Primer walking

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Primer walking is a sequencing method of choice for sequencing DNA fragments between 1.3 and 7 kilobases. Such fragments are too long to be sequenced in a single sequence read using the cyclic dideoxy terminator method (also known as PCR based dideoxy sequencing or Automated Sanger method). The DNA of interest may be a plasmid insert, a PCR product or a fragment representing a gap when sequencing a genome.

The fragment is first sequenced as if it were a shorter fragment — sequencing will be performed from each end using either universal primers or primers designated by the customer. This should identify the first 1000 (approx.) bases. In order to completely sequence the region of interest design and synthesis of new primers — complementary to the final 20 bases of the known sequence is necessary to

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