Quantitative Real-Time PCR Assay for Determining Transgene Copy Number in Transformed Plants

Ingham, David J.; Beer, Sandra; Money, Stephanie; Hansen, Geneviève

doi:10.2144/01311rr04

Cited by 219 publications

(154 citation statements)

References 14 publications

(18 reference statements)

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“…Gene duplications were verified by rtPCR by using the 2 -⌬⌬Ct method (37). Primers were designed to amplify 100-bp fragments internal to either lacY or ymfD (a single-copy reference gene) using the SYBR green PCR core reagent kit (PE Biosystems).…”

Section: Methodsmentioning

confidence: 99%

Evolutionary genomics of ecological specialization

Zhong

Khodursky

Dykhuizen

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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We used a combination of genomic techniques to monitor chromosomal evolution across hundreds of generations as Escherichia coli adapted to growth-limiting concentrations of either lactulose, methyl-galactoside, or a 72:28 mixture of the two. DNA microarrays identified 8 unique duplications and 16 unique deletions among 42 evolvants from 23 chemostat experiments. Each mutation was confirmed by sequencing PCR-amplified flanking genomic DNA and, except for one deletion, an insertion sequence was found at the break point. vPCR of insertion sequences identified these same mutations and 16 additional insertions (all confirmed by sequencing). The pattern of genomic evolution is highly reproducible. Statistical analyses show that duplications at lac and mutations in mgl are adaptations specific to lactulose and to methyl-galactoside, respectively. Adaptation to mixed sugars is characterized by similar mutations, but lac duplications and mgl mutations usually arise in different backgrounds, producing ecological specialists for each sugar. This suggests that an antagonistic pleiotropic tradeoff between duplications at lac and mutations in mgl retards the evolution of generalists. Other mutations that repeatedly appear in replicate experiments are adaptations to the chemostat environment and are not specific to one or the other sugar.W e have taken advantage of the repeatability of laboratory adaptation to investigate the roles of insertion sequences (IS) in the evolution of resource specialization.The repeatability of laboratory adaptation is well documented (1-11) and is perhaps attributable to using large populations with simple ecologies. Large populations reduce the role of chance in evolution: random genetic drift is minimized, whereas mutation, always erratic in small populations, produces numerous allelic variants with each generation. In simple constant environments, selection becomes focused with great intensity on a few key genes. This makes experimental evolution far more reproducible than typically envisioned for small populations inhabiting complex natural environments (12). Reproducibility makes possible rigorous tests of adaptive hypotheses. For example, Cooper et al. (13) used DNA expression macroarrays to identify parallel changes in the expression profiles of two evolvants. Guided by the observation that many of the genes with changed expression belong to the ppGpp and CRP regulons, they identified a mutation in spoT in one population that, when reintroduced into the ancestral genetic background, increased fitness as well as reproducing many of the changes in expression.IS elements are small (1-to 2-kb) segments of DNA capable of transposing within and between prokaryotic replicons (14). A major source of insertional mutations and chromosomal rearrangements (15), their evolutionary significance is a subject of perennial interest (16)(17)(18)(19)(20). Patterns of sequence polymorphism among natural isolates of Escherichia coli suggest a brisk turnover of elements (21), with both the numbers and location...

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Section: Methodsmentioning

confidence: 99%

Evolutionary genomics of ecological specialization

Zhong

Khodursky

Dykhuizen

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…The number of transgene copies has traditionally been estimated by Southern analysis, in which a blot of digested genomic DNA is hybridized with a radioactive DNA probe corresponding to the transgene to produce an informative band pattern. The drawbacks of this method are laborious and time-consuming, especially when a large number of samples need to be estimated (Ingham et al, 2001). It requires a great amount of DNA and hazardous radio-isotopes (Mason et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

“…Recently, the development of quantitative Polymerase Chain Reaction (qPCR) methods for determining the transgene copy number has overcome the limitations of Southern analysis (Ingham et al, 2001;Mason et al, 2002). To date, qPCR technology has been applied to analyze copy number of transgenic plants, including soybean and peanut (Schmidt and Parrott, 2001), tomato (Mason et al, 2002), maize (Ingham et al, 2001;Shou et al, 2004;Song et al, 2002), rapeseed (Weng et al, 2004), wheat (Doshi et al, 2007;Li et al, 2004), rice (Jiang et al, 2009;Yang et al, 2005), tobacco (Subr et al, 2006), cotton (Yi et al, 2008), citrus (Omar et al, 2008), grape (Costa et al, 2009) and cassava (Beltran et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Use of Quantitative Polymerase Chain Reaction for Determining Copy Numbers of Transgenes in <i>Lesquerella fendleri</i>

Chen¹

2010

American Journal of Agricultural and Biological Sciences

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Problem statement:In transgenic plants, the number of transgene copies could greatly influence the level of expression and genetic stability of the target gene, thus it is important to develop an efficient method for accurate estimation of transgene copies. The quantitative Polymerase Chain Reaction (qPCR) technique is becoming more efficient nowadays to determine copy numbers of transgenes in transgenic plants, being used here, for the first time in quantifying copy numbers of transgenes in Lesquerella fendleri. Approach: The system utilized a known one copy gene, LfKCS4/5, from L. fendleri as an endogenous calibrator and the threshold Crossing point (Ct) measured by Applied Biosystem 7500 system to calculate the copy numbers of transgenes in primary transgenic lines (T0 generation). Results: The qPCR condition was optimized and each primer set had a PCR efficiency of 0.99 or 1.01. Our data demonstrated unambiguous 2-fold discrimination of the copy number of ß-glucuronidase gene (gusA) and hygromycine phosphotransferase II (hptII) genes in 12 T0 lines. Most of the lines contained one or two copies of each gene. Eight out of 12 samples (66.7%) showed more copies of gusA gene than that of hptII gene, suggesting rearrangements of the Transferred (T)-DNA. Possible modifications of the T-DNA cassette in L. fendleri are discussed based on main models of T-DNA integration in the plant genome. Conclusion: The qPCR described in this study is an efficient method and it is particularly useful in identification and selection of transgenic plants with desirable copy numbers at early stage.

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“…The transgene used was the BAR gene (BAR F: 5'ACA GCG ACC ACG CTC TTG A-R3' / BAR R: 5'GCT CTA CAC CCA CCT GCT GAA3') and the reference was the adh1 gene (Adh1F: 5' GTA ACA TGC TCC AGC ACT GCT ATT3' / Adh1R: 5'TCG TAT GAT GTG TTC AGC CAG ACT 3') (Ingham et al 2001). The qPCR reactions were carried out in 10 μl reaction mixtures containing 50 ng template DNA, Fast SYBR Green Master Mix 1X (Applied Biosystems, Foster City, CA, USA), and 0.5 μM primers.…”

Section: Molecular Analyses Of Transgenic Eventsmentioning

confidence: 99%