In many research projects, cDNA isolation forms the basis for detailed analyses at the molecular level. However, when cDNA libraries are screened using conventional plaque hybridization techniques, cDNA clones with an incomplete 5' end are frequently obtained. Since a complete cDNA, or at least a complete open reading frame, is required for many applications (such as expression of functional proteins or sequence-based prediction of sub-cellular location), the missing 5' sequence has to be obtained at a later point in time. However, isolation of full-length cDNAs by plaque screening is extremely labor-intensive when the sought-after cDNA is present at low abundance in a given library.Direct amplification
One alternative is direct PCR amplification of the missing sequence using the cDNA library as template [1,2]. In this case, a vector primer (VP) and a gene-specific primer (GP) are created as shown in the scheme in Fig. 1. As all cDNA clones in the library act as a template for the vector primer, nonspecific products are formed very easily in this PCR reaction. It is therefore important to use highly-specific primers and to ensure that reaction conditions are as stringent as possible, with one crucial factor being the setting of the annealing temperature. An example of this strategy is given below.
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.
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 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.
Fig. 3 A. Diagram of the isoamylase cDNA clone. The PCR-amplified 5' end consists of an overlapping sequence and an additional 5' area which completes the open reading frame. B. At the 5' end, the GC content of the isoamylase sequence is up to 80%, whereas the average GC content of the overall sequence is merely 52%.Discussion
With the aid of a simple PCR reaction, it was possible to amplify the 5' end of a cDNA coding for isoamylase directly from a ?ZAPII library. Conventional plaque screening of the same cDNA library produced several short isoamylase cDNAs, but no clone with a complete coding region. This finding may be attributable to the extremely high GC content of the 5' region of the isoamylase cDNA (Fig. 3b). In regions with high GC content, RNA is able to form stable secondary structures, which abort the cDNA synthesis and lead to shorter cDNA clones. Similarly, single-stranded DNA templates in a PCR reaction can form secondary structures that may interfere with amplification [3,4]. The higher the annealing temperature selected for the PCR reaction, the more likely secondary structures will be suppressed. This may explain why annealing temperatures of 69C or even 72C lead to more specific product than a temperature of 63C, although 63C is much closer to the theoretically optimal temperature of 61C. Determining a suitable stringent annealing temperature is crucial for obtaining specific products in the PCR. In this case, temperature gradients such as those on Mastercycler gradient enable systematic, time-saving experiments.Bibliography
Ohara, O., Dorit, R., and Gilbert, W. (1989) One-sided polymerase chain
reaction: the amplification of cDNA. Proc. Natl. Acad. USA 86, 5673-5677.
Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. USA 85, 998-1002.
Moreau, A., Duez, C., and Dusart, J. (1994) Improvement of GC-rich template amplification by inverse PCR. BioTechniques 17, 233-234.
Schuchard, M., Sarkar, G., Ruesink, T., and Spelsberg, T. C. (1993) Two-step hot PCR amplification of GC-rich avian c-myc sequences. BioTechniques 14, 390-394.