Single-Stranded DNA Ligation by T4 RNA Ligase for PCR Cloning of 5′-Noncoding Fragments and Coding Sequence of a Specific Gene

Construction and screening of DNA libraries for genes of interest represent the standard but tedious cloning procedures (1). The advent of PCR technology has greatly enhanced the efficiency of traditional gene isolation and cloning techniques. Various strategies have been developed for PCR amplification of an unknown DNA fragment that flanks one end of a known sequence which include inverse PCR (2), panhandle PCR (3,4), vectorette PCR (5,6), ligation PCR (7,8) and capture (oligo-cassette) PCR (9,10). However, the procedures involved in these techniques are generally time-consuming, high-cost, and suffer from technical difficulties such as inefficient or low-specific PCR amplification. We have devised a simple method for cloning 5′-noncoding regions as well as coding sequences of a gene in a straightforward manner without going through library construction and screening. This technique has been used to isolate a phenylalanine ammonia-lyase (PAL) gene including its 5′-noncoding region from loblolly pine (Pinus taeda L.). In our method, a gene-specific primer was first extended by PCR using genomic DNA as a template and single-stranded DNA extension products were ligated to an arbitrary oligonucleotide with T4 RNA ligase to create templates with 5′and 3′-ends of known sequences. These templates were then re-amplified to make double-stranded DNAs for further cloning and sequencing (Fig. 1). Using this simple technique, ≤2.0 kb 5′-noncoding fragments for PAL gene have been cloned. Genomic DNA of loblolly pine was isolated according to a modified CTAB method (11). Based on the sequence of our partial PAL cDNA clone, a primer (primer A in Fig. 1), 5′-CAGTTCAGTGGATCGCTGCC located near the 5′-end coding region of PAL cDNA clone, was synthesized (Integrated DNA Technologies). The pine genomic DNA was used as a template for extending the PAL gene-specific primer. The PCR was carried out in 100 μl reaction mixture containing 200 ng genomic DNA, 0.2 μM primer A, 200 μM of each dNTP in Taq DNA polymerase buffer and 2.5 U Taq DNA polymerase (Fisher Scientific) with a Perkin-Elmer DNA thermal cycler 480. The reaction mixture was first denatured at 94 C for 3 min, followed by 40 cycles of denaturing at 94 C (1 min), annealing at 60 C (45 s) and extension at 72 C (2 min). After passing through a PCR select-III column (5 Prime–3 Prime, Inc) to remove primers and polymerase, the primer extension products were precipitated with ethanol. The precipitate was dissolved in distilled water and mixed with T4 RNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 10 mM DTT, 1 mM ATP and 60 μg/ml BSA), 25 nM of 5′-phosphorylated oligodeoxyribonucleotide B (oligo B in Fig. 1) and 10 U T4 RNA ligase (Boehringer Mannheim) in a final volume of 20 μl. The oligo B, 5′-AGGGTGCCAACCTCTTCAAG, is an arbitrary 20mer. The mixture was incubated at 22 C overnight. The following PCR was carried out in 50 μl reaction mixture containing 1 μl ligation mixture, 200 μM of each dNTP, 0.2 μM of primers A and C in Taq DNA polymerase buffer and 2.5 U Taq DNA polymerase. Primer C, 5′-CTTGAAGAGGTTGGCACCCT, is complementary to oligo B. PCR was carried out at 94 C for 1 min, 58 C for 45 s and 72 C for 2 min for 30 cycles, followed by a final extension at 72 C for 10 min. Ten microliters of the PCR products were taken for agarose gel electrophoresis and Southern blot hybridization to confirm the identity of the PCR products (Fig. 2). Then the PCR products (1–2 μl) were used directly for ligation to vector pCR II, using TA cloning kit (Invitrogen) according to manufacturer’s protocol. Plasmid clones containing recombinant DNAs were then analyzed by restriction enzyme digestion, gel electrophoresis, and sequencing. With this method, we have isolated a pine PAL gene with the complete coding region and ∼2 kb of the 5′-noncoding region. This simple method uses T4 RNA ligase to attach an arbitrary oligomer to the extended target DNA fragments for a subsequent PCR amplification to clone the target DNA for its 5 -noncoding fragments and coding sequence (Fig. 1). Using an oligonucleotide from the 3 -region of a gene as a primer (Primer A in Fig. 1) and genomic DNA as a template, a mixture of single-stranded DNA primer extension products can be readily obtained. Since many genes, especially those in animal genomes, contain introns, the design of primer A should avoid intron junctions to ensure the primer specificity. Thus, our technique should also be applicable to cloning those genes with numerous or extremely long introns in their coding or 5 -noncoding regions. In this case, it is advisable that several PCR amplification and T4 RNA ligase-driven ligation should be carried out in a walking fashion so that overlapping fragments can be obtained to cover the whole region. The T4 RNA ligase-based cloning method was originally designed by Edwards et al. (12) for constructing cDNA libraries and adapted by others (13) for cloning cDNAs. However, it has not been applied to genomic DNAs. T4 RNA ligase has low substrate specificity toward DNAs as compared with its affinity to RNA molecules. When coupled with highly sensitive PCR amplifica-