Detection of T-DNA by Polymerase Chain Reaction PCR

Many suppliers of Taq polymerase supply a concentrated buffer solution to which nucleotides, primers and DNA and enzyme need only be added. The final buffer typically contains 10 mM Tris HCl (pH 8.3 at 25 °C) 1.5 mM 

Oligonucleotides should be chosen such that the last two to three nucleotides at their 3 ends are not complementary and ideally should have a similar GC%. The formula Tm = 2(A + T) + 4(G + C) is a reasonably accurate estimate of Tm for 20-21-mers and the formula Tm = 69.3 + 0.41(G + C)% — 650/N, where N = length of oligonucleotide, appears to give a reasonable estimate of Tm for oligos of 20-30 nucleotides in length (Hamill et al. 1991). 

Inverse PCR can, in theory, be used to estimate the number of insertions of T-DNA into a plant genome. A recent report by Does et al. (1991) described the use of this approach to detect the presence and number of T- 

DNA/plant junction sites in transformed tobacco plants. Although described for binary vector-mediated transformation, the method could be used to determine the number of integrations of Ri T-DNA into transformed root lines. 

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