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

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.

Vertical distribution and diel vertical migration of krill beneath snow-covered ice and in ice-free waters.

A bottom mounted upward looking Simrad EK60 120-kHz echo sounder was used to study scattering layers (SLs) and individuals of the krill Meganyctiphanes norvegica. The mooring was situated at 150-m depth in the Oslofjord, connected with an onshore cable for power and transmission of digitized data. Records spanned 5 months from late autumn to spring. A current meter and CTD was associated with the acoustic mooring and a shore-based webcam monitored ice conditions in the fjord. The continuous measurements were supplemented with intermittent krill sampling campaigns and their physical and biological environment. The krill carried out diel vertical migration (DVM) throughout the winter, regardless of the distribution of potential prey. The fjord froze over in mid-winter and the daytime distribution of a mid-water SL of krill immediately became shallower associated with snow fall after freezing, likely related to reduction of light intensities. Still, a fraction of the population always descended all the way to the bottom, so that the krill population by day seemed to inhabit waters with light levels spanning up to six orders of magnitude. Deep-living krill ascended in synchrony with the rest of the population in the afternoon, but individuals consistently reappeared in near-bottom waters already <1 h after the ascent. Thereafter, the krill appeared to undertake asynchronous migrations, with some krill always being present in near-bottom waters even though the entire population appeared to undertake DVM.

Application of blocking oligonucleotides to improve signal-to-noise ratio in a PCR.

"Universal" or group-specific PCR primers have a tendency to predominately hybridise with the common sequences in samples with mixed templates. The result is that the rarer sequences are seldom retrieved by cloning or sequencing. The use of a blocking oligonucleotide (oligo) designed to specifically prevent amplification of dominant or unwanted DNA templates is an easy way to improve the amplification of rarer sequences. Here, we describe the different types of blocking principles and the different types of blocking oligos and give guidelines and examples of their application.

Vertical migration, feeding and colouration in the mesopelagic shrimp Sergestes arcticus.

Intraspecific variation in vertical distribution, timing of vertical migration, and colouration of the mesopelagic shrimp Sergestes arcticus were studied in the >400 m deep part of Masfjorden, Norway. Very few individuals were caught in the upper strata during daytime, and larger individuals occurred deeper during the day than smaller ones. Vertical migration was prominent and no overall trend of increasing length with depth was found at night. Small individuals arrived in the upper layers earlier than larger ones. Animal colouration assessed by digital photography revealed significant variance in individual redness. Depth of capture was the most important factor explaining colouration, with increasing degree of redness with depth. Assessing the gut fullness of the transparent shrimps provided a rapid way of estimating feeding activity and showed that feeding took place mainly at night.