Editing Primer walking (section)

== Primer walking versus shotgun sequencing ==
Primer walking is an example of [[directed sequencing]] because the primer is designed from a known region of DNA to guide the sequencing in a specific direction. In contrast to directed sequencing, shotgun sequencing of DNA is a more rapid sequencing strategy.<ref>{{Cite web|title=ScienceDirect.com {{!}} Science, health and medical journals, full text articles and books.|url=https://www.sciencedirect.com/|access-date=2021-11-20|website=www.sciencedirect.com}}</ref>

There is a technique from the "old time" of genome sequencing. The underlying method for sequencing is the Sanger chain termination method which can have read lengths between 100 and 1000 basepairs (depending on the instruments used). This means you have to break down longer DNA molecules, clone and subsequently sequence them. There are two methods possible.<ref>{{Cite web|title=genetics - Why do we use DNA sequencing methods such as shotgun?|url=https://biology.stackexchange.com/questions/21251/why-do-we-use-dna-sequencing-methods-such-as-shotgun|access-date=2021-11-20|website=Biology Stack Exchange}}</ref>

The first is called chromosome (or primer) walking and starts with sequencing the first piece. The next (contiguous) piece of the sequence is then sequenced using a primer which is complementary to the end of the first sequence read and so on. This technique doesn't require much assembling, but you need a lot of primers and it is relatively slow.<ref>{{Cite web|title=genetics - Why do we use DNA sequencing methods such as shotgun?|url=https://biology.stackexchange.com/questions/21251/why-do-we-use-dna-sequencing-methods-such-as-shotgun|access-date=2021-11-20|website=Biology Stack Exchange}}</ref>

To overcome this problem the shotgun sequencing method was developed. Here the DNA is broken into different pieces (not all broken at the same place), cloned and sequenced with primers specific for the vector used for cloning. This leads to overlapping sequences which then have to be assembled into one sequence on the computer. This method allows for the parallelization of the sequencing (you can prepare a lot of sequencing reactions at the same time and run them) which makes the process much faster and also avoids the need for sequence specific primers. The challenge is to organize sequences into their order, as overlaps are not as clear here. To resolve this problem, a first draft is made and then critical regions are resequenced using other techniques such as primer walking.<ref>{{Cite web|title=genetics - Why do we use DNA sequencing methods such as shotgun?|url=https://biology.stackexchange.com/questions/21251/why-do-we-use-dna-sequencing-methods-such-as-shotgun|access-date=2021-11-20|website=Biology Stack Exchange}}</ref>