BB211: Cell and Molecular Biology

Dr Eve Lutz
Department of Bioscience
Recombinant DNA technology:
Lecture 5
DNA Sequencing: sequence analysis
Background reading:
Reference for this lecture please read the following:
Chapter 16
Klug, WS & Cummings, MR Essentials of Genetics, 4th ed.

Nucleic acid sequencing

Determining the sequence of the nucleotide bases (A, T, G, C) in
molecules of DNA. This became possible once techniques for
producing and isolating DNA restriction fragments were available.

Two main methods of DNA sequencing are:

1. Chemical cleavage - Maxam & Gilbert method

2. Enzymic chain termination method - Sanger method

Fred Sanger is one of Britain's greatest scientists. Developed

methods for sequencing both DNA and proteins. Won the Nobel
prize twice, once for each of these breakthroughs.

What can the DNA sequence tell us?

1. Predict the sequence of amino acids of proteins encoded by the

DNA

• Ascertained by translating the codons in open reading

frames into their encoded amino acids
2. Determine the composition of RNA molecules encoded by the
DNA
a) rRNAs
b) tRNAs

3. Locate the position and determine the composition of introns in

genes from eukaryotes

4. Characterise the complete genetic make-up of an organism

(genome sequencing)

Both the Maxam & Gilbert method and the Sanger method
require the production of fragments of DNA from the piece of
DNA which is being sequenced. For both of these methods these
fragments are separated (resolved) from each other - this usually
means resolving DNA fragments which differ in length by one
nucleotide. This is done by polyacrylamide gel electrophoresis
(PAGE)

For PAGE, the gel is composed of polymerised acrylamide.

Acrylamide is a neurotoxin and needs to be handled with care.
Gels are very thin and usually quite long, ~0.5m - 2m in length.
Polyacrylamide gels give excellent resolution - allowing
discrimination between fragments that differ in size by only one
nucleotide

• example of M&G sequencing reactions resolved on a

polyacrylamide gel
• example of Sanger sequencing reactions resolved on a
polyacrylamide gel

Automatic sequencing
Recently, Sanger sequencing has been automated. This method
doesn't require radioactivity. Instead, 4 different fluorescently
labelled dyes are attached to the 4 different ddNTPs. This
means that strands that terminate at G's will have a different
label to those that terminate at A's etc. This in turn means that
the reaction can be done in 1 tube (not 4) and loaded into one lane
of a polyacrylamide gel. The data is read by a fluorescence
scanner during electrophoresis, as bands pass close to the bottom
of the gel.

Automated sequencing is cheaper, safer (no radioactivity) and

allows more bases to be read, usually 750-1000. Automated DNA
sequencers and robotic workstatons are much used for genome
sequencing projects

webpage last updated 21/2/03

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