2.4.5.2. Mitochondrial DNA analyses

The mitochondrion, a maternally inherited organelle, provides numerous genetic polymorphisms that can be useful for inferring sources for new SHB populations and for some small-scale studies of populations. Mitochondrial DNA (mtDNA) sequences for this species have been analysed for the gene encoding the Cytochrome oxidase I enzyme, a gene often used for DNA ‘barcoding’ (species-level and population-level identification) in insects. Evans et al. (2000) described two oligonucleotide primers, AT1904S (5’-GGTGGATCTTCAGTTGATTTAGC-3’) and AT2953A (5’-TCAGCTGGGGGATAAAATTG-3’) that amplify a ca. 1,000 base-pair region of the SHB mitochondrion. These can be amplified and analysed as below:

  1. Amplify using a thermal cycling protocol of
    • 93°C (1 min),
    • 54°C (1 min),
    • 72°C (2 min) for 35 cycles,
    • followed by a 5 min elongation step at 72°C.
  2. Prepare a 1% agarose gel in 1X TAE buffer by mixing:
    2.1. 10X solution of 48.4 g of Tris base [tris(hydroxymethyl)aminomethane],
    2.2. 11.4 ml of glacial acetic acid (17.4 M),
    2.3. 3.7 g of EDTA, disodium salt.
    2.4. Bring to a final volume of 1 l with deionized water.
    2.5. Mix 1 g agarose/100 ml gel buffer.
    2.6. Microwave in an Erlenmeyer flask for ca. 1 min.
    2.7. Swirl the liquids.
    2.8. Microwave again for ca. 1 min.

    Solution should reach a rapid boil and be fully dissolved.
    2.9. Let cool for 10 min.
    2.10. Add 1 µl of 10 mg/ml Ethidium bromide per 100 ml of gel volume.
  3. Prepare sucrose loading buffer by mixing:
    • 4 g sucrose,
    • 25 mg bromophenol blue or xylene cyanol (0.25%),
    • dH2O to 10 ml.
  4. Mix 3 µl of each sample with 2 µl sucrose loading buffer.
  5. Load each sample in an individual well.
  6. Include a DNA size standard in a well alongside the products (e.g. the 100 base-pair ladder from Invitrogen).
  7. Electrophorese the products at ca. 100V/25 amps (depending on gel apparatus) in a horizontal gel rig.
  8. Visualize under UV light.
  9. There should be a single strong band at ca. 1,000 base pairs.
  10. Purify the resulting products (e.g. PCR Purification Kit, Qiagen).
  11. Sequence from each direction via standard Sanger dideoxy sequencing (see the BEEBOOK paper on molecular methods, Evans et al., 2013) and the amplification primers.

 Sequences can be aligned against numerous SHB haplotypes in public sequence databases (e.g., http://www.ncbi.nlm.nih.gov/gquery/?term=aethina+tumida) to determine novelty or placement into a described haplotype.