Why nucleic acid isolation is necessary
DNA and RNA occur inside cells together with:
- proteins
- lipids
- carbohydrates
- salts and ions
- metabolites
- polysaccharides
- phenolic compounds
- nucleases
- cell-wall components
The objective of nucleic acid extraction is therefore not simply to break the cell. It involves four major operations:
Cell disruption → release of nucleic acids → removal of contaminants → recovery of purified nucleic acid
A good purification method should provide:
- high yield
- high purity
- intact nucleic acid
- minimal degradation
- suitability for downstream applications
The exact method differs because genomic DNA, plasmid DNA and RNA have different physical and chemical properties.
Genomic DNA Isolation
What is genomic DNA?
Genomic DNA is the complete DNA content of an organism or cell.
In eukaryotes, it is predominantly present in the nucleus, while additional genomes occur in organelles such as:
- mitochondria,
- chloroplasts in plants.
In bacteria, genomic DNA is generally present as a large circular chromosome in the nucleoid region.
The major challenge in genomic DNA isolation is obtaining high-molecular-weight DNA without mechanical shearing.
General principle of genomic DNA isolation
Cell disruption
Cells must first be broken open. Methods include:
- mechanical disruption,
- grinding,
- homogenization,
- sonication,
- enzymatic digestion,
- detergent-mediated lysis.
For plant tissues, disruption can be particularly important because the cell wall must also be broken.
Plant tissues contain:
- cellulose,
- pectin,
- polysaccharides,
- polyphenols,
- pigments,
- secondary metabolites.
These compounds can interfere with DNA purification and downstream enzymatic reactions. Therefore, plant DNA extraction often requires additional precautions.
Grinding in liquid nitrogen is commonly used because it:
- rapidly freezes the tissue
- makes the tissue brittle
- facilitates mechanical disruption
- reduces enzymatic activity during grinding
Detergents in DNA extraction
Detergents disrupt lipid membranes. Common examples include:
1. Sodium dodecyl sulfate (SDS):
- disrupts lipid bilayers
- denatures proteins
- helps release DNA
Because SDS also disrupts protein structure, it is particularly useful during cell lysis.
2. Cetyltrimethylammonium bromide (CTAB):
CTAB is especially important in plant DNA isolation. It helps remove:
- polysaccharides
- membrane components
- certain proteins
A high-salt CTAB protocol is widely used for plant tissues containing large amounts of polysaccharides.
Protein removal
Proteins must be removed because they can interfere with downstream applications. Two major strategies are:
- Proteinase K is commonly used.
- It digests proteins, including many nucleases.
- This is useful because nucleases can otherwise degrade nucleic acids.
A classical method uses phenol : chloroform.
Phenol denatures proteins, while chloroform improves phase separation and removes lipid-soluble contaminants.
After centrifugation:
- aqueous phase → nucleic acids
- interphase → denatured proteins
- organic phase → phenol/chloroform and lipid-soluble contaminants
The principle of phenol extraction is that proteins become denatured and partition away from nucleic acids, which remain predominantly in the aqueous phase.
Removal of RNA from genomic DNA
During DNA extraction, RNA is often co-purified. Therefore, RNase A may be added.
RNase A:- degrades RNA
- does not degrade DNA under appropriate conditions
- reduces RNA contamination of genomic DNA preparations
Thus:
DNA + RNA → RNase treatment → DNA-rich preparation
DNA precipitation
DNA can be precipitated using alcohol. Common alcohols include:
- Ethanol: DNA is commonly precipitated using 2-2.5 volume ethanol in the presence of salt.
- Isopropanol: Isopropanol requires a smaller volume (0.6 volume) than ethanol and efficiently precipitates nucleic acids, but it can also precipitate more contaminants.
DNA is negatively charged because of its phosphate backbone. In the presence of appropriate cations, the negative charges are neutralized sufficiently to reduce DNA solubility in alcohol.
Why salt?Cations such as Na⁺ and NH₄⁺ help neutralize the negative phosphate charges. This promotes nucleic acid precipitation.
Washing the DNA pellet
A DNA pellet is commonly washed with 70% ethanol.
Purpose:
- remove residual salts
- remove some contaminants
- retain DNA in the precipitated state
The pellet is then dried sufficiently and dissolved in:
- TE buffer
- Tris buffer
- nuclease-free water
Over-dried DNA can become difficult to redissolve.
Silica-column purification
Modern commercial DNA extraction kits frequently use silica membranes. The fundamental principle is:
High salt/chaotropic conditions → nucleic acid binds silica → washing → removal of contaminants → low-salt aqueous buffer → nucleic acid elution
Silica-based purification exploits the strong interaction between nucleic acids and silica under appropriate high-salt/chaotropic conditions.
DNA does not simply bind silica under every condition. Binding depends strongly on:
- salt concentration
- pH
- chaotropic conditions
Genomic DNA: critical precautions
Because genomic DNA is very large, several precautions are to be followed:
- Avoid vigorous vortexing: Vortexing can mechanically shear high-molecular-weight genomic DNA.
- Avoid repeated freeze–thaw cycles: These can contribute to DNA damage/shearing.
- Use wide-bore tips when necessary: This reduces mechanical stress on high-molecular-weight DNA.
- Avoid nucleases: Use clean, nuclease-free equipment and appropriate conditions.
Plasmid DNA Isolation
What is plasmid DNA?
Plasmids are generally small, covalently closed circular, double-stranded DNA molecules, capable of autonomous replication in suitable hosts. They are extensively used as:
- cloning vectors
- expression vectors
- reporter constructs
- sequencing templates
How plasmid DNA is separated from bacterial genomic DNA?
