ABSTRACT
Polymerase chain reaction (PCR) technology has advanced during the last decade, becoming the most popular experimental tool in molecular plant sciences. There is a wide variety of PCR applications covering both basic and applied research. For example, PCR-detectable genetic markers, namely SSLP (simple sequence length polymorphism) and AFLP (amplified fragment length polymorphism), are frequently used for genotype mapping of plant populations. In addition, analyzing contamination by genetically modified crops (Germini et al., 2004) and detecting pathogenic bacteria (e.g., Escherichia coli O157; Maurer et al., 1999) are commonly performed using PCR during inspection of food safety. PCR coupled with reverse transcription (RT-PCR) is also a key technology in plant molecular biology, including transcript analysis and other derivative procedures for identifying specific gene expression such as cDNA subtraction and differential display techniques. The PCR procedures can also be applicable in the quantification of target DNA and transcripts in biological samples. Moreover, quantitative PCR (Q-PCR) procedures, because of their extremely high sensitivity and specificity (Gachon et al., 2004; Ingham et al., 2001)
have also become extremely common, replacing conventional blotting technologies such as northern and Southern blotting. For example, the quantification of integration of gene copies into transgenic Arabidopsis genome through Southern blotting requires >200 ng of genomic DNA, whereas Q-PCR procedures require only 10 ng (Honda et al., 2002). Further, quantitative RT-PCR allows accurate and specific determination of target transcripts in the presence of highly homologous subfamilies (Ohno et al., 2004), which is usually difficult using northern blot analysis. In the present chapter, we focus on the application of Q-PCR in the analysis of transgenic plants; for example, in isolation of single-copy transgenic plants using quantitative genomic PCR and quantification of ectopic expression of integrated genes using quantitative RT-PCR.
