PCR with End Trimming and Cassette Ligation: A Rapid Method to Clone

We described a method for PCR amplification of unknown flanking genomic DNA fragments. This method is a combination of PCR with "endtr imming method" and "cassettes and cassette-primers method". In this method, genomic DNA was digested with three different groups of restriction enzymes. DNA in group 1 was digested with BamHI, Bglll, Fbal, or Mbol. DNA in group 2 was digested with Blnl, Nhel, Spel, or Xbal. DNA in group 3 was digested with $011 or Xhol. Digested DNA in each group was end-tr immed with Klenow fragment of DNA polymerase I in the presence of only one dNTP; dGTP, dCTP, and dTrP for group 1, 2, and 3, respectively. The synthesized cassettes, C1, C2, and C3, had S' protruding sequences of S'-ATC-3', S'-TAG-3', and $'-CGA-3', respectively. Each compatible cassette was ligated to the end-trimmed DNAs in group 1-3, respectively. Nested PCR was then performed using an end-tr immed and cassette-Iigated DNA as a template. Primers annealing to known sequences and cassettes were used for the nested PCR. The amplified DNA fragments were electrophoresed on a polyacrylamide gel and purified. The sequences of the DNA fragments were determined after cloning into pBluescript. T h e PCR method, which permits specific in vitro amplification of DNA fragments, has found many applications in molecular biology. However, the PCR amplification of unknown flanking DNA fragments is difficult, because PCR requires two target-specific primers annealing to both ends of the DNA fragment to be amplified. To overcome this limitation, several methods have been developed.(1-1~ 13--15) Inverse PCR (~) involves restriction enzyme digestion and circularization by intrastrand end-to-end ligation. The following PCR using primers directing toward an unknown sequence amplifies the flanking sequence. Panhandle PCR (2) and targeted inverted repeat amplification (3) involve restriction enzyme digestion of genomic DNA and ligation of a complementary oligonucleotide to the known core sequence. Denaturation and reannealing form a stem-loop within complementary strands with a 3' recessed end, followed by filling with template-directed polymerization or ligation of an oligomer. The presence of an inverted repeat at two sites permits PCR amplification of the unknown flanking DNA. Anchored PCR (4) involves attachment of a homopolymer tail to the firststrand cDNA by terminal deoxynucleotidyl transferase that undergoes templateindependent polymerization. PCR is performed using one primer annealing to a known sequence and another primer annealing to a homopolymer tail. In ligation-anchored PCR, (s) T4 RNA ligase is used to attach a designed DNA oligomer instead of forming a homopolymer strand to the first-stand cDNA. An end-trimming method (6,7) amplifies a cDNA fragment using one compatible primer to a known sequence and another oligo(dT) primer. The PCR product is end-trimmed by T4 DNA polymerase in the presence of only one dNTP to create a sticky end to a corresponding vector. Cassettes and cassette-primers method,(7) cassette-ligation mediated PCR, (8~ capture PCR, (9~ and vectorette PCR, ~176 involve restriction enzyme digestion of cDNAs or genomic DNAs and their ligation to oligonucleotides or vector sequences to create universal primerannealing sites. PCR, in these methods, utilizes one specific compatible primer to a known sequence and another universal primer that anneals to the universal sequence. Roberts et al. have successfully defined the exon structure of the entire human dystrophin gene, which consists of 79 exons, using vectorette PCR and direct sequencing. ~ PCR in other methods makes use of nonspecific or partially specific primers. Targeted gene walking ~ makes use of sets of specific and nonspecific primers to amplify an unknown flanking DNA sequence. A