PCR Amplification of cDNA Libraries for Cloning and Screening

Screening a cDNA library is a common practice for cloning genes of interest in many laboratories. Success of screening is largely dependent on the abundance of cDNAs representing genes of interest and the availability of suitable probes. The procedure involves several rounds of screening to obtain true-positive clones, which might take a week or longer. Typically, each round of screening requires transfer of the clones to a membrane, denaturation and neutralization of membrane-bound DNA and hybridization with radioactive or nonradioactive probes to obtain positive signals. This procedure is not only laborious and time-consuming but also can give rise to false positives and high backgrounds despite stringent hybridization and washing conditions. Polymerase chain reaction (PCR) has made it possible to screen libraries and clone genes more quickly and easily. By using PCR techniques, rapid screening of genomic libraries has been achieved (2). A nonisotopic screening by PCR also has been used for rapid gene cloning (1). It is also possible to isolate a fulllength cDNA from a cDNA library using a PCR technique (3). To replenish and maintain a library, it must be amplified. Library amplification can pose problems when it is not performed carefully. The ratio of phages and bacterial host cells for a proper multiplicity of infection (MOI) is crucial, as is proper storage after amplification. Otherwise, the library can become biased, underrepresenting or overrepresenting certain genes. To avoid the lengthy procedure of amplification and its potential pitfalls, we directly amplified cDNA inserts in the libraries using PCR and tested usability of the procedure for PCR-based screening or cloning. This simple procedure can be used in amplification of libraries when PCR-based screening or cloning is performed. The basic idea of the procedure stems from reverse transcription (RT)PCR. When certain messages are abundant, more copies of first-strand cDNA are synthesized in an RT reaction, which in turn results in better amplification of a target fragment in the following PCR. Similarly, if either strand of cDNA inserts in a library is linearly amplified, the following PCR will be more effective. The procedure consists of two rounds of amplification. The first round of amplification is a linear pre-amplification of the inserts using a vector primer, and the second round involves amplification of the target fragment using a specific primer and the vector primer. In the first round, a small aliquot of a library is amplified by PCR using M13 forward or reverse primers and relatively low annealing temperatures to obtain good annealing of primers. The amplified DNA can be stored at 4° or -20°C or freeze-thawed many times without affecting the PCR outcome, in contrast to the vulnerable storage and handling conditions of the library. In the present experiment, a lambda ZAP II (Stratagene, La Jolla, CA, USA) flower cDNA library of Arabidopsis thaliana was used. The cDNA library was obtained as an aliquot of the amplified library from Arabidopsis Biological Resource Center (ABRC, Columbus, OH, USA). One microliter of the library (2 × 108 plaque-forming units [pfu]/mL) was needed to amplify 1050 bp A. thaliana ribosomal protein L3 gene (ARP1). One microliter of the phage library stock was diluted in 50 μL distilled water in a 0.5-mL microcentrifuge tube and subsequently freeze-thawed. The tube was then placed in boiling water for 3 min, followed by the addition of PCR mixture (Life Technologies, Gaithersburg, MD, USA), and sterile water was added to a 100-μL final volume. The PCR mixture contained 1 μM M13 forward primer, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.5 mM each dNTP and 10 U of Taq DNA polymerase (Life Technologies). The inserts were amplified by initial denaturation at 94°C for 3 min and 94°C for 30 s, annealing at 39°C for 2 min and extension at 72°C for 3 min for 30 cycles in a thermal cycler (Perkin-Elmer, Norwalk, CT, USA). A final extension of 5 min at 72°C was added. Varied amounts (1, 2, 5 μL) of the amplified library were used as templates in the second-round PCR in conjunction with an M13 forward primer and an ARP1-specific primer, CD1 (5′-GCTGGTATGACTCACAT-3′) (Figure 1, lanes 2–4). The PCR products showed that 1 μL of the amplified cDNAs out of 100 μL of PCR product was sufficient to amplify the specific 1-kb ARP1 fragment (Figure 1, lane 2). The intensity of the band was comparable to that of the product from the direct amplification with 1 μL of library stock without preamplification (Figure 1, lane 5). Because the orientation of cDNA inserts is not known, two separate amplifications might be necessary, one with M13 forward primer, the other with M13 reverse primer. Alternatively, with one amplification reaction, specific sense or antisense primer can be used in combination with appropriate vector primers.