Nucleic Acid Extraction Principles

CH 2. PRINCIPLES OF NUCLEIC ACIDS EXTRACTION

I. INTRODUCTION

DNA and RNA isolation are essential for molecular biology to analyze genome structure, gene expression, and function.
Nearly all molecular biology techniques start with nucleic acid extraction.
The quantity, quality, and integrity of DNA and RNA directly affect the results of any investigation.
There is no universally perfect extraction method; each has advantages and disadvantages.
Method choice depends on the type of nucleic acid needed (genomic, plasmid, mitochondrial, chloroplast DNA, total RNA, mRNA, nuclear RNA, etc.) and the required quantity and quality.
An effective extraction method should:
- Yield nucleic acid without major contaminants.
- Be efficient and nonselective, isolating all species with equal efficiency.
- Not physically or chemically alter or break the purified molecules.
- Be relatively fast and simple.

II. DNA EXTRACTION

DNA extraction separates DNA from all other cellular components to prepare a homogenous DNA solution.
DNA constitutes about 1% of cell components and is usually enveloped by membranes (except in prokaryotes).
In eukaryotes, most DNA (~90%) is in the nucleus with an equal mass of proteins.
In viruses and bacteriophages, DNA is encapsulated by a protein coat and represents 30-50% of the total mass.
Separating DNA from small molecules (amino acids, glucose, metabolites) is straightforward due to the large difference in molecular weight.
The main cellular components that are difficult to remove during DNA purification are proteins and RNA.
DNA isolation involves four essential steps:
1. Cell breakage
2. Removal of protein and RNA
3. Concentration of DNA
4. Quantitative and qualitative analysis of the sample

1. Cell lysis

Cell breakage can be achieved chemically, mechanically, or enzymatically.
Mechanical methods (sonication, grinding, blending, high pressure) are not suitable for intact DNA preparations because they shear DNA into small fragments.
Chemical (detergents) and/or enzymatic lysis are preferred for obtaining intact DNA.
Lysis is performed in a buffer (e.g., Tris) to maintain a neutral pH (6-9).
- Low pH can cause DNA depurination and DNA transfer into the phenol phase during deproteinization.
- High pH (>12) results in DNA denaturation.
Lysis buffer should include DNase inhibitors such as EDTA and detergents.
EDTA is a Mg^{2+} chelator and a powerful DNase inhibitor because most cellular DNases require Mg^{2+} as a cofactor.
- EDTA inhibits Mg^{2+} ion-induced aggregation of nucleic acids to each other and to proteins.
- EDTA destabilizes proteins and other cell structures that require calcium ions, facilitating cell lysis.
Detergents solubilize lipids in membranes and inhibit cellular DNases.
- Many detergents cause protein denaturation.
Lipids are easily removed during phenol/chloroform extraction.
Animal cell lysis is usually performed using anionic detergents like sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate (LDS), sodium dodecyl sarcosinate (Sarcosyl), sodium 4-aminosalicylate, sodium tri-isopropylnaphthalene sulfonate, or hexadecyltrimethyl ammonium bromide (CTAB, a cationic detergent).
Non-detergent denaturing agents (e.g., guanidinium thiocyanate, B-mercaptoethanol) can also be used to denature proteins.
The ionic strength of the lysis solution is usually maintained by adding NaCl to preserve DNA structure, as hydrogen bonds are broken in low ionic strength medium.
Breaking tough plant and bacterial cell walls requires vigorous physical force that can shear large DNA molecules.
Plant cells are pre-treated with enzymes to make the cell membrane accessible to detergents.
Bacterial cells are treated with lysozyme before detergent application.
Plant cell walls can be removed by treatment with xanthogenates or enzymes that degrade cellulose.

