Kinetic bias in estimates of coastal picoplankton community structure obtained by measurements of small-subunit rRNA gene PCR amplicon length heterogeneity

Abstract

Marine bacterioplankton diversity was examined by quantifying natural length variation in the 5' domain of small-subunit (SSU) rRNA genes (rDNA) amplified by PCR from a DNA sample from the Oregon coast. This new technique, length heterogeneity analysis by PCR (LH-PCR), determines the relative proportions of amplicons originating from different organisms by measuring the fluorescence emission of a labeled primer used in the amplification reaction. Relationships between the sizes of amplicons and gene phylogeny were predicted by an analysis of 366 SSU rDNA sequences from cultivated marine bacteria and from bacterial genes cloned directly from environmental samples. LH-PCR was used to compare the distribution of bacterioplankton SSU rDNAs from a coastal water sample with that of an SSU rDNA clone library prepared from the same sample and also to examine the distribution of genes in the PCR products from which the clone library was prepared. The analysis revealed that the relative frequencies of genes amplified from natural communities are highly reproducible for replicate sets of PCRs but that a bias possibly caused by the reannealing kinetics of product molecules can skew gene frequencies when PCR product concentrations exceed threshold values.

FIG. 1

Electropherogram of DNA fragments amplified by PCR with primer set A from genomic DNA isolated from seawater subsample 2. The letters A to W correspond to the peaks detected by the Genescan 2.1.2 software in at least one of triplicate reactions. The x axis represents the size of domains in base pairs estimated by comparison to the size standard GS2500 (Applied Biosystems Inc.). The y axis represents relative fluorescence units.

FIG. 2

Comparison between LH-PCR and SSU rDNA clone library. The figure shows the percentage of integrated fluorescence of each individual domain A produced by the optimized LH-PCR (final product concentration, <1.5 nM) from genomic DNA isolated from subsample 2 (A) or nearly full-length SSU rDNA PCR amplicons used to construct the clone library (B). The x axis represents the size of domains in base pairs, estimated by comparison to the size standard GS2500 (Applied Biosystems Inc.). The relative abundance of clones recovered in the OCS clone library classified by the length of domain A is shown in panel C. Error bars each represent one standard deviation from the mean of triplicate reactions.

FIG. 3

An example of PCR bias fitting the kinetic model, with PCR amplicons obtained from natural community DNA. The molar ratio of the dominant fragment (317 bp) to total products is plotted as a function of the final product concentration. Primer set A was used for the amplification from environmental DNA subsamples 1 (solid circles) and 2 (open circles).