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. As the traditional chain termination method does not allow long DNA strands to be sequenced, this method works by dividing the long sequence into several consecutive short ones.

The term "primer walking" is used where the main aim is to sequence the genome. The term "chromosome walking" is used instead when we know the sequence but don't have a clone of a gene. For example the gene for a disease may be located near a RFLP on DNA.

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 obtain contiguous sequence information.

The basic technique 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.

That way, the short part of the long DNA that is sequenced keeps "walking" along the sequence. The method can be used to sequence entire chromosomes (thus, chromosome walking). A different method with the same purpose which becomes more popular for large-scale sequencing (e.g., the Human Genome Project) is shotgun sequencing.