10 min readPathway sheet2 or 3-mark question on the enzymes used to break cells and purify DNA; it is also the major experiment in the practical exam

Isolating DNA, with the plant DNA practical

To get pure DNA, break the cells with enzymes and remove RNA and proteins with ribonuclease and protease. Then add chilled ethanol and spool out the DNA.

Cell-breaking enzymesLysozyme (bacteria), cellulase (plant cells), chitinase (fungi)

Removing other moleculesRNA by ribonuclease; proteins (such as histones) by protease

Precipitating DNAChilled ethanol; DNA appears as fine threads, collected by spooling

Why pure DNARestriction enzymes need DNA free from other macromolecules

Why DNA must be purified

DNA lies inside membranes. When a cell is broken, DNA comes out mixed with RNA, proteins, polysaccharides and lipids. Genes lie on long DNA molecules wound around proteins called histones. A restriction enzyme can cut DNA properly only when it is pure, so everything else must be removed first.

Isolating DNA in the laboratory

1
Break open the cell wall with an enzyme suited to the cell.
Lysozyme (bacteria), cellulase (plants), chitinase (fungi)
2
The cell releases DNA along with RNA, proteins, polysaccharides and lipids.
3
Remove RNA.
Ribonuclease
4
Remove proteins, including histones.
Protease
5
Remove other molecules by suitable treatments.
6★
Add chilled ethanol. Pure DNA precipitates as a collection of fine threads in the suspension; wind them out on a rod (spooling).

★ key step

Which enzyme breaks which cell wall

Source	Cell wall made of	Enzyme used
Bacteria	Peptidoglycan	Lysozyme
Plant cells	Cellulose	Cellulase
Fungi	Chitin	Chitinase

Activity: isolate DNA from spinach, green peas, papaya or banana

1
Take a small amount of fresh spinach leaves (or soaked green pea seeds, ripe papaya or banana). Cut it into small pieces.
2
Grind it in a mortar with a little cold extraction buffer: water with common salt and a few drops of liquid detergent (or shampoo).
3
Filter the paste through cheesecloth (or a fine strainer) into a test tube. Keep the filtrate.
4
Add a pinch of meat tenderiser (papain) or a few drops of fresh pineapple juice (bromelain). Mix gently and wait a few minutes.
These are proteases; they break down histones and other proteins
5
Tilt the tube and pour ice-cold ethanol, about equal in volume to the filtrate, slowly down the side so it forms a layer on top. Do not shake.
6★
Observe: white, fine, thread-like, slimy material collects where the two layers meet. Wind it onto a glass rod and lift it out. Record: this is DNA.

★ key step

Why each item is used in the practical

Item	Role
Grinding	Breaks the cell walls so the contents come out
Detergent	Dissolves the lipids of the cell membrane and nuclear membrane, releasing DNA
Common salt (NaCl)	Its sodium ions neutralise the negative charges on DNA, so the strands can clump together and precipitate when ethanol is added
Meat tenderiser or pineapple juice	Protease (papain or bromelain) digests histones and other proteins
Ice-cold ethanol	DNA is insoluble in cold ethanol, so it precipitates; the cold also slows enzymes that would break DNA

Precautions in the practical

Use fresh plant material and keep the ethanol ice-cold.
Pour the ethanol slowly along the side of the tube so the two layers do not mix.
Do not shake the tube after adding ethanol; the long DNA threads break.
Wind the threads gently with a clean glass rod.

In real life

Indian forensic and hospital labs use the same steps, with purer chemicals, to extract DNA. They use it for DNA fingerprinting and to test for genetic disorders. Papain from papaya and bromelain from pineapple, the proteases in this practical, are also used to tenderise meat.

Exam angle

2 marks: “Name the enzymes used to break open (i) bacterial cells (ii) plant cells (iii) fungal cells when isolating DNA. How is RNA removed?” (i) Lysozyme (ii) cellulase (iii) chitinase. RNA is removed with ribonuclease.

3 marks: “Describe how pure DNA is isolated from a cell. How is the DNA finally collected?” Break the cell wall with lysozyme, cellulase or chitinase; remove RNA with ribonuclease and proteins with protease; remove other molecules; add chilled ethanol so DNA precipitates as fine threads; collect by spooling.

2 marks: “In the DNA isolation practical, why are detergent and ice-cold ethanol used?” Detergent dissolves the lipid membranes to release DNA. DNA is insoluble in cold ethanol, so it precipitates as white threads at the junction of the layers.

Remember it

Little Cats Chew: Lysozyme (bacteria), Cellulase (plants), Chitinase (fungi). Then RNase, protease, cold alcohol.