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qPCR Methods for the Quantification of Transgene Insert Copy Number and Zygosity Using the Comparative Ct Method in Transgenic Sorghum bicolor L. Moench

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Accelerated Breeding of Cereal Crops

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Abstract

The development and analysis of transgenic plants is a costly and time-consuming process. A major focus of this analysis is a determination of the structure and number of T-DNA insertions in each transgenic event. Traditionally, this has been accomplished via a combination of standard end-point PCR and laborious Southern blotting techniques. Whereas end-point PCR can generally only yield presence/absence information regarding transgenes, Southern blot analysis can yield highly accurate information regarding the structure of transgene insertions as well as the number of insertions. While this process is reliable and routine, it requires relatively large amounts of high-quality high-molecular-weight plant genomic DNA (gDNA), several days to obtain a result, and sometimes multiple probes overlapping the sequence(s) of interest. In contrast, the quantitative real-time polymerase chain reaction (qPCR) requires relatively little gDNA, which does not need to be high-molecular weight; is easy to set up, requiring less labor than blotting techniques; and can deliver a result inside of 1 h. Herein, we describe a simple and rapid method using a fluorogenic 5′ nuclease “TaqMan®” assay in conjunction with the comparative (2−ΔΔCt) quantification method to determine transgene copy number in transgenic Sorghum bicolor. The same assay can be used to determine the zygosity of homozygous and hemizygous transgenic segregants in a T1 population. A method to extract large amounts of good-quality high-molecular-weight gDNA suitable for Southern blotting is also provided for additional validation of the qPCR assay.

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Correspondence to Albert P. Kausch .

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Hague, J., Nelson, K., Yonchak, A., Kausch, A.P. (2022). qPCR Methods for the Quantification of Transgene Insert Copy Number and Zygosity Using the Comparative Ct Method in Transgenic Sorghum bicolor L. Moench. In: Bilichak, A., Laurie, J.D. (eds) Accelerated Breeding of Cereal Crops. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1526-3_13

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  • DOI: https://doi.org/10.1007/978-1-0716-1526-3_13

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1525-6

  • Online ISBN: 978-1-0716-1526-3

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