2. Protein removal

Deproteinization depends on the physical differences between nucleic acids and proteins, such as solubility in organic solvents, partial specific volume (density), and sensitivity to digestive enzymes.

a. Extraction with organic solvents

Nucleic acids are hydrophilic and soluble in water, while proteins have hydrophobic residues, making them partially soluble in organic solvents like phenol or chloroform.
When an aqueous protein solution is mixed with phenol, phenol molecules tend to be more soluble in the hydrophobic cores of proteins, causing them to swell and denature.
Denatured proteins, with exposed hydrophobic groups surrounded by phenol micelles, are more soluble in the phenol phase than in the aqueous phase.
Most proteins partition into the phenol phase or precipitate at the interphase.
Nucleic acids lack strong hydrophobic groups and are insoluble in phenol.
Ionic detergents unfold proteins, exposing hydrophobic regions to phenol micelles, aiding partitioning into the phenol phase.
Chloroform is not miscible with water and its deproteinization action is based on its ability to cause denatured polypeptide chains to enter or be immobilized at the water-chloroform interphase, leading to protein precipitation.
Phenol extraction is often followed by chloroform extraction to remove phenol traces from the aqueous phase.
Remaining chloroform traces are easily removed due to its high volatility.
Phenol and chloroform extractions remove lipids, which are more soluble in the organic phase.

b. Other deproteinization methods

Complex formation: Some compounds or detergents form insoluble complexes with proteins or DNA/RNA in a selective manner; these complexes are removed by centrifugation.
DNA binding to silica: DNA binds to silica surfaces in high salt concentrations, while proteins do not due to their hydrophobic characteristics. DNA is eluted by lowering the salt concentration.
Isopycnic (density gradient) sedimentation: Separates based on differences in partial specific volume (proteins: 0.70-0.73 cm³/g; DNA: 0.55 cm³/g).
Enzymatic treatment: Uses enzymes for specific protein degradation.

3. Removal of RNA

RNA is usually removed from DNA preparations using enzymatic procedures (RNase treatment), although isopycnic sedimentation and mild alkaline treatment can also be used.
Two widely used ribonucleases are RNase A and RNase T1.
RNase A: Isolated from bovine pancreas, it is an endoribonuclease that cleaves RNA after Cp and Up residues, generating oligonucleotides ending with 3'P pyrimidine nucleotide. It is active under a wide range of conditions but is difficult to inactivate. At NaCl concentrations below 0.3 M, it cleaves both ss and dsRNA, but at high salt (above 0.3 M), it digests only ssRNA.
RNase T1: Isolated from Aspergillus oryzea, it is very similar to RNase A in its reaction conditions but cuts after Gp residues, generating oligonucleotides ending in a 3'P guanosine nucleotide. At high NaCl concentration, it is active only on ssRNA.

4. Concentrating the nucleic acid sample

a. Selective precipitation of nucleic acids

After protein and lipid removal, small organic molecules and mineral salts contaminate the nucleic acid sample.
Alcohol and salts are used to concentrate DNA from deproteinized solutions and remove small organic molecules and low-molecular-weight impurities.
Ethanol and isopropanol can be used for nucleic acid precipitation.
Alcohol precipitation decreases the solubility of nucleic acids in water.
Polar water molecules solvate nucleic acids in aqueous solutions, with positively charged dipoles interacting strongly with the negative charges on phosphate groups.
Ethanol, being less polar, cannot interact as strongly, making it a poor solvent for nucleic acids, causing precipitation.
Adding salt (sodium or ammonium salts) along with ethanol reduces water activity, facilitating precipitation at 70% ethanol.
Ethanol-salt precipitation is carried out at -20°C or lower to further decrease water's solubilizing activity.
Isopropanol precipitation requires a lower concentration (50%) to precipitate DNA, but small DNA fragments may precipitate poorly at room temperature.
Isopropanol is less volatile than ethanol.
Centrifugation after precipitation yields a nucleic acid pellet, which is washed with 70% ethanol to remove precipitated salts. The pellet is then dried and resuspended in an appropriate buffer.

b. Other concentrating techniques:

Dialysis is particularly used for very high molecular weight DNA (200 kb or more).
Ion-exchange chromatography.

