Screening cloned PCR fragments by restriction endonuclease finger-printing to obtain wild-type sequences.

A major application of polymerase chain reaction (PCR) is the cloning of DNA fragments with known sequences, e.g., full-length cDNAs and genomic fragments. Although the introduction of the long PCR procedure (2) has facilitated this PCR-based cloning, its usefulness is limited by the high rates of faulty nucleotide incorporation that is seen in all heat-stable DNA polymerases (6). Although several high-fidelity, heat-stable DNA polymerases are commercially available, they sometimes fail to amplify the target sequences, and their error rates are still considerable when amplifying fragments longer than 1 kb (6). After cloning PCR-amplified fragments, screening for clones with the wild-type sequence is mandatory. Restriction endonuclease fingerprinting (REF) (7) is a variant of the single-strand conformation polymorphism (SSCP) method (9) and was reported as being suitable for the detection of mutations in fragments as long as 1 kb (7). We have modified this REF for the screening of wild-type cloned PCR fragments. Using this method, we successfully obtained wild-type cDNAs close to 2 kb in length. Figure 1 shows the protocol schematically. The actual procedure we used when cloning the full-length, wild-type human Smad4 cDNA (1.8 kb) (4) into the expression vector pcDNA1.1/Amp (Invitrogen, Carlsbad, CA, USA) is detailed below. The full-length Smad4 cDNA was PCR-amplified from reverse-transcribed human placental mRNA using the following primers: P1: 5′-TGAATTCTTTTCCTTGCAACGTTAGCTGTTG-3′ and P2: 5′-CGAGTCTCCAACGGTAAAAGACCTCAGTC-3′. P1 has an EcoRI site, and P2 has an XhoI site; each is tagged at the 5′ end. Four different heat-stable DNA polymerases were tested, and only the rTth DNA Polymerase, XL (Perkin-Elmer, Norwalk, CT, USA) gave good amplification. The PCR cocktail contained 1× XL Buffer II (provided with the enzyme from PerkinElmer), 1.1 mM Mg(OAc)2, 200 μM dNTPs and 300 nM each of P1 and P2. Two units of the rTth DNA polymerase, XL were added after the reactions were heated to 94°C just before PCR cycling (this “hot start” is as recommended by the manufacturer). Thirty PCR cycles were performed as follows: 94°C for 40 s, 55°C for 30 s and 68°C for 3 min. The amplified fragment was then purified using the Wizard PCR Preps DNA Purification System (Promega, Madison, WI, USA), digested with EcoRI and XhoI, ligated into EcoRI-XhoI-digested