Bio 423 Lecture 5

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 Back to Recombinant DNA Technology Lecture List