An NCBE / Unilever educational guide

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illuminating

| ILLUMINATING DNA | Version 1.0 | June 20002

Introduction | Equipment | Acknowledgements | Copyright

equipment and materialsIn this booklet we have tried to describe practical protocolsthat are relatively inexpensive and require little specialistequipment.

The NCBE endeavours to develop and supply at low costseveral of the items required, such as electrophoresisapparatus, restriction enzymes, plasmids and so on.

Because the details of suppliers and their products can change,we have decided not to list them in this booklet. Instead, asupplement giving up-to-date information is available fromthe NCBE’s Web site, or it can be obtained on request fromthe NCBE. Our address is given on the back page.

Investigating Plant DNA and The Transformer Protocol. Thelatter publication also provides an introduction to ethical,social and other issues raised by DNA technology. All ofthese publications, the other practical guides in this series,and supplementary resources are available from the NCBE’sWeb site:

http://www.ncbe.reading.ac.uk

An adequate treatment of the wider issues raised by DNAtechnology (particularly those associated with humangenetics, environmental concerns or food biotechnology)cannot be given in this slim booklet, nor can it take the placeof the many excellent school textbooks covering basicbiochemistry and molecular biology. It aims merely to providesufficient information for you to understand the practicalexercises. References to other materials are providedthroughout and on the back cover. The NCBE’s Web siteoffers a more comprehensive list.

to the readerWork with DNA is central to many, if not most, developmentsin modern biotechnology. There is growing public awarenessof DNA technologies, their possible applications and widerimplications. However, much of the essential debate aboutcurrent genetics has generated more heat than light.

Fortunately, the basic science upon which DNA technologiesare founded features in nearly every school examinationsyllabus in biology or science. This booklet is intendedprimarily for post-16 students of biology and their teachers.

The practical exercises described here provide an introductionto some of the classical techniques of molecular biology in aform suitable for the school laboratory. For reasons of safetyand expense, some the work in this booklet is not particularlysuited to open-ended practical investigations, but some ideasthat may provide starting points for additional work of yourown are given on pages 36–37. More ideas are provided inother NCBE publications such as: The Lambda Protocol;

COPYRIGHTThe material in this booklet is copyright.

The author and illustrator, Dean Madden, has asserted his moralright to be identified as the copyright holder under Section 77 of

the Copyright, Designs and Patents Act, UK (1988).

PHOTOCOPYINGPhotocopies of this booklet, or selections from it may be made

for educational use, provided that the copies are distributed free-of-charge or at the cost of reproduction, and the author of the

materials is credited and identified as the copyright holder.

acknowledgementsThe production of this booklet was very kindly supportedby Unilever plc. The contents are entirely the responsibilityof the NCBE however. Unilever’s only conditions insponsoring this booklet were that we made the publicationfreely available via the Internet, and that we consulted ourcolleague, Dr Malcolm Thomas, in the Department ofEducation at the University of Aberystwyth. Malcolm mademany helpful comments, for which we are grateful. MarjorieSmith of Dollar Academy was very supportive too, giving ustips for making this booklet more palatable to those inScotland. The molecular structure data for several of thefigures came from the Protein Data Bank and the NucleicAcid Database; accession numbers are given by the figures.Any errors in this booklet are the author’s, of course.

Some of the investigationsdescribed here require ahigh-speed microcentrifuge.An inexpensive, award-winning microcentrifugefor school use has beendeveloped by our friendProfessor John Cave ofMiddlesex University andthe Technology EnhancementProgramme (TEP®), with helpfrom Science and Plants forSchools (SAPS) and the NCBE.

www.ncbe.reading.ac.uk |3

contents04 DNA: molecule of the century

DNA, genes and chromosomes | The genetic code and protein synthesis | Enzymes, precisemolecular tools | Bacterial transformation | Genetically modified plants and animals | Thepolymerase chain reaction | Marker genes and gene regulation | DNA gel electrophoresis| Gene mapping | DNA sequencing | Using genetic data

14 Safety guidelines

Good microbiological laboratory practice

18 Practical activities

Modelling DNAPhysical and computer models are useful for studying molecular structures 18

Extracting DNASimple extraction of DNA from onions or fish sperm 20

DNA quantificationDiphenylamine gives a blue-coloured complex with deoxyribosethat can be used to measure the amount of DNA in a sample 22

β-galactosidase inductionE. coli makes the enzyme β-galactosidase — but only when induced to 24

Extraction of plasmid DNAPlasmid DNA can be extracted from bacteria and run on an electrophoresis gel 26

Bacterial transformationProve that DNA is the genetic material by transforming bacteria 28

Restriction and ligationRestriction enzymes and DNA ligase can be used to ‘cut and paste’ DNA 30

Restriction site mappingFind out where the restriction sites are in phage Lambda’s DNA 32

Amplifying Lambda DNAThe polymerase chain reaction has been likened to a ‘genetic photocopier’;use it here to amplify a fragment of Lambda’s DNA 34

36 Ideas for investigations

38 Running gels | Units of measurement

39 Staining DNA on electrophoresis gels

40 Recipes

42 Glossary

44 Other sources of information