Use of DHPLC for rapid screening of recombinant clones.

Denaturing HPLC (DHPLC) is a relatively novel technique that has been mostly applied to molecular detection of pathological gene alterations (1,2). The method relies on the identification of heteroduplex molecules, due to mismatches between wild-type and mutant alleles, that are visualized in the form of altered chromatograms. Other nonconventional applications of DHPLC have also been reported, including quantification of mitochondrial heteroplasmy (3) and somatic mosaicism (4), gene mapping (5), and bacterial species identification (6). More recently, DHPLC reliability in mutation detection was validated by two different studies, based on PCR-mediated mutagenesis (7,8). In these two reports, several mutant bacterial clones were cloned, sequenced, and then confirmed by DHPLC for the presence of genomic alterations in MET and p53 genes, respectively. Here we describe a different application of DHPLC that speeds up the screening step of recombinant clones after site-directed mutagenesis (SDM), by avoiding needless sequencing of non-mutant clones. SDM is a versatile molecular tool for the introduction of specific mutations into target DNA to study gene expression and functional significance of genomic alterations. Several applications of the SDM strategy showed a variable mutagenesis efficiency of 50%–90% (9). Selection of mutant recombinant clones by colony hybridization or sequencing analysis represents the most tedious, time-consuming, and expensive step of the entire procedure. To overcome this cumbersome process, several expedients have been conceived, such as the insertion of silent restriction enzyme cleavage site in mutagenic primers (10). However, even this improvement still requires plasmid DNA isolation/or PCR amplification from several colonies and subsequent screening by restriction enzyme digestion. Here we report a DHPLC-based method for the rapid discrimination of recombinant bacterial clones versus non-mutant parental clones, after performing two different SDM protocols: one based on “megaprimer PCR” (10) and one based on a commercial kit (QuickChange® Site-Directed Mutagenesis Kit; Stratagene, La Jolla, CA, USA). The first protocol was carried out to create a single point mutation (39C→T) in exon 2 of the β-globin gene (HBB), using two PCR amplifications and three primers, as described (10). The amplified products were then cloned into pGEM® vector (Promega, Madison, WI, USA), according to manufacturer’s instructions. Conversely, the QuickChange-based protocol was used to create four different missense changes in exons 2 (124A→G), 7 (836A→G), 12 (1403 C→T), and 13 (1508 G→C) of the PTPN11 gene. From both experiments, several bacterial colonies were isolated and grown into 100 μL LB for 30 min at 37°C. Two microliters of growing colonies were then lysed by heating at 98°C for 10 min and used as template for PCR amplification. The reaction was carried out in a GeneAmp® PCR System 9700 (Applied Biosystems, Foster City, CA, USA) in a total volume of 25 μL containing 250 μM dNTPs, 0.5 μM each specific HBB and PTPN11 primers, 1.25 U AmpliTaq Gold® DNA polymerase (Applied Biosystems), in 1× reaction buffer (10 mM Tris HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2). The PCR mixture was held at 94°C for 11 min and then cycled 25 times at 94°C for 30 s, 57°C for 30 s, and 72°C for 45 s, followed by 7 min at 72°C in the final cycle. Since each bacterial clone contains only a single allele, to originate DHPLC-detectable heteroduplex DNA molecules, each single colony PCR product was mixed with a homozygous wild-type (HBB or PTPN11) PCR product. These PCR mixtures were denatured at 95°C for 5 min and allowed to cool at 65°C for 30 min. Samples were then analyzed in WAVE DHPLC and DNASep Column (both from Transgenomic, Crewe, UK) as described previously (11). The gradient was formed by mixing buffer A (0.1 Benchmarks