Isolation of DNA

The right sample affects DNA yield and purity. In exams link sample quality with the first step.

The governing concept is preservation of nucleic-acid integrity before and during extraction. A biological sample can contain nucleases, microorganisms, and other factors that progressively break DNA into smaller fragments. Keeping the sample cold, using suitable storage conditions, and applying approved protective reagents can slow nuclease activity and microbial growth. Consequently, more of the original DNA remains intact and available for purification, amplification, or analysis. Option B is correct because preservation aims to reduce degradation. Staining is a later visualization step, burning destroys DNA, and conversion of protein into starch is unrelated to nucleic-acid isolation.

In fresh tissue, DNA has usually undergone less degradation by nucleases and other breakdown processes. Therefore, it can yield longer, less fragmented, more intact DNA. Ethanol is used to precipitate DNA; tissue does not precipitate ethanol. Exam tip: Fresh or properly preserved samples generally give better-quality DNA.

Poor storage can increase DNA degradation and contamination. Therefore sample handling is very important.

During homogenization, tissue or cells are ground or disrupted to form a fine, uniform suspension. This exposes more cells to the lysis buffer and makes release of DNA easier. Ethanol is used later for DNA precipitation, not for homogenization. Exam tip: remember homogenization as tissue disruption that prepares the sample for lysis.

Liquid nitrogen quickly freezes tissue. This makes grinding easier and can reduce nuclease activity.

The governing concept is cell lysis, the first major step in making intracellular DNA accessible. A lysis buffer contains chemicals that disrupt the plasma membrane and, for many samples, the nuclear membrane as well. Depending on its composition, it can also denature proteins, dissolve membrane lipids, and inhibit nucleases that might otherwise degrade DNA. Thus option C is correct: opening the cells releases DNA into the surrounding solution so it can subsequently be separated and purified. DNA staining is a later detection procedure. Turning a sample into a plant or converting protein into glucose is unrelated to the chemical purpose of a lysis buffer.

Detergent dissolves or disrupts the lipid components of cell and nuclear membranes, causing membrane breakdown. This helps release DNA from the cell. A ribosome is involved in protein synthesis, not the main target of membrane disruption. Exam tip: Link detergent in DNA isolation with cell lysis, the membrane-breaking step.

In eukaryotic cells nuclear DNA remains in the nucleus. When the nuclear membrane breaks DNA enters the solution.

Lysozyme breaks bonds between sugar units in peptidoglycan, the major component of the bacterial cell wall. This weakens the wall and makes bacterial lysis easier during DNA isolation. RNase degrades RNA, not the cell wall. Exam tip: associate lysozyme with bacterial cell-wall breakdown in DNA isolation.

The main component of a plant cell wall is cellulose. Cellulase breaks the bonds in cellulose, so it is useful for breaking the plant cell wall during DNA isolation. Protease breaks proteins, not cellulose. Exam tip: choose the enzyme according to the main cell-wall component—cellulase for cellulose.

Chitin can be a major structural component of the fungal cell wall. Chitinase breaks down chitin and helps disrupt the cell wall, making it easier to release DNA. RNase is used to remove RNA, whereas detergents help dissolve membrane lipids. Exam tip: Match the enzyme to the cell-wall component—chitinase is used for fungi.

Proteinase K helps reduce protein contamination and nuclease activity. This improves DNA purity and protection.

RNase is an enzyme that degrades RNA. Therefore, treating a DNA sample with RNase reduces RNA contamination and improves DNA purity. Protein contamination is generally removed using a protease, not RNase. Exam tip: the name “RNase” indicates that the enzyme targets RNA.

DNase is an enzyme that cuts phosphodiester bonds in DNA. It can break DNA into smaller fragments, making the sample unsuitable for isolation or analysis. Ethanol is used for DNA precipitation; DNase does not convert DNA into ethanol. Exam tip: Use nuclease-free reagents and equipment during DNA isolation to prevent DNA degradation.

Many DNases and other nucleases require divalent metal ions such as \(Mg^{2+}\) for their activity. EDTA chelates these ions, making them unavailable to the enzyme; this reduces nuclease activity and protects the DNA. Cutting proteins is the role of proteases, not EDTA. Exam tip: Remember EDTA as a metal-ion chelator used to protect DNA during isolation.

Tris is a buffer whose main role is to maintain a stable pH in the DNA solution. A stable pH helps protect DNA from damage under excessively acidic or alkaline conditions. Restriction enzymes, not Tris, cut DNA into smaller fragments. Exam tip: When Tris appears in a DNA isolation or storage buffer, associate it primarily with pH buffering.

In TE buffer Tris maintains pH and EDTA gives protection from nucleases. Link it with storage buffer.

The phosphate groups in DNA carry negative charges. Cations from the salt neutralise or shield these charges, reducing repulsion between DNA molecules and allowing them to aggregate and precipitate in the presence of alcohol. Alcohol lowers DNA solubility, but it does not neutralise the phosphate charges. Exam tip: remember that salt mainly neutralises the negative charges on the DNA phosphate backbone.

Sodium acetate provides ions and helps DNA precipitation. With alcohol DNA recovery can improve.

Adding ethanol or isopropanol decreases the solubility of DNA. Cations from salt partly neutralise the negative charges on DNA phosphate groups, allowing DNA to separate as a precipitate. DNA translation occurs on ribosomes, not because of alcohol. Exam tip: in DNA extraction, the main role of alcohol is DNA precipitation.

Cold alcohol further decreases the solubility of DNA, allowing DNA to come out of solution and be recovered more effectively as a precipitate. Low temperature also reduces DNase activity, helping to limit DNA degradation. Converting DNA into protein is incorrect because DNA is a nucleic acid, not a protein. Exam tip: In DNA isolation, cold ethanol or isopropanol is added after salt treatment to precipitate DNA.

A 70 percent ethanol wash removes salts and small impurities. The pellet is then dried properly.

Remaining ethanol can inhibit downstream reactions. Therefore dry the pellet without over-drying.

An over-dried DNA pellet can become hard and compact, making it slow and difficult to redissolve in TE buffer or water. Ethanol is not formed from DNA, and cell lysis does not increase during pellet drying. Exam tip: After DNA precipitation, dry the pellet only enough to remove residual alcohol; do not let it become excessively hard.