High through-put analysis of transgene borders

A method for finding an unknown polynucleotide to a known polynucleotide sequence in isolated DNA, which comprises:

• a) digesting the isolated DNA that contains a portion or all of the known polynucleotide sequence and an adjacent unknown polynucleotide sequence with one or more suitable restriction enzymes to produce a plurality of digested polynucleotide restriction fragments;
• b) synthesizing a complementary strand of the digested polynucleotide restriction fragments using an oligonucleotide primer sequence having an attachment chemistry bound to the 5′ end of the oligonucleotide primer sequence;
• c) isolating the complementary strand by binding the attachment chemistry to a suitable isolation matrix, then denaturing the complementary strand from the digested polynucleotide restriction fragments;
• d) ligating a single stranded adapter to the isolated complementary strand bound to the isolation matrix to produce a ligated isolated complementary strand;
• e) performing a first PCR amplification of the ligated isolated complementary strand using a first PCR primer designed to bind to the known polynucleotide sequence and a second PCR primer designed to bind to the single stranded adaptor to produce a first PCR amplicon, wherein said first PCR primer comprises SEQ ID NO:
• 3 and said second PCR primer comprises SEQ ID NO:
• 4;
• f) performing a second PCR amplification of said first PCR amplicon, wherein the second PCR amplification amplifies an internal sequence of said first PCR amplicon to produce a second PCR amplicon;
• and
• g) sequencing the second PCR amplicon to ascertain the sequence of the unknown polynucleotide sequence.