Several real-time PCR assays have also been developed to target different Alu repetitive sequences for highly sensitive nuDNA quantification. Nicklas and Buel (2003) have described a SYBR Green real-time PCR assay using specific primers to target a 124 bp Alu sequence with primate specificity and 1 pg sensitivity. Walker et al. (2003) presented two alternative SYBR Green real-time PCR designs to target inter-Alu (including a complex pool of sequences of different sizes) and intra-Alu (200-226 bp) sequences, respectively. Inter-Alu assay was found to be not completely human-specific while intra-Alu PCR was demonstrated to be a specific and sensitive method for human nuDNA quantification in the range of 10 ng to 1 pg.
The main advantages of SYBR Green real-time PCR designs to target Alu assays are simplicity and high sensitivity. The disadvantages are the necessity of optimization to avoid the possibility of unspecific PCR artefacts and the possible inaccuracy in the DNA quantification as a consequence of a hypothetical individual variation in the copy number of Alu sequences.
Walker et al. (2005) have described a TaqMan-MGB triplex real-time PCR assay for simultaneous quantitation of human nuDNA (based on an intra-Alu sequence design), mtDNA and Y chromosome DNA (see Table 4.1, Autosomal DNA, Y chromosome and mtDNA quantification by triplex PCR).
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