Modified inverse PCR method for cloning the flanking sequences from human cell pools.

Inverse polymerase chain reaction (PCR) is a powerful approach for cloning the DNA sequences lying outside the boundaries of known sequences (7). This approach can be used to determine the integration site of a virus or a transposon (2,4). The procedure described by Triglia (7) can be directly used to amplify the flanking sequence in a plasmid or in a bacterial genome. However, it has been difficult to apply this method in cloning the flanking sequence in a mammalian genome. Since the target sequence is usually rare, the nonspecific amplification often masks the target products. Several groups overcame the difficulty by enriching the target sequence, in which the size of the restriction fragment has to be determined by Southern blot (3), or by using oligonucleotide hybridization to determine the target products containing the known sequence (2,3). Here, we modified the classical inverse PCR procedure and successfully applied it in cloning the flanking sequence of a known sequence integrated in the human genome in two days. Four primers were designed in a 130-bp known sequence of the Tc1 transposon (Table 1). A nested PCR was used to amplify the rare sequence, and 5% formamide was used to eliminate the nonspecific amplification (1,5). The NEOmarked transposon (pRP466-NEO) and the transposase expression plasmid (pCEP4-Tc1A) were constructed by the similar strategy described before (6). First, the pRP466-NEO alone was introduced into KB cells by electroporation, so that the flanking sequence would be the same as that of the plasmid because it is randomly integrated in the genome. After being selected with G418 for two weeks, one clone, KB-466, proved to have the same flanking sequences as the plasmid pRP466-NEO by Southern blot and by PCR analysis and was used as a positive control. The DNAs from KB466 and KB cells were digested with TaqI at 65°C and with SspI at 37°C, then extracted with phenol/chloroform and chloroform, precipitated in ethanol and washed with 70% ethanol. Approximately 10 ng digested DNA were ligated in a total volume of 50 μL, containing 1 U T4 DNA Ligase (Promega, Madison, WI, USA) at 12°C overnight, and 1 μL of the ligated product was used as the template in the first-round PCR on an UNO II Thermal Cycler (Biometra, Gottingen, Germany). After 5 min denaturing at 94°C, the PCR was performed in a total volume of 50 μL, containing 10 mM Tris, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTPs, 1 U Taq DNA polymerase, 5% formamide and 30 pmol primers 2 and 3, for 27 cycles of 30 s at 94°C, 30 s at 55°C and 50 s at 72°C. Then, 1 μL PCR product was diluted in 1000 μL double-distilled (dd)H2O, and 1 μL was used in the second round PCR amplification, in which the reaction condition was the same as that of the first round except that the primers 1 and 4 were used. The result (Figure 1A) indicated that the modification was effective and that the reactions were very efficient and highly specific. In most cases, only one band could be detected after the nested PCR, while many bands were observed if no 5% formamide were added in the PCR. The fragment sizes were the same as predicted, and the sequencing result indicated that the cloned fragments were correct. In one reaction, the sequence of a cloned fragment (Figure 1A, lane 2) showed that there was a 53-bp TaqI fragment inserted in the flanking seBenchmarks