5. Purification of plasmid DNA

Plasmid purification differs from genomic DNA preparation as it involves removing proteins and bacterial chromosomal DNA.
Most methods start with a crude bacterial lysate and standard protein removal procedures.
Methods exploit structural differences between plasmid (circular, supercoiled dsDNA) and chromosomal DNA to achieve separation.
Alkaline lysis is widely used.
Cells are lysed by SDS, and DNA is denatured by NaOH.
Neutralization results in rapid reannealing of circular plasmid DNA due to the interconnection of both ssDNA circles.
More complex bacterial chromosomal DNA cannot reanneal in this short time and forms a large DNA precipitate.
Lowering the temperature results in precipitation of protein-SDS complexes.
Both complexes (DNA and protein) are removed by centrifugation.
The remaining plasmid sample is contaminated with some proteins and RNA, which are then removed by previously described methods.

III. PRINCIPLES OF RNA EXTRACTION AND PURIFICATION

Preparation of pure RNA is essential for analyzing gene expression patterns and mechanisms via Northern blotting, nuclease protection assays, RT-PCR, DNA arrays, cDNA library preparation, and in vitro translation.

1. Principles of RNA extraction

A typical eukaryotic cell contains about 20 pg of RNA, mostly in the cytoplasm, whereas a prokaryotic cell contains 1000 times less.
About 80-85% of eukaryotic RNA is rRNA, 15-20% is stable low molecular weight species (tRNA and snRNA), and 1-3% is mRNA, which is heterogeneous in size and primary structure.
The poly-A tail at the 3' end of mRNA allows its separation and isolation from other RNA classes.
Various RNA purification techniques yield RNA samples of different quantity and quality.
- The choice of technique depends on the RNA required (quantity, quality, and nature).
- For quantitative RT-PCR, RNA intactness is not critical, but it is required for cDNA library preparation or Northern blot analysis.
- Complete removal of DNA contamination is critical for RT-PCR but not important for in vitro translation.
Physical and chemical properties of RNA and DNA are similar, so basic RNA purification procedures are similar to those of DNA.
All RNA purification methods include:
1. Cell or tissue lysis
2. Effective denaturation of nucleoprotein complexes and removal of proteins
3. Removal of contaminating DNA
4. Concentration of RNA molecules
5. Determination of purity and integrity of isolated RNA
Physical forces during lysis are allowed since RNA molecules are much smaller and more flexible than DNA molecules.
Precautions should be taken to inactivate internal and external RNases, which are omnipresent, very stable, and do not require cofactors.
Irreversible inactivation of endogenous RNases and protection against exogenous RNase contamination are necessary.
Extraction buffers include powerful RNase inhibitors (e.g., diethyl pyrocarbonate, RNasin), and all solutions and equipment are treated to remove exogenous RNases.
Frequent sources of exogenous RNase contamination are manipulator's hands and bacteria and fungi on dust particles in the air.
Strong protein denaturation agents (e.g., guanidinium hydrochloride, guanidinium isothiocyanate combined with 2-mercaptoethanol) during cell lysis quickly and irreversibly inactivate endogenous RNases and contribute to denaturing nucleoprotein complexes.

2. Examples of techniques

A widely used method is a single-step procedure that breaks cells and fragments DNA into small fragments (<10 kb).
Lysis is performed in an acidic buffer (pH 5.5) containing 4M guanidine thiocyanate and high sarcosyl concentration, then phenol-chloroform is added.
Under these conditions, RNA remains in the aqueous phase, while DNA and proteins are distributed into the organic phase.
Centrifugation yields an aqueous phase containing RNA and other small molecules.
Further purification and concentration are achieved by alcohol precipitation.
The high-salt lithium chloride method involves cell breakage in low pH, high salt buffer in the presence of RNase inhibitors.
Protein and DNA are removed by acidic phenol/chloroform extraction, and RNA is recovered by lithium chloride precipitation.
At 4M LiCl, large RNA molecules precipitate while remaining DNA and small molecular weight RNAs are kept in solution.
Centrifugation yields an RNA pellet.
DNA contamination may be avoided by performing soft lysis that destroys the plasma membrane and keeps the nuclear envelope intact.
Nuclei are removed by centrifugation, and RNA extraction is carried out on the supernatant, which corresponds to the cytoplasm.
Ultracentrifugation on a CsCl gradient separates RNA from DNA after cell lysis in a denaturing medium, yielding two independent fractions since DNA and RNA have different densities.
All the RNA are in the pellet because it is denser than DNA, forming bands near the middle of the tube.
Commercially produced RNA isolation kits provide a simple and fast method for preparing total RNA from bacteria, cultured cells, and tissues.

