( annealing temperature (Fig. 2a). A PC...)Optimizing the Direct Amplification of Missing cDNA 5' Ends,,, Using the Eppendorf Mastercycler gradient

annealing temperature (Fig. 2a). A PCR blot analysis using an isoamylase-specific oligonucleotide probe was carried out to determine which of these products are specific, i.e. which contain an isoamylase sequence (Fig. 2b). Low annealing temperatures lead to mixtures of reaction products which do not hybridize with the isoamylase probe, whereas increasingly specific products are obtained at higher annealing temperatures.

Fig. 2 A. Agarose gel of the PCR products (ethidium bromide). The individual reactions are labeled with the corresponding annealing temperature. The theoretically optimal annealing temperature in this PCR reaction would have been 61C. The following PCR program was carried out on Mastercycler gradient: 4 minutes, 95C, (45 sec., 95C, 45 sec., 62 10C, 90 sec. 72C) x 35, 10 min 72C.

Fig. 2 B. DNA blot analysis of PCR products from Fig. 2a with an oligonucleotide probe of the 5' end of the isoamylase cDNA (see Fig. 1).

The PCR products obtained at an annealing temperature of 72C were cloned without further purification (TA Cloning Kit, Invitrogen). Ten clones selected at random with inserts were sequenced and tested for the presence of the isoamylase sequence. This procedure identified one PCR product with overlapping isoamylase sequence and additional 5' sequence as shown in Fig. 3.

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