Use of real-time PCR for determining copy number and zygosity in transgenic plants

Abstract

This review examines how real-time PCR can be used to determine copy number and zygosity in transgenic plants. Distinguishing between plants that harbor one and two copies of a transgene or are hemizygous and homozygous requires the ability to routinely distinguish twofold differences, a detection difference which approaches the resolution of PCR-based quantification methods. After explaining the basic principles, especially the threshold cycle (Ct value) as the basic measuring unit in real-time PCR, we introduce three quantitation methods currently in use. While the absolute and relative standard curve approaches are qualitative methods that distinguish high-copy from low-copy transformants, the comparative (2(-DeltaDeltaCt)) method with double-dye oligonucleotides (TaqMan probes) is able to detect twofold differences. In order to obtain reliable results, Ct values for an amplicon should be below 25 and the standard deviation below 0.3. Although real-time PCR can deliver exact copy number determinations, the procedure is not fail-safe. Therefore, real-time PCR should to be viewed as complementary to--rather than as a replacement of--other methods such as Southern analysis, but it is particularly useful as a preliminary screening tool for estimating copy numbers of a large number of transformants.

Effects of a neuregulin 1 variant on conversion to schizophrenia and schizophreniform disorder in people at high risk for psychosis

“…We adapted the comparative method with double-dye oligonucleotides (TaqMan probes, Applied Biosystems, Foster City, CA, USA) of Bubner and Baldwin. 8 We found no evidence for hemizygosity in our sample.…”

Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice

“…The wild type (DJ or HJ) was used as the recipient for Agrobacterium-mediated transformation as described previously to generate the transgenic rice (Hiei et al, 1994). The T-DNA copy numbers of relative transgenic rice plants were confirmed by RT-qPCR described previously (Bubner and Baldwin, 2004;Yang et al, 2005), and the results are presented in the Supplemental Table 3. Homozygous T3 or T4 plants were taken for the following field test.…”

MOLECULAR GENOTYPING OF CHICKEN (Gallus gallus L.) FEATHERING GENES IN CONNECTION WITH SEPARATION BY SEX

“…Southern hybridization is laborious, takes a lot of time and requires a variety of equipment. When quantitative PCR (PCR-RT) is used to solve the task, the results may be inadequate if the necessary conditions are not met during the test and subsequent statistical processing of the data [15,18,19]. However, with appropriate execution, this method is highly technological and allows for a short time to accumulate a large array of information.…”

“…The determination of genotypes КК and Kk by PCR-RT is reduced to the problem of discrimination of two copies of a gene in the genome from one [8,15,16]. It can be solved by quantitative PCR [8,17], as well as by hybridization of nucleic acids [15,18,19].…”

“…It can be solved by quantitative PCR [8,17], as well as by hybridization of nucleic acids [15,18,19]. Each of these approaches has advantages and disadvantages.…”