Isolation of DNA

In recombinant DNA work the gene or DNA fragment must first be obtained. Therefore DNA isolation is an initial step.

DNA is found in biological cells. Therefore cell-containing samples are suitable for DNA isolation.

Animal cells do not possess a rigid cell wall or chloroplasts, but their plasma membrane encloses the cytoplasm and a nuclear envelope encloses the DNA-containing nucleus. Efficient isolation therefore requires disruption of the cellular membrane and access to the nucleus; lysis conditions and detergents help solubilize lipid membranes. Additional treatment may remove proteins associated with DNA, but that does not change the basic structural requirement. Option C is correct because both barriers must be opened for nuclear DNA to be released. Option A is incomplete, while soil layers and chloroplast walls are not normal structures of animal tissue and cannot explain animal DNA extraction.

Plant tissues can have a tough cell wall and polyphenols polysaccharides. Therefore special buffers are useful.

Lysozyme hydrolyses the β-1,4 glycosidic bonds in peptidoglycan of the bacterial cell wall. This weakens the wall and makes cell lysis easier for releasing DNA. Phospholipids of the cell membrane are generally disrupted by detergents, not by lysozyme. Exam tip: associate lysozyme in bacterial DNA isolation with the peptidoglycan cell wall.

Fungal cell walls contain chitin. Chitinase breaks chitin bonds, weakening or disrupting the cell wall so that DNA can be released and isolated. Option C is incorrect because DNase, not chitinase, degrades DNA. Exam tip: In DNA isolation, an enzyme named after a cell-wall component usually breaks down that component.

Grinding breaks the tissue into smaller pieces and disrupts cells mechanically. This increases the contact of lysis buffer and enzymes with the cells, making it easier to release DNA. Option B is incorrect because DNA isolation aims to release DNA from cells, not keep it stable inside them. Exam tip: associate grinding with tissue disruption before cell lysis.

Plant tissues often contain rigid cell walls and fibrous material, so mechanical disruption is an important early step in DNA isolation. A mortar and pestle physically grind the tissue into a fine homogenate, increasing the exposed surface area and helping lysis reagents reach more cells. For some tough or frozen tissues, liquid nitrogen keeps the material brittle and limits heat generation during grinding, although it must be handled with suitable safety precautions. Option A is correct because the apparatus performs mechanical tissue disruption. It does not sequence DNA, translate RNA, or serve only as a protein-digestion tool; those processes require different methods or reagents.

Homogenization converts the tissue or cell sample into a more uniform mixture. This allows the lysis buffer to contact cells more evenly, so cell lysis can occur more uniformly and DNA extraction may improve. It does not automatically purify proteins or remove the need for ethanol during DNA precipitation. Exam tip: Link homogenization with uniform sample preparation and efficient lysis.

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

In total DNA extraction mitochondrial DNA can come along with nuclear DNA. It depends on sample type.

Plant total DNA can include nuclear mitochondrial and chloroplast DNA. Organelles also contain DNA.

High molecular weight DNA indicates less broken genomic DNA. It is useful in some sequencing and library work.

High molecular weight DNA consists of long strands that can break into smaller fragments during vigorous vortexing, harsh pipetting, or repeated freeze-thaw cycles. Gentle pipetting and careful mixing minimise DNA shearing, so this is the correct practice. Exam tip: For intact long DNA, remember “gentle handling; avoid vigorous mixing.”

The goal of plasmid prep is to obtain plasmid DNA. Chromosomal DNA contamination can affect downstream analysis.

In alkaline lysis genomic DNA can precipitate with debris. Plasmid DNA is recovered in solution.

After cell lysis, vigorous vortexing or pipetting can shear the long chromosomal DNA into small fragments. These fragments may remain in solution and co-purify with plasmid DNA, increasing genomic DNA contamination. A plasmid cannot turn into protein, and ethanol cannot become DNA. Exam tip: during plasmid isolation, mix by gentle inversion rather than harsh vortexing.

After centrifugation the upper liquid layer is called supernatant. Depending on protocol it may contain DNA or impurities.

Centrifugation separates a mixture into a pellet at the bottom and a supernatant above it, but the location of DNA depends on the particular isolation protocol and step. DNA may be precipitated into the pellet, remain dissolved in the supernatant, or be present in a fraction that must be retained for a later precipitation or purification step. Correctly identifying the two layers prevents accidental disposal of the fraction containing the target nucleic acid. Option A is correct because discarding the wrong layer can cause substantial or complete DNA loss. The other choices concern sensory observations that have no role in deciding which fraction should be kept.

In phenol-chloroform extraction proteins and debris can be at the interface. The aqueous phase should be collected carefully.

After centrifugation during DNA isolation, DNA is usually present in the aqueous phase, while the interface may contain proteins, cellular debris, and other impurities. Carefully removing the aqueous phase prevents the interface from being carried over and keeps the DNA purer. Protein synthesis and cell growth are not purposes of this step. Exam tip: keep the pipette tip away from the interface while collecting the aqueous layer.

Na⁺ ions supplied by sodium acetate shield or neutralise the negative charges of DNA phosphate groups. This reduces repulsion between DNA molecules, allowing DNA to precipitate when ethanol or isopropanol is added. Breaking the sugar-phosphate backbone would damage DNA, so it is not the role of sodium acetate. Exam tip: remember that salt reduces the effective negative charge on DNA during precipitation.

Cold conditions, especially when cold ethanol is used, reduce the solubility of DNA. DNA therefore comes out of the solution and can be collected more easily as a precipitate. Converting DNA into RNA is transcription, not DNA precipitation. Exam tip: In DNA isolation, alcohol and cold conditions are used mainly to improve DNA recovery.

With low DNA the pellet can be invisible. Tube orientation and careful pipetting prevent DNA loss.

A DNA pellet may be loosely attached to the bottom of the tube and can be difficult to see. If it is disturbed while removing the supernatant, DNA may be carried away with the supernatant, causing DNA loss. Option B describes a possible event during disturbance, but DNA loss is the important outcome. Exam tip: remove the supernatant slowly with a pipette and keep the tip away from the pellet.