Primer walking (or Directed Sequencing) 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 chain termination method. This method works by dividing the long sequence into several consecutive short ones. The DNA of interest may be a plasmid insert, a PCR product or a fragment representing a gap when sequencing a genome. The term "primer walking" is used where the main aim is to sequence the genome. The term "chromosome walking" is used instead when the sequence is known but there is no clone of a gene. For example, the gene for a disease may be located near a specific marker such as an RFLP on the sequence. The fragment is first sequenced as if it were a shorter fragment. Sequencing is performed from each end using either universal primers or specifically designed ones. This should identify the first 1000 or so 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 obtain contiguous sequence information.
The overall process is as follows:
A primer that matches the beginning of the DNA to sequence is used to synthesize a short DNA strand adjacent to the unknown sequence, starting with the primer (see PCR).
The new short DNA strand is sequenced using the chain termination method.
The end of the sequenced strand is used as a primer for the next part of the long DNA sequence, hence the term "walking".
The method can be used to sequence entire chromosomes (hence "chromosome walking"). Primer walking was also the basis for the development of shotgun sequencing, which uses random primers instead of specifically chosen ones.
Shotgun sequencing â€“ an alternative method, using random, rather than consecutive, sub-strands.