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Friedrich Miescher
salmon

Friedrich Miescher.

In 1868, Johann Friedrich Miescher travelled from his native Switzerland to Tübingen in Germany. The 24-year-old had come to study in the laboratory of Ernst Felix Hoppe-Seyler, a pioneering biochemist who coined the modern name for the red pigment in blood, haemoglobin. After several months of toil in a laboratory in the cellar of Tübingen Castle, Miescher managed to isolate a previously-unknown acidic substance from white blood cells (leucocytes) washed from pus-laden bandages donated by a nearby hospital. Miescher called his discovery 'nuclein' because it was found in the nuclei of the cells. This substance was impure however, and Hoppe-Seyler insisted on repeating the work himself before he would allow an account to be published in his newly-formed journal of biochemistry.

Upon returning to his home in Basel in 1870, Miescher refined the method and was able to extract nuclear material from the sperm of the salmon for which, in those days, the Rhine was famed. Like those in leucocytes, the nuclei in sperm cells are relatively large. From these Miescher first extracted pure nuclein. In 1889 a pupil of his, Richard Altmann, gave us the modern term for nuclein, 'nucleic acid'. Thus, this year, we celebrate the Golden Jubilee of the Double Helix and not '50 years of DNA'.

To the science teacher, an account of Miescher's methods makes fascinating reading, not least because of the crude techniques available and the striking resemblance they bear to many of today's classroom protocols. Compared to Miescher, however, today's teachers have an easy time of it. With no refrigeration, Miescher had to start work at 5 am to ensure that the reagents were cold enough to precipitate DNA, and he had to prepare his own protease enzymes from the stomachs of freshly-slaughtered pigs! (Judson, 1996).

School Science Review cover

This article appeared in the March 2003 issue of the School Science Review.

Miescher's lab in Tübingen

Miescher's lab in the cellar of Tübingen Castle.

CLASSROOM PROTOCOLS

The isolation of DNA from everyday materials has become a popular and widespread activity in school laboratories over the past 17 years. Although similar practical protocols had been described previously (e.g., Sands, 1970), these were not adopted widely owing to the complexity of the procedures involved and the hazardous nature of several of the reagents required (Falconer and Hayes, 1986). Simpler methods of isolating DNA first appeared in American textbooks in the mid-1980s (e.g., Helms, et al, 1986) and subsequently made their way into specialist school biotechnology projects (e.g., Rasmussen and Matheson, 1990). By the early '90s these methods had crossed the Atlantic, featuring in German and English publications (Bayrhuber, et al, 1990; NCBE, 1991). With the arrival of simple and inexpensive methods, what was an undergraduate or post-16 practical exercise moved down the age range and even into the primary classroom (Assinder, 1998).

DNA YOUR ONIONS?

The most commonly-used (if not, for obvious reasons, the most popular) method of extracting DNA requires little more than onions, household detergent and salty water. This method became widespread in the UK due to the practical workshops and publications of the NCBE (NCBE, 1991; 1993). More sophisticated methods of extracting DNA from cress and dried peas were developed by Science and Plants for Schools and the Australian CSIRO education centre 'The Green Machine' (NCBE, 1995) but their association with DNA gel electrophoresis restricted these protocols to use by older students.

In the search for sweeter-smelling alternatives to onions, several have suggested applying the 'onion method' to a variety of fruits, including kiwi fruit, bananas and strawberries (see, for example, the BBC's 'Science Shack' Web site). Although these fruits seem to yield copious amounts of DNA, the 'DNA' produced is in fact little more than pectin. This can be demonstrated simply by adding pectinase to the preparation (it's also quite easy to precipitate pectin in alcohol; an old jam-maker's trick).

EXTRACTING THE PEA (DNA)

The protocol described here uses frozen peas. This has several advantages over the traditional onion method. Firstly, no blender is needed to break up the plant tissue. Provided the peas have thawed, they can be squashed with the back of a spoon or a glass rod. Secondly, supplies of peas can be stored easily in the freezer and taken out in suitable amounts when required. And last, but not least, the peas don't smell! Isolating the DNA (and RNA) takes about 35 minutes, including an incubation period of 15 minutes.

ADVANCE PREPARATION

The ethanol used must be ice cold. Place it in a plastic bottle in a freezer at least 24 hours before you attempt this activity. Please read the safety note, below.

MATERIALS AND EQUIPMENT
Needed by each person or group

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Peas, about 50 g (frozen ones are suitable, but thaw them first)

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Washing-up liquid, 10 mL (use a watery type, not the thicker, concentrated variety)

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Table salt, 3 g

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Distilled water, 90 mL

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Very cold ethanol, about 10 mL, straight from the freezer (industrial methylated spirit, IMS, is suitable, but please see the safety note on this page)

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Novozymes Neutrase (a protease), 2-3 drops

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Ice, in a jug with cold water

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Coffee filter paper (do not use laboratory filter paper, as liquid takes too long to pass through it)

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1 mL plastic syringe (without a needle)

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Large plastic funnel

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250 mL beakers, 2

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Boiling tube or plastic graduated tube

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Glass rod with a flattened end or a spoon for stirring the mixture

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Water bath, maintained at 60 °C

frozenpeas

PROCEDURE

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Dissolve the salt in 90 mL of distilled water. Add the washing-up liquid and mix gently.

