separated by electrophoresis under denaturing conditions, and analysed separately.
DNA labelled at one end is divided into four aliquots and each is treated with
chemicals which act on specific bases by methylation or removal of the base. Condi-
tions are chosen so that, on average, each molecule is modified at only one position
along its length; every base in the DNA strand has an equal chance of being modified.
Following the modification reactions, the separate samples are cleaved by piperidine,
which breaks phosphodiester bonds exclusively at the 5^0 side of nucleotides whose
base has been modified. The result is similar to that produced by the Sanger method,
since each sample now contains radioactively labelled molecules of various lengths,
all with one end in common (the labelled end), and with the other end cut at the same
type of base. Analysis of the reaction products by electrophoresis is as described for
the Sanger method.
5.12 Suggestions for further reading
Augen, J. (2005).Bioinformatics in the Post-Genomic Era. Reading, MA: Addison-Wesley.
Brooker, R. J. (2005).Genetics Analysis and Principles, 2nd edn. New York: McGraw-Hill.
Hartwell, L. et al. (2008).Genetics: From Genes to Genomes, 3rd edn. New York: McGraw-Hill.
Lodish, H. et al. (2008).Molecular Cell Biology, 6th edn. San Francisco, CA: W. H. Freeman.
Lewin, B. (2007).Genes IX. Sudbury, MA: Jones & Bartlett.
Strachan, T. and Read, A. P (2004).Human Molecular Genetics, 3rd edn. Oxford, UK: Bios.
Walker, J. M. and Rapley, R. (2008).Molecular Biomethods Handbook, 2nd edn. Totowa, NJ:
Humana Press.
5 – – – TACGCTCG –^32 P3 Single-stranded DNA,
labelled only at its 3 end
Modification of C using hydrazine,
this removes base, leaving ribosyl urea
Cleavage at modified bases,
using piperidine
G–^32 P
TCG–^32 P
GCTCG–^32 P
plus non-radioactive fragments
Separation on sequencing gel alongside products of
other modification/cleavage reactions
- – – TA GCTCG–^32 P
- – – TACG TCG–^32 P
- – – TACGCT G–^32 P
Fig. 5.41Maxam and Gilbert sequencing of DNA. Only modification and cleavage of deoxycytidine is shown,
but three more portions of the end-labelled DNA would be modified and cleaved at G, GþA, and TþC, and
the products would be separated on the sequencing gel alongside those from the C reactions.
194 Molecular biology, bioinformatics and basic techniques
