Sequence Analysis of the Variable Number Tandem Repeat in Staphylococcus aureus Protein A Gene - Bacterial Pathogenesis: Methods and Protocols - page 273

and a value of >1 is indicative of purifying or stabilizing selection (21) . This
result strongly suggests that the variation seen in the VNTR region is not the
result of outside influences and that the polymorphism is reflective of intrinsic
changes in the evolution of the species. Therefore, spa typing while appropriate
for short-term investigations can also be useful for global epidemiological studies.
2. Materials
2.1. DNA Isolation
1. Solid media for isolation of S. aureus .
2. Sterile pipet tips.
3. Sterile water.
4. 1.5-mL tube microfuge tubes (or 96-well microtiter plate).
5. Boiling water bath (or thermocycler).
6. Centrifuge.
7. Lysostaphin (optional)
2.2. PCR
1. PCR primers as follows ( see Fig. 1B ):
a. (TIGR-F) 5´-GCCAAAGCGCTAACCTTTTA-3´
b. (TIGR-R) 5´-TCCAGCTAATAACGCTGCAC-3´
2. PCR reaction components (dNTPs, buffer, etc.).
3. 0.2-mL PCR reaction tubes (or 96-well microtiter plates).
4. Thermocycler.
5. Materials and apparatus for agarose electrophoresis.
6. PCR purification method (user-specific).
2.3. DNA Sequencing
1. Either or both primers used for PCR ( see Subheading 2.2, step 1 ).
2. User-defined DNA sequencing platform and materials.
3. Software for trace chromatogram viewing.
3. Methods
3.1. DNA Isolation
spa typing is a simple, fast, and accurate method that involves the isolation,
amplification, and sequencing of the VNTR region near the 3´ end of the
protein A gene. The efficient lysis of S. aureus cells requires incubation with
lysostaphin, an endopeptidase that specifically cleaves the penta-glycine cross