PCR bias in ecological analysis: a case study for quantitative Taq nuclease assays in analyses of microbial communities

Abstract

Succession of ecotypes, physiologically diverse strains with negligible rRNA sequence divergence, may explain the dominance of small, red-pigmented (phycoerythrin-rich) cyanobacteria in the autotrophic picoplankton of deep lakes (C. Postius and A. Ernst, Arch. Microbiol. 172:69-75, 1999). In order to test this hypothesis, it is necessary to determine the abundance of specific ecotypes or genotypes in a mixed background of phylogenetically similar organisms. In this study, we examined the performance of Taq nuclease assays (TNAs), PCR-based assays in which the amount of an amplicon is monitored by hydrolysis of a labeled oligonucleotide (TaqMan probe) when hybridized to the amplicon. High accuracy and a 7-order detection range made the real-time TNA superior to the corresponding end point technique. However, in samples containing mixtures of homologous target sequences, quantification can be biased due to limited specificity of PCR primers and probe oligonucleotides and due to accumulation of amplicons that are not detected by the TaqMan probe. A decrease in reaction efficiency, which can be recognized by direct monitoring of amplification, provides experimental evidence for the presence of such a problem and emphasizes the need for real-time technology in quantitative PCR. Use of specific primers and probes and control of amplification efficiency allow correct quantification of target DNA in the presence of an up to 10(4)-fold excess of phylogenetically similar DNA and of an up to 10(7)-fold excess of dissimilar DNA.

FIG. 1

TaqMan probes used in this study. Shown are the positions of reporter (R) and quencher (Q) of probes S8807 (A) and S8807A (B) and alignment with complementary strands of target sequences in Synechococcus (Syn.) sp. strains BO 8807, BO 8808, BO 9404, and BO 8805. Mismatches are in boldface and in italics.

FIG. 2

Ethidium bromide-stained PCR products from end point TNAs. Two-microliter volumes were analyzed in a 1% agarose gel. Lanes: M, λ DNA digested with PstI; 1 to 4, DNAs from Synechococcus sp. strains BO 8807, BO 8808, BO 8805, and BO 9404; 5, A. variabilis strain ATCC 29413; 6, Synechocystis sp. strain BO 8402; 7, Microcystis sp.; 8, E. coli strain K-12; 9, Anabaena sp. strain PCC 7120; 10, no-template control; 11 to 14, Synechococcus spp. Assay mixtures (25 μl) contained 0.625 U of Taq polymerase and primers PITSANF and PITSEND (200 nM each), amplifying the ITS-1 in the ribosomal operon of many cyanobacteria. The assay mixtures analyzed in lanes 1 to 9 contained 10 ng of DNA, 1.5 mM Mg²⁺, and 20 nM probe S8807. The assay mixtures analyzed in lanes 11 to 14 contained 1 ng of DNA, 2.5 mM Mg²⁺, and 50 nM probe S8807A. Templates were amplified in 30 cycles using a two-step PCR program (3 min of annealing and polymerization at 59°C, denaturation at 94°C).

FIG. 3

Melting curves of probes S8807 and S8807A. The melting behavior of two TaqMan probes hybridizing to template DNAs from Synechococcus spp. strains BO 8807 and BO 8808 was analyzed by end point TNA. PCR conditions: 1 ng of template DNA, 50 nM probe (S8807 or S8807A), 2.5 mM Mg²⁺, 0.625 U of Taq polymerase, 50 nM primers PITSANF and PITSEND at annealing-extension temperatures of 56 to 65°C, 200 nM at 66 and 67°C, 400 nM at 68 and 69°C, and 800 nM at 70°C. Cycling conditions: 1.5 min of annealing-polymerization, 30 cycles.

