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

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Screening methods. Schematic illustration of different ways to screen away a dominating DNA template in a PCR mixture. 1. Universal primers amplifying target and non-target DNA and removal of non-target DNA prior or after PCR with restriction enzymes. 2. Species-specific PCR primers amplifying only target-DNA. 3. Group-specific PCR primers amplifying groups excluding non-target sequence. 4. Blocking non-target amplification, only target sequences amplified using universal PCR primers a) annealing inhibiting blocking primer, b) elongation arrest blocking primer c) annealing inhibiting DPO blocking primer.

Figure 2

Primers. A 203 bp region of Euphausia superba 28S sequence showing the location of the different primers applied in this study.

Figure 3

Sequence similarity tree. Sequence similarity tree of sequences amplified from krill stomachs and related sequences. The krill stomach sequences are named so as the first three letters means sampling date, i.e. M24 = March 24. 2007, S17 and S20 = September 17. and September 20. 2007, St. means Stomach number and the number in parenthesis equals the number of identical sequences of each different sequence found in the different stomachs. The tree is unrooted.