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Optimizing the Direct Amplification of Missing cDNA 5' Ends,,, Using the Eppendorf Mastercycler gradient

>Isoamylase cDNA in ZAPII



Fig. 1 Diagram of the isoamylase cDNA in the ?ZAPII vector (Stratagene). The white rectangle represents the existing cDNA clone. The missing 5' region has a gray background. VP = vector primer (30-mer, Tm = 59C, 5'-GCT CAC TCA TTA GGC ACC CCA GGC TTT ACA), GP = gene-specific primer (30-mer, Tm = 66C, 5'-CAC CGG CTC GTC CTC CTC CCC CTC ATC CTC), S = isoamylase-specific probe. Method and results

The 5' end of a cDNA which encodes an isoamylase involved in starch metabolism, was isolated from wheat. A ?ZAPII cDNA library from wheat caryopses was used (approx. 2.5 x 107 pfu per reaction). In addition, the reaction mixture contained primers (0.3 mM each), nucleotides (0.2 mM each), 1.5 mM Mg2+ and Taq Polymerase (0.5 units). Aliquots (50 l) of a homogeneous reaction mix were used in a PCR-cycle with annealing temperatures ranging from 54C to 72C using the Mastercycler gradient. In order to identify specific products, the PCR products were separated (Fig. 2a) and hybridized against a DIG-labeled oligonucleotide probe of the 5' end of the original cDNA (Fig. 2b).

The annealing temperature for the PCR reaction described here was calculated to be 61C (PrimerSelect Software, DNA-Star Sequence Analysis Package). Both lower and much higher annealing temperatures were tested in order to find optimal reaction conditions. The size and complexity of the PCR product or mixture of products obtained was strongly dependant on the annealing temperature (Fig. 2a). A PCR blot analysis using an isoamylase-specific oligonucleotide pr
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