volume DNA samples; Disadvantages: prone to coprecipitating salts with DNA; needs multiple washes with 70% ethanol) Ethanol (Prednosti: minimal precipitation with salts, residual ethanol in DNA precipitation easily evaporates, doesn’t affect subsequent experiments; Disadvantages: larger overall volume, needs prolonged storage at -20°C for effective precipitation, requires 70% ethanol wash)

Sodium acetate/Ammonium acetate/Potassium acetate/Sodium chloride/Lithium chloride/Potassium chloride

Salts in extraction procedures function by neutralizing the charges on the DNA sugar-phosphate backbone. Sodium acetate at pH 5.2 is commonly used alongside ethanol for nucleic acid precipitation. In solution, sodium acetate dissociates into Na+ and [CH3COO]- ions. The positively charged sodium ions neutralize the negative charges on the PO3- of the nucleic acid sugar-phosphate backbone, reducing repulsion between DNA molecules. This significantly decreases the hydrophilicity of DNA molecules, hence greatly reducing their solubility in water.

Ethanol

Wash DNA precipitates with 70% ethanol to remove excess salt, centrifuge, and discard the ethanol, leaving the DNA in the precipitate. Air-dry or vacuum-dry the precipitate. Avoid excessive drying as this may make it challenging to dissolve the DNA later.

Tris-EDTA (TE) Buffer/Sterile Water

In earlier DNA isolation methods, DNA needed to be dried and stored, then diluted as needed. Nowadays, for long-term storage, it’s prudent to store DNA in a buffer that maintains its pH and prevents degradation. TE buffer contains Tris (10 mM) and EDTA (1 mM), where Tris acts as a buffer and EDTA as a chelating agent. For DNA isolation, the pH is typically set to 7.5-8.5. The weak alkalinity of TE buffer helps prevent the chance of acid hydrolysis, further stabilizing DNA in water.

The Tris amino component in TE buffer shields DNA chains from radiation damage in solid and liquid solutions. When radiation generates free radicals, it may harm DNA chains. Thus, in fluid solutions at room temperature, Tris acts by clearing hydroxyl free radicals.

EDTA aims to chelate Mg2+ ions in solutions necessary for DNase or RNase activity, shielding DNA from DNase or RNase attacks.

Sterile water can be used for short-term DNA storage. If TE buffer is used for DNA storage, sterile water should further dilute it to reduce the EDTA concentration. This ensures magnesium ions are available for polymerase activity during PCR. Buffer components in TE might impede DNA amplification if the DNA needs to be sequenced later.