Plasmid purification takes advantage of differences in:
- DNA size
- DNA topology
- response to alkaline denaturation
- physical behavior during neutralization
The classical method is alkaline lysis.
Alkaline lysis method
Resuspension
The bacterial pellet is resuspended in a buffer containing components such as:
- Tris: Maintains pH.
- EDTA: Chelates divalent cations such as Mg²⁺ and Ca²⁺. This inhibits many DNases because these enzymes require divalent metal ions.
- RNase A: Removes RNA released during bacterial lysis.
Alkaline/SDS lysis
The lysis solution contains:- SDS: Disrupts the bacterial membrane and denatures proteins.
- NaOH: Raises the pH and causes denaturation of DNA.
Neutralization
The alkaline lysate is neutralized using an acidic potassium salt solution (usually potassium acetate), commonly potassium acetate.
Neutralization:
- lowers the pH
- allows plasmid DNA to renature
- causes SDS/protein/chromosomal DNA complexes to precipitate
Potassium ions contribute to precipitation of SDS as an insoluble potassium dodecyl sulfate complex.
This helps remove:
- SDS
- denatured proteins
- cellular debris
- much of the chromosomal DNA
The crucial plasmid–chromosome distinction
Centrifugation
After neutralization, the mixture is centrifuged.
Pellet contains mainly:- cellular debris
- denatured proteins
- precipitated SDS
- much of the chromosomal DNA
- plasmid DNA
- RNA if RNase treatment was insufficient
- salts
- some soluble contaminants
The supernatant is therefore transferred carefully without disturbing the pellet.
Purification of plasmid DNA
After alkaline lysis, plasmid DNA can be purified by:
- silica-column chromatography
- alcohol precipitation
- anion-exchange chromatography
- CsCl–ethidium bromide density-gradient centrifugation
The silica-column approach is common in modern laboratory kits while alcohol precipitation is used for manual extraction methods.
RNA Isolation
Why RNA isolation is more difficult than DNA isolation
RNA is chemically less stable than DNA. The major reason is the presence of a 2′-OH group on ribose. DNA lacks this 2′-OH group. RNA is therefore more susceptible to alkaline hydrolysis.
Furthermore, RNA is extremely vulnerable to degradation by RNases. RNases are widespread, relatively stable, present on skin and laboratory surfaces, and are, therefore, often difficult to eliminate completely. Therefore, RNA isolation requires strict RNase-free practices.
Common precautions
- RNase-free tubes
- RNase-free water
- clean gloves
- dedicated reagents
- appropriate RNase inhibitors where required
- rapid processing
- avoidance of contamination
General strategy for RNA isolation
Guanidinium-based RNA extraction
A classical approach uses strong chaotropic agents such as guanidinium thiocyanate. This reagents:
- denature proteins
- disrupt cellular structures
- strongly inhibit RNases
This is crucial because RNA must be protected from RNase-mediated degradation immediately after cell disruption.
Phenol–guanidinium extraction
A widely used principle is use of a mixture of phenol and guanidinium reagent (often marketed under trade name of Trizol reagent).
The lysate is extracted with an organic phase, followed by centrifugation. The phases separate.
Under appropriate conditions:
- RNA is recovered from the upper aqueous phase
- DNA/protein contaminants are partitioned differently
The precise phase behavior depends on pH and extraction conditions.
Acidic phenol conditions favor partitioning of DNA away from RNA, which is one reason acidic phenol-based systems are useful for RNA purification.
RNA precipitation
RNA can be precipitated using:
- isopropanol
- ethanol
A salt or appropriate ionic environment facilitates precipitation.
After centrifugation, RNA forms a pellet.
The pellet is washed, commonly with 70% ethanol, and subsequently dissolved in RNase-free water or an appropriate buffer.
Silica-column RNA purification
RNA can also be purified using silica membranes.
General principle:
Lysate + chaotropic/high-salt conditions → RNA binds silica → Wash → Elution under low-salt conditions
The same broad solid-phase principle used for DNA purification therefore also applies to many RNA extraction kits.
Removal of genomic DNA from RNA preparations
A common problem is RNA preparation is contaminated with genomic DNA. The solution is treatment with DNase. DNase degrades DNA while preserving RNA under appropriate conditions.
RNA quality assessment
RNA quality can be assessed using:
A. Spectrophotometry
Nucleic acids absorb strongly around 260 nm. Proteins absorb strongly around 280 nm. A commonly used indicator is A260/A280.
For relatively pure RNA, the ratio is generally around 2.0, while for pure DNA it is around 1.8.
A260/A230 provides information about contamination by substances such as:
- phenol
- guanidinium salts
- carbohydrates
- other organic compounds
A relatively pure nucleic-acid preparation generally has an A260/A230 ratio around 2–2.2. A contaminated sample will have lower value.
B. Agarose gel electrophoresis
For intact eukaryotic total RNA, abundant ribosomal RNA species produce prominent bands.
For example:
- 28S rRNA
- 18S rRNA
A degraded RNA sample shows:
- diffuse smearing
- loss of distinct rRNA bands
C. Bioanalyzer/TapeStation-type systems
These provide quantitative RNA integrity measures such as an RNA integrity score.
Why RNA samples are often stored at very low temperature
RNA is susceptible to:
- RNase degradation
- hydrolysis
Therefore, purified RNA is generally stored under conditions that minimize degradation, commonly at −80°C for long-term storage. Repeated freeze–thaw cycles should be minimized.