FIG. 4

Quantification of DNA from Synechococcus sp. strain BO 8807 by end point TNA. Twenty-five-microliter Taq nuclease assay mixtures contained 10⁻³ to 10³ ng of DNA, representing approximately 3 × 10² to 3 × 10⁸ genome copies of Synechococcus sp. strain BO 8807; 50 nM primers PITSANF and PITSEND; 50 nM probe S8807A; 2.5 mM Mg²⁺; and 0.625 U of Taq polymerase. PCR conditions: annealing-polymerization at 60°C for 1.5 min, 30 cycles. The insert shows a 1% agarose gel containing 2 μl of each assay mixture per lane. PCR products were stained with ethidium bromide. Lanes M, λ DNA digested with PstI.

FIG. 5

Standard curves obtained by the C_T method in real-time PCR. For TNA, 25-μl assay mixtures contained approximately 10⁰ to 10⁷ (10⁸) copies of genomes, 12.5 μl of TaqMan universal PCR master mix (5 mM [final concentration] Mg²⁺), 300 nM primers, and 50 nM probe. Target DNA, primers (P), and probes (S) in panel A: A. nidulans, P100PA and P3, S100A. Target DNA, primers, and probes in panel B, Synechococcus sp. strain BO 8807, P8807AP and P8807AM, S8807A. The PCR comprised 45 cycles with 1 min at 60°C for annealing-polymerization and 15 s of denaturation at 95°C. Fluorescence threshold (ΔRQ) = 0.04; s = slope. Amplification efficiency was calculated as follows: ɛ_c = 10^−1/s − 1.

FIG. 6

Competitive end point TNA. Assay mixtures contained 1 ng of genomic DNA from Synechococcus sp. strain BO 8807 and 10⁻³ to 10³ ng of DNA from Synechococcus sp. strain BO 8808, A. nidulans, or herring sperm. The PCR assay conditions comprised 30 cycles with annealing and extension at 60°C (1.5 min), 50 nM primers PITSANF and PITSEND was used for amplification of ITS-1, and 50 nM probe S8807A was used to detect strain BO 8807. Controls: Synechococcus sp. strain BO 8807 only.

FIG. 7

For quantitative PCR, a 25-μl TNA mixture contained 1 ng of genomic DNA from Synechococcus sp. strain BO 8807 and 10⁻³ to 10² ng of DNA from A. nidulans (lanes 1 to 6) or 1 ng of DNA from Synechococcus sp. strain BO 8807 and A. nidulans alone (lanes 7 and 8, respectively). The assay conditions are described in the legend to Fig. 6. A 2-μl volume of each assay mixture was analyzed on a 1% agarose gel and stained with ethidium bromide. λ DNA digested with PstI was used as molecular size markers (lanes M). Lane C, no-template control.

FIG. 8

Real-time competitive PCR (A) and corresponding C_T values (B). Assay mixtures contained approximately 10³ copies of Synechococcus sp. strain BO 8807 DNA (control) and approximately 10⁰ to 10⁸ copies of Synechococcus sp. strain BO 8809 as a competitor, 300 nM primers P8807AM and P8807PE, 50 nM probe S8807A, and 12.5 μl of TaqMan universal PCR master mix (5 mM [final concentration] Mg²⁺). The assays were run for 45 cycles with 1 min of annealing and extension at 60°C and 15 s of denaturation at 95°C. The threshold was set to ΔRQ = 0.04. The control was Synechococcus sp. strain BO 8807 only.

FIG. 9

Detection of Synechococcus sp. strain BO 8807 DNA in the presence of similar DNA (7% sequence divergence in the ITS-1) using PCR primer P8807AP. Approximately 10⁰ to 10⁸ copies of Synechococcus sp. strain BO 8809 DNA were added to approximately 10¹ (■), 10² (□), or 10³ (●) genome copies of Synechococcus sp. strain BO 8807. The control was Synechococcus sp. strain BO 8807 only. A 25-μl TNA mixture contained the DNA, 300 nM primers P8807AP and P8807AM, 50 nM probe S8807A, and 12.5 μl of TaqMan universal PCR master mix (5 mM [final concentration] Mg²⁺). The PCR comprised 45 cycles of 1 min of annealing-polymerization at 60°C and 15 s of denaturation at 95°C. The threshold value ΔRQ was 0.04.