3. Extraction of mRNA

For applications like cDNA library construction or analysis of low-abundance mRNAs, isolation of mRNA instead of total RNA is indispensable.
Most eukaryotic mRNAs have a 3'-poly-A tail (~200 nucleotides in higher eukaryotes and ~50 nucleotides in lower eukaryotes).
Poly-A mRNA is prepared in two steps: total RNA isolation, followed by purification by affinity chromatography to select poly-A mRNA.
A total RNA sample is passed through a column filled with cellulose or latex beads coupled to oligo-dT or oligo-U.
Poly-A tracks of mRNA hybridize to oligo-dT oligonucleotides covalently linked to the beads at high salt concentration.
mRNA is retained in the column, and other RNAs are excluded.
After washing with the same high salt solution, the retained mRNA is eluted with water or low-salt buffer (which disrupt hydrogen bonds), giving a 20-50-fold enrichment of mRNA.
A second round of purification may be applied for better enrichment.
Oligo-dT can be directly or indirectly coupled to magnetic beads for easy collection of bound mRNA.
Magnetic beads are coupled to streptavidin.
Biotin, a ligand of streptavidin, is coupled to oligo-dT.
Biotin-oligo-dT and streptavidin-beads are added to a total RNA sample in salt condition to allow annealing.
mRNA anneals with oligo-dT, which is linked to biotin, which binds to streptavidin coupled to the magnetic beads.
A metallic needle takes out the mRNA population in a selective manner.
After washing, mRNA is separated from the remaining components by elution.
If elution uses a large volume of water or buffer, the sample is diluted and then concentrated by alcohol precipitation or dialysis.

IV. PRIMARY ANALYSES OF PURIFIED NUCLEIC ACIDS

After preparation, a nucleic acid sample must be controlled quantitatively and qualitatively before starting further experiments.
Concentration, potential protein contamination, and intactness must be determined before specific analyses like Northern blotting, Southern blotting, PCR, or RT-PCR.

1. Optical density

UV spectrophotometry is used to determine nucleic acid concentration and purity regarding protein contamination.
Resonance structures of pyrimidine and purine bases are responsible for UV absorption.
DNA has maximum and minimum absorbance at 260 nm and 234 nm, respectively.
These are affected by the degree of base ionization and pH.
Beer-Lambert law correlates light absorption (optical density) with concentration: A{260} = \epsilon{260} [C] l where "A" is OD (absorbance) at 260 nm, "\epsilon " is the extinction coefficient, and "l" is the path length (1 cm).
The extinction coefficient for dsDNA is usually taken as 0.02 \mu g^{-1}cm^{-1} at neutral or slightly basic pH.
An OD of 1 unit at 260 nm corresponds to a DNA concentration of 50 \mug/ml (1/0.02 = 50 \mug/ml).
The extinction coefficient of ssDNA is 0.027 \mu g^{-1}cm^{-1}, giving a ssDNA concentration of 37 \mug/ml for an OD of 1 (1/0.027 = 37 \mug/ml).
The linear relationship between absorbance at 260 nm and DNA concentration holds between 0.1 and 2 absorbance units.
Reliable measurements can be made for solutions of 0.5 to 100 \mug/ml using a standard UV spectrophotometer.
Samples with an OD > 2 should be diluted before measuring.
Measurements at a lower range (A_{260} <0.2) can be affected by light scattering on dust particles.
Absorbance at 320 nm should assess the degree of such contamination. DNA doesn't absorb at this wavelength, so any absorbance is due to light scattering.
The absorbance at 320 nm should be less than 5% of the A_{260}.
Absorbance measurements at 280 nm and the A{260}/A{280} ratio are useful for determining DNA purity re: protein contamination.
Proteins absorb maximally at 280 nm due to tyrosine, phenylalanine, and tryptophan.
The ratio A{260}/A{280} for pure dsDNA is 1.8-2. A ratio < 1.8 indicates significant protein contamination.

