Chapter Five - Yeast-Gene Replacement Using PCR Products
Section snippets
Theory
Perhaps one of the most useful aspects of the baker's yeast Saccharomyces cerevisiae as a model organism for molecular biology is its ability to very efficiently carry out homologous recombination between transformed linear pieces of DNA and its genome. A stretch of as little as 40 base pairs of homology at each end of a linear piece of DNA is sufficient to target it for integration at a specific genomic locus. Because the required regions of homology are so short, they can simply be
Equipment
PCR thermocycler
Microwave oven
Agarose gel electrophoresis equipment
Transilluminator or other UV light source
Micropipettors
Micropipettor tips
0.2-ml thin-walled PCR tubes
Materials
PCR primers
Plasmid or other source of DNA encoding selectable marker
Selective yeast media
Taq (or similar thermostable DNA polymerase)
dNTP mix (may be supplied with Taq enzyme, or contains: dATP, dGTP, dTTP, and dCTP)
PCR buffer (may be supplied with Taq enzyme, or contains: Tris–HCl, pH 8.8, (NH4)2SO4, Tween-20, and MgCl2)
Agarose
Tris base
Boric acid (H3BO3)
EDTA
Ethidium bromide
Ficoll
Bromophenol blue
Xylene cyanol
DNA ladder
Purified water (can be distilled and autoclaved, or filtered by Milli-Q or
Duration
Preparation Variable Protocol 5–7 h
Preparation
A source of template DNA encoding the selectable marker and any other sequences to be inserted into the genome must be obtained, and PCRs must be designed (see also Explanatory chapter: PCR -Primer design) and synthesized prior to starting this protocol. In many ways, obtaining or generating the appropriate template DNA and designing PCR primers are the most difficult and critical parts of this protocol.
There are many plasmids carrying sequences encoding
Overview
PCR, using the primers and template discussed above, is used to generate a linear DNA construct that is then transformed into yeast. The goal of this step is to generate ~ 1 μg of the desired construct. Optimal PCR conditions must be determined empirically for each pair of primers (see Explanatory Chapter: Troubleshooting PCR), but an example of commonly used conditions is described. Because the introduction of mutations is undesirable, it may be worthwhile to use a thermostable polymerase with
Overview
A small sample of the PCR product that has been generated is now separated by size on an agarose gel containing a DNA-intercalating dye such as ethidium bromide (see Agarose Gel Electrophoresis), to ensure that a sufficient quantity of a single product of the correct size has been obtained.
Duration
1.5 h
- 2.1
Cast the agarose gel mix into a gel tray after boiling in a microwave oven to completely dissolve the agarose. Take care while heating the gel mix not to allow it to boil over. Fill the gel tank with 1×
Overview
A standard lithium chloride transformation of yeast can be used to introduce the PCR product generated above for integration into the yeast genome (see Chemical Transformation of Yeast).
Duration
1 h
Tip
Transformation of yeast by a linear piece of DNA that must integrate into the genome is far less efficient than transformation by a plasmid. Thus, it generally requires more DNA and yields fewer colonies. Use 0.2–1.0 μg DNA per transformation, but not more than this. Using too much DNA can actually inhibit
Overview
Colony PCR can be used to ensure that colonies coming up on selective media do in fact carry the selectable marker integrated at the appropriate genomic locus (see Colony PCR). A PCR product generated by colony PCR and containing the entire region affected by the gene replacement can also be sequenced to ensure that no unintended mutations were introduced.
Tip
Follow the instructions of the sequencing facility to prepare a sample for sequencing. Generally, the PCR product must be column purified to