Well, it's been a little while since I've updated. These things happen. I've been playing a bit with this full genome amplification kit called GenomiPHI (
www.genomiphi.com). The kit contains a DNA polymerase (Phi29) and random primers. The polymerase is quite nice because it is less sensitive to environmental contaminants (humics, etc) than can be co-extracted with DNA. It works at low temperatures (30 deg C), does not require thermal cycling and is a proofreading enzyme.
The enzyme is useful because it makes copies (up to 5000x) of total genomic DNA. Apparently this is an unbiased reaction (I haven't tested this myself). I have so far employed this technique to enhance PCR amplification of the mcrA gene from methanogenic archae (see below). The kit is about $150 for 25 reactions - not cheap, but not out of reach.
In high sulfate environments such as marine microbial mats, methanogens do not compete well with
sulfate-reducing bacteria and are generally not abundant. Thus, the detection of methanogens by PCR with mcrA gene primers can be difficult. I tried to circumvent this problem by performing a pre-amplification with the genomiPHI kit. I took several DNA extracts from high sulfate mats and diluted them either 1/10th or 1/100th. 1 ul of these dilutions were then added to the genomiPHI reaction with random primers and run overnight at 30C. It turned out that diluting the initial DNA sample was not a great idea. The first figure shows the DNA after genomiPHI amplification on a 0.8% agarose gel (first 7 lanes, left to right) and a standard DNA extract (lane 8).
No obvious difference between dilutions was observed after the overnight pre-amplification (the first three lanes are the same sample, undiluted, 1/10 and 1/100th). These samples were then subject
to standard PCR with mcrA primers. The first seven samples (left to right) are the PCR yield from the reaction containing DNA pre-amplified with the genomiPHI kit. The next 3 are the same samples amplified without pre-amplification. The next 1 (strong yield) is a PCR yield from a low-sulfate mat sample generating high levels of methane. The final lane is the negative control that unfortunately has a slight bit of contamination. Anyway, sample #1 yielded strong PCR amplification when the undiluted DNA extract was subject to GenomiPHI pre-amplification. At a 1/10th dilution the PCR yield was weaker and at a 1/100th dilution, no PCR product was detected even after pre-amplification. The same sample gave only a weak PCR yield when amplified directly from the original DNA extract. A dilution of the DNA extract prior to the pre-amplification turned out to be a poor idea, and these samples did not amplify well (except for #16) even after GenomiPHI amplification. More work to be done, but still, it is interesting.
If interested, check out this article:
Gonzalez et al. 2005. Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments. Environmental Microbiology. 7(7):1024-1028.
