BIOL463 2014
A technique in five minutes
TECHNIQUES SPEED-DATING PRESENTATIONS: WRITE-UP TEMPLATE
Names and contributions of group members:
Andrew Moffatt – research for mini-report, power point presentation
Saikrishna Venakatsubramanian – group coordination, research for mini-report
and presentation, crate power point presentation
Jelena Grabeljsek – research for mini-report, put mini-report together
Tracy Pham – research for mini-report, power point presentation, preparation of
props for “speed-dating” session
Yao Liu – research for mini-report and presentation
Technique chosen: Site directed mutagenesis a.k.a. site specific mutagenesis or
oligonucleotide-directed mutagenesis
What does this technique ‘do’?
Introduces a specific mutation point (point mutation) or couple-bases mutation with
insertions or deletions, frame shift, nonsense mutations into a gene to alter the
primary amino acid sequence of proteins
What applications is this technique employed for?
 Engineer gene products with a certain functionality (or loss of)
 Investigate the effects of specific mutations on gene product function
 Screen an array of mutants to determine sequence in question
The methods used for these applications include: PCR extension for an insertion
and/or deletion, cassette mutagenesis, inverse PCR, oligonucleotide
heteroduplex and random point mutations/chemical alterations
What questions (give a couple of examples) relating to gene regulation
and/or development can be addressed using this technique?
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Identifying function of genes and mechanisms of gene regulation in
development
Roles of different genes at different stages in development
What critical reagents are required to use this technique?
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Synthetic oligonucleotide (RNA/DNA) primer that contains desired mutation
Template DNA sequence: any vector, DNA fragment or product of PCR that
comprises of complementary DNA (to be mutated) can be used as the initial
template
BIOL463 2014


A technique in five minutes
Thermostable DNA polymerase (pfu)
Buffer for DNA polymerase (pfu, ligase)
What critical information is required to be able to employ this technique?

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Knowledge of the wild type sequence and protein structure
Knowledge of wild-type protein function
References:
Adereth, Y., Champion, K.J., Hsu, T., and Dammai, V. 2005. Site-directed
mutagenesis using Pfu DNA polymerase and T4 DNA ligase. Biotechniques, 38(6):
864-868
Bachman, J. 2013. Site-Directed Mutagenesis. Methods in Enzymology, 529: 241248
Liu, L., and Lomonossoff, G. 2006. A site-directed mutagenesis method utilising
large double-stranded DNA templates for the simultaneous introduction of multiple
changes and sequential multiple rounds of mutation: Application to the study of
whole viral genomes. Journal of Virological Methods, 137: 63-71.
Smith, M. 1982. Site-Directed Mutagenesis. Trends In Biochemical Sciences, 7(12):
440-442
Reikofski, J., and Tao, B.Y. 1992. Polymerase Chain Reaction (PCR) Techniques for
Site Directed Mutagenesis. Biotechnology Advances, 10: 535-547
Smith, M. 1986. Site-Directed Mutagenesis. Philosophical Transactions of the Royal
Society of London, 317: 295-304
Wagner, C.R., and Benkovic, S.J. 1990. Site Directed Mutagenesis: A Tool For
Enzyme Mechanism Dissertation. Trends In Biotechnology, 8: 263 - 270