2. Electrophoresis

Quality (re: degradation) is determined by electrophoresis on an agarose gel.
The migration velocity of a molecule in an electric field depends on the potential difference (V, range between 2 to 15 V/cm), the net charge of the molecule, the distance between electrodes (cm), viscosity of the solution and radius of the molecule (size).
Smaller molecules penetrate the meshwork more easily than larger ones.
Near neutral pH, phosphate groups in the sugar-phosphate backbone of DNA and RNA carry negative charges.
Nucleic acid molecules migrate toward the positive electrode (anode) when placed in an electric field during gel electrophoresis.
Due to the repetitive nature of the sugar-phosphate backbone, the net charge/mass ratio is approximately constant.
Nucleic acid molecules move through the gel according to their sizes and shapes (conformation), but also according to the dimension of the gel slots and the voltage applied.
Smaller molecules move through the gel matrix more readily than larger molecules, so that molecules of different lengths migrate as distinct bands sorted according to size.
Band thickness and intensity are proportional to the amount.
Agarose gels are used for separating DNA fragments from 0.2 to 50kb in length.
Large molecules (>5kb) are run at low voltage for better resolution, small ones are run at high voltage to avoid diffusion.
DNA shape (conformation) also influences migration rate. Plasmid DNA can exist in three conformational states: closed circular supercoiled; circular relaxed and linear.
Supercoiled DNA migrates faster than linear DNA of the same size since it is more compact.
The relative mobility of the three forms is dependent on agarose concentration, the strength of the current applied, and the ionic strength of the buffer.
Nucleic acids are transparent and require staining to be seen. A common method is to incubate the gel in ethidium bromide (ETB) solution.
This planar molecule binds to DNA by intercalating between the base pairs.
When the gel is illuminated with UV light (excitation at 305 nm), ETB emits orange light.
The regions of the gel containing nucleic acids fluoresce more brightly.
Non-isotopic visualization procedures include chemiluminescence and silver staining. Silver stains are 100-fold more sensitive than ETB for the detection of nucleic acids on gels.
Silver staining depends on the reduction of silver ions to form metallic silver images.

Interpretation of gel electrophoresis results:

Lane P1 refers to a perfect plasmid sample with supercoiled linear and relaxed forms. Supercoiled form is more compact and therefore moves faster than the linear and relaxed forms, which move slowly although they have the same nucleotide content as the supercoiled form
P2 corresponds to a degraded sample of plasmid (or any DNA).
P3 is contaminated with genomic DNA which is unable to migrate fast in the gel because of its large size. Therefore, it stays near the origin of migration.
Determination of plasmid size is based on the position of the linear form in the gel.
Lane G refers to a sample of genomic DNA whose size is large. It consists of a smear rather than a sharp band since there are molecules of different sizes (random breaks occur along the DNA molecules during extraction).
Lane R1 is a perfect sample of total RNA. Only two bands are observed corresponding to the 18s (2.3 kb) and 28s (4.2 kb) ribosomal RNA (the most abundant in the cell). The 28s RNA band stains with approximately twice the intensity of 18s RNA.
R2: Occasionally, a diffuse band is visible near the front of migration and corresponds to the 5s (0.12 kb) and 5.8s (0.156 kb) RNA. The other RNA classes are present and sorted according to their sizes but not visible because of their low amount (below sensitivity of ETB staining).
Lane R3 corresponds to an RNA sample which is contaminated by genomic DNA (near the origin of migration).
Lane R4 corresponds to a degraded RNA sample.
Lane R5 corresponds to mRNA sample which forms a smear of slight intensity.