Torres Anthony, Gaudino Reginald
Cannabis Research Institute, Discovery Partners Institute, University of Illinois System, Chicago, IL, USA.
Front Range Biosciences, Lafayette, CO, USA.
Methods Mol Biol. 2025;2943:103-115. doi: 10.1007/978-1-0716-4642-7_9.
This chapter presents a protocol for probe-based quantitative polymerase chain reaction (qPCR) genotyping, focusing on male-specific genotype identification in plant samples. DNA is isolated from raw tissue or FTA cards and subjected to a preamplification enrichment reaction. The diluted product is then used as input for qPCR detection, employing specific probes to identify the presence or absence of the target genotype. This robust and high-throughput method offers increased specificity for genotyping various sample types, including crude nucleic acid extracts. An advantage of qPCR is that genotyping can be performed on various sample types, including from crudely prepared nucleic acid extracts used as input with inhibitor-tolerant polymerases. In this workflow, we describe an extraction and probe-based qPCR method used to detect male-specific genotype identification in plant sample DNA extracted from raw tissue or FTA imprinted cards.
本章介绍了一种基于探针的定量聚合酶链反应（qPCR）基因分型方案，重点是植物样本中雄性特异性基因型的鉴定。从原始组织或FTA卡中分离DNA，并进行预扩增富集反应。然后将稀释后的产物用作qPCR检测的输入，使用特异性探针来鉴定目标基因型的存在与否。这种强大的高通量方法为包括粗核酸提取物在内的各种样本类型的基因分型提供了更高的特异性。qPCR的一个优点是可以对各种样本类型进行基因分型，包括用作输入的粗制核酸提取物，这些提取物可与耐抑制剂的聚合酶一起使用。在本工作流程中，我们描述了一种提取和基于探针的qPCR方法，用于检测从原始组织或FTA印记卡中提取的植物样本DNA中的雄性特异性基因型鉴定。
