Frontiers in Zoology/ Use of blocking primers in prey DNA

1. Frontiers in Zoology

This may be a journal worth considering for some papers. This is one of the BMC journals and claims an unofficial impact factor of 3.24 (cf BMC Genetics at 1.58, but the Frontiers factor will be based on a small sample). It used to be cheaper to publish in than the other BMC journals but not anymore. As with other BMC journals, they will publish some papers for free and you can ask about this before submission.

2. The following paper is technically clever in using blocking primers to inhibit the amplification of “host” DNA. They analyse diet in krill using universal primers for 28S RNA that they claim will amplify in all eukaryotes. The blocking primers are designed from the krill sequence – the only sequence that they detect when they don’t use the blocking primer. The method, and blocking primers more generally, may have multiple applications.

Blocking primers to enhance PCR amplification of rare sequences in mixed samples – a case study on prey DNA in Antarctic krill stomachs

Hege Vestheim and Simon N. Jarman

Frontiers in Zoology 2008, 5:12doi:10.1186/1742-9994-5-12
Published:     20 July 2008
Abstract (provisional)


Identification of DNA sequence diversity is a powerful means for assessing the species present in environmental samples. The most common molecular strategies for estimating taxonomic composition depend upon PCR with universal primers that amplify an orthologous DNA region from a range of species. The diversity of sequences within a sample that can be detected by universal primers is often compromised by high concentrations of some DNA templates. If the DNA within the sample contains a small number of sequences in relatively high concentrations, then less concentrated sequences are often not amplified because the PCR favours the dominant DNA types. This is a particular problem in molecular diet studies, where predator DNA is often present in great excess of food-derived DNA.

We have developed a strategy where a universal PCR simultaneously amplifies DNA from food items present in DNA purified from stomach samples, while the predator’s own DNA is blocked from amplification by the addition of a modified predator-specific blocking primer. Three different types of modified primers were tested out; one annealing inhibiting primer overlapping with the 3′ end of one of the universal primers, another annealing inhibiting primer also having an internal modification of five dI molecules making it a dual priming oligo, and a third elongation arrest primer located between the two universal primers. All blocking primers were modified with a C3 spacer. In artificial PCR mixtures, annealing inhibiting primers proved to be the most efficient ones and this method reduced predator amplicons to undetectable levels even when predator template was present in 1000 fold excess of the prey template. The prey template then showed strong PCR amplification where none was detectable without the addition of blocking primer. Our method was applied to identifying the winter food of one of the most abundant animals in the world, the Antarctic krill, Euphausia superba. Dietary item DNA was PCR amplified from a range of species in krill stomachs for which we had no prior sequence knowledge.

We present a simple, robust and cheap method that is easily adaptable to many situations where a rare DNA template is to be PCR amplified in the presence of a higher concentration template with identical PCR primer binding sites.

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