This page contains more detailed protocols and primer sequences from Dean, BALLARD, GLASS and BallarD (2003) showing that Wolbachia uninfected Drosophila simulans retain greater amounts of mitochondrial DNA polymorphism than infected populations in east Africa. Genetics 16: 159-169.

Cytotype distribution and abundance: Shown below are schematic and empirical results of competitive PCR assays used in the paper above.  Bold lines indicate DNA strands, boxes indicate primers, arrows indicate direction of elongation, + and – indicate whether the primer anneals to the major or minor strand, respectively.  Figures not drawn to scale.

Distinguishing between siIIA or siIIB:  These primers amplify a 1226 bp fragment common to both siIIA and siIIB, and either a 740 bp fragment specific to siIIA or a 531 bp fragment specific to siIIB.

MtDNA sequencing:  Three regions were amplified and sequenced with the primers 1128+ and 1815-, 3182A+ and 3929-, and 7780+ and 8475-.  Primer 3182A+ has a two base pair mismatch to siIII, while 3182B+ has a two base pair mismatch to siII.  The regions yielded sequence of 599 bp, 601 bp, and 576 bp in length and included portions of the ND2, COI, COII, ATP8, ND5, and ND4 genes, the transfer RNAs tRNA_trp, tRNA_cys, tRNA_tyr, tRNA_lys, tRNA_asp, and tRNA_his, and four intervening spacer regions. 

Period DNA sequencing: We gathered 1029 continuous bp of the per locus.  This region contains 1 complete exon, 2 partial exons, and 2 introns. PCR reactions were carried out with the external primers 3768+ and 4856-.  For sequencing, we used the external primers and two additional internal primers, 4348+ and 4286-. 

PCR precipitation/purification:  To purify PCR reactions and remove unincorporated primers, we employed a simple salt precipitation method, modified from [Kreitman, M., 1991 Molecular techniques in taxonomy, pp. 357-367 in NATO ASI Series, edited by G. M. Hewitt, A. W. B. Johnson and J. P. W. Young. Springer-Verlag, Berlin].  This is an inexpensive and efficient method in terms of both money and time.  We precipite in the same tubes that the PCR reactions were performed in, thus the method is best used in a 96 well tray format.  We describe the method for a 50 μl reaction, but it is easily modified for different volumes.

1.         From 50 μl PCR reaction, visualize 4 μl on a gel.
2.         To remaining 46 μl PCR reaction, add 23 μl of 7.5 M Ammonium Acetate (CH₃CO₂NH₄).
3.         Add 69 μl cold 100% ethanol.
4.         Replace caps and gently invert a few times to mix.  Be mindful that the caps remain
            sealed during mixing.
5.         Spin at 4ºC, 2200g for 15 minutes.
6.         Remove caps, invert tray, spin at 100g for 1 minute.
7.         Add 200 μl 70% ethanol to wash the pellet (which you probably will not see).
8.         Replace caps, spin at 4ºC, 2200g for 1 minute.
9.         Remove caps, invert tray, spin at 100g for 1 minute.
10.       Resuspend as desired (we usually resuspend in 25 μl, then use 1-4 μl for sequencing
            reactions).