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Mash the peas using a glass rod or a spoon. Add the pea pulp to a beaker with the salty washing-up liquid solution.

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Stand the beaker in a water bath at 60 °C for exactly 15 minutes. This treatment causes the pea cell membranes to break down. The detergent forms complexes surrounding the membrane phospholipids and proteins, causing them to precipitate. In addition, the sodium ions from the salt shield the negatively-charged phosphate groups of the DNA molecules, causing them to coalesce. At 60 °C, DNase enzymes, which would otherwise start to cut the DNA into fragments, are partially denatured.

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Cool the mixture by placing the beaker in an ice water bath for 5 minutes, stirring frequently. This slows the breakdown of the DNA which would occur if a high temperature was maintained.

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Filter the mixture into a second beaker. Ensure that any foam on top of the liquid does not contaminate the filtrate. The filtrate contains the soluble proteins and DNA.

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OPTIONAL: Add 2-3 drops of protease to about 10 mL of the pea extract in a boiling tube and mix well. The protease will degrade some of the proteins in the preparation.

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Very carefully pour ice cold ethanol or IMS down the side of the boiling tube, to form a layer on top of the pea extract.

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Leave the tube, undisturbed, for a few minutes. Nucleic acids (DNA and RNA) are insoluble in cold ethanol and will precipitate into the upper (ethanol) layer.

Pea DNA precipitates into ethanol.

The DNA is the white material in the clear alcohol layer above the pea extract.

FURTHER INVESTIGATIONS

A hook for recovering the DNA can be made by briefly heating the tip of a Pasteur pipette in a Bunsen burner flame, then bending the tip round before allowing the glass to cool. To electrophorese the DNA extract, simply dissolve some of it in about 0.5 mL of bromophenol blue loading dye, then load about 20 µL into a well in a 1% agarose gel. Staining with 0.04% (w/v) Azure A solution after electrohoresis will reveal the nucleic acids (RNA shows up a lighter pink colour) (Madden, 2000).

Variations of this extraction procedure can be used for other food items, e.g., fish sperm (milt or soft roe) or fish eggs (Strömberg, 2001). Several publications refer to the use of calf thymus tissue, but its use in schools is no longer recommended (see Safety note, below).

SAFETY

Ethanol in freezers
Most freezers are not spark-proof. Consequently, you must ensure that any ethanol placed in a freezer is in a sealed, vapour-tight container. An alternative to using a freezer is to stand the sealed bottle of ethanol in ice for several hours before use. For more information about safety in schools when working with DNA, teachers in the UK should consult
Topics in Safety (ASE, 2001).

Use of animal tissue
Since the advent of BSE and vCJD in the United Kingdom, school safety authorities there advise that calf thymus should no longer be used in schools, as there is a risk (albeit small) of accidental exposure to the infectious agent while the extract is being prepared.

Safety guidelines for practical work with DNA
can be found here.

SUPPLIERS

Most of the items required for this procedure can be obtained from a supermarket.

Novozymes Neutrase can be bought in small volumes from the UK's National Centre for Biotechnology Education.

REFERENCES

Judson, H. F. (1996) The eighth day of creation. Makers of the revolution in biology. New York; Cold Spring Harbor Laboratory Press.

Sands, M. K. [Ed] (1970) Nuffield Advanced Science Laboratory Guide. London; Longman.

Falconer, A. C. and Hayes, L. J. (1986) The extraction and partial purification of bacterial DNA as a practical exercise for GCE Advanced level students. Journal of Biological Education 20 (1) 25-26

Helms, D. et al (1986) Biology in the laboratory. New York; W. H. Freeman and Company.

Rasmussen, A. M. and Matheson R. H. (1990) A sourcebook of biotechnology activities. North Carolina; National Association of Biology Teachers.

Bayrhuber, H., Gliesche, Ch. and Lucius, E. R. (1990) DNA-Isolierung mit einfachen Mitteln (Isolation of DNA using simple methods). Unterricht Biologie 14 (151) 44.

NCBE (1991) DNA your onions? NCBE Newsletter, Spring 1991

Assinder, S. (1998) Discovering DNA. 'The recipe of life' Swindon; Biotechnology and Biological Sciences Research Council

NCBE (1993) Practical biotechnology. A guide for schools and colleges. Reading; National Centre for Biotechnology Education.

NCBE [Ed.] Investigating plant DNA. Reading; National Centre for Biotechnology Education.

Madden, D. (2000) Illuminating DNA. Reading; National Centre for Biotechnology Education.

Strömberg, E. (2001) DNA from caviar. Bioscience Explained 1 (1) http://www.bioscience-explained.org

Delpech, R. and Madden, D. (2001) 'Working with DNA'. In Topics in Safety (Third Edition) pp. 99-105. Hatfield; Association for Science Education.

WEB SITES

National Centre for Biotechnology Education
All NCBE publications are available free-of-charge (as PDF files) from this site. The Centre also supplies the protease which is used in the procedure.
http://www.ncbe.reading.ac.uk

Kiwi fruit 'DNA' [BBC web page no longer available]
How to extract pectin from kiwi fruit!
http://www.bbc.co.uk/science/scienceshack/backcat
/experiments/madna.shtml

Friedrich Miescher Institute
Useful accounts (in English) of Miescher's work.
http://www.fmi.ch

kiwi

Copyright © National Centre for Biotechnology Education, 2006 | www.ncbe.reading.ac.uk