PCR with end trimming and cassette ligation: a rapid method to clone exon-intron boundaries and a 5'-upstream sequence of genomic DNA based on a cDNA sequence

PCR Methods Appl. 1994 Aug;4(1):19-25. doi: 10.1101/gr.4.1.19.


We described a method for PCR amplification of unknown flanking genomic DNA fragments. This method is a combination of PCR with "end-trimming 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, BglII, FbaI, or MboI. DNA in group 2 was digested with BlnI, NheI, SpeI, or XbaI. DNA in group 3 was digested with SalI or XhoI. Digested DNA in each group was end-trimmed with Klenow fragment of DNA polymerase I in the presence of only one dNTP; dGTP, dCTP, and dTTP for group 1, 2, and 3, respectively. The synthesized cassettes, C1, C2, and C3, had 5'protruding sequences of 5'-ATC-3',5'-TAG-3', and 5'-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-trimmed and cassette-ligated 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amidophosphoribosyltransferase / genetics
  • Animals
  • Base Sequence
  • Cloning, Molecular / methods*
  • DNA Restriction Enzymes
  • DNA, Complementary
  • Exons*
  • Genome
  • Introns*
  • Male
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • Promoter Regions, Genetic
  • Rats
  • Sequence Alignment


  • DNA, Complementary
  • Amidophosphoribosyltransferase
  • DNA Restriction Enzymes

Associated data

  • GENBANK/D10853