Two-fold differences are the detection limit for determining transgene copy numbers in plants by real-time PCR

Abstract

Background: After transformation, plants that are homozygous and contain one copy of the transgene are typically selected for further study. If real-time PCR is to be used to determine copy number and zygosity, it must be able to distinguish hemizygous from homozygous and one-copy from two-copy plants. That is, it must be able to detect two-fold differences.

Results: When transgenic Nicotiana attenuata plants which had been previously determined by Southern analysis to contain one or two copies of the transgene, were analyzed by real-time PCR (2-delta delta Ct method), the method failed to confirm the results from the Southern analysis. In a second data set we analyzed offspring of a hemizygous one-copy plant, which were expected to segregate into three groups of offspring in a 1:2:1 ratio: no transgene, hemizygous, homozygous. Because it was not possible to distinguish homozygous from hemizygous plants with real-time PCR, we could not verify this segregation ratio.

Conclusions: Detection of two-fold differences by real-time PCR is essential if this procedure is to be used for the characterization of transgenic plants. However, given the high variability between replicates, a detection of two-fold differences is in many cases not possible; in such cases Southern analysis is the more reliable procedure.

Figure 1

Calculation of efficiency A) ΔCt (ampNAT1-ampGSP1) for as-hpl plant A422-4-1 over a 1:5 dilution series, ampNAT1 and ampGSP1 are amplified in the same well (multiplex) with conventional TaqMan^® probes, DNA extracted with a miniprep (Ariel) method. B) Ct of ampNAT1 and ampGSP1 (same PCR as A) for calculation of efficiency. C) Ct of ampNOT1 for calculation of efficiency for as-hpl plant A434-1-12 over a 1:2 dilution series, amplification as singleplex reaction with Minor-Groove-Binder probe.

Figure 2

Southern analysis of chromosomal DNA of N. attenuata transformed with binary vectors containing antisense-cDNA for N. attenuata hydroperoxide lyase (hpl), allene oxide synthase (aos), lipoxygenase (lox) and digested with SspI. A) T-DNA of transformation vector pNATHPL: P_CaMV/T_CaMV, CaMV 35S promoter/terminator: P_NOS/T_NOS NOS promoter/terminator, as-hpl, antisense hpl; sat-1, nourseothricin resistance gene; LB/RB, left/right border of T-DNA; SspI, recognition site for restriction enzyme; s, amplicon ampNAT1 (conventional TaqMan^® probe); n, amplicon ampNOT2 (Minor-Groove-Binder probe); Southern probe, 260 bp radioactive PCR-labeled probe for Southern blot; vectors pNATAOS, pNATLOX have the same organisation as pNATHPL except that that as-hpl is replaced by as-aos or as-lox, respectively. B) Southern blots of progeny of the hemizygous line A443-1 transformed with pNATHPL, 21 plants were examined (plants 1–24, except 10 and 17; *, DNA of plant Nr. 16 was blotted, but not included into the segregation ratio, because of low concentration); 5 plants without T-DNA, 16 plants with T-DNA: result corresponds to the sensitive/resistant ratio of 1:3 in segregration analysis of a hemizygous line; P (plasmid), 2 ng pNATHPL; WT, wild-type DNA. C) Southern blots for progeny of homozygous lines (2 from each line) transformed with pNATHPL, pNATAOS or pNATLOX; 1 as-hpl line and 2 as-lox lines show two bands on the blot, indicating two insertion sites for the T-DNA; P (plasmid), 2 ng pNATHPL; WT, wild-type DNA.