Primer walking
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Primer walking is a technique used to clone a gene (e.g., disease gene) from its known closest markers (e.g., known gene). As a result, it is employed in cloning and sequencing efforts in plants, fungi, and mammals with minor alterations. This technique, also known as "directed sequencing," employs a series of Sanger sequencing reactions to either confirm the reference sequence of a known plasmid or PCR product based on the reference sequence (sequence confirmation service) or to discover the unknown sequence of a full plasmid or PCR product by designing primers to sequence overlapping sections (sequence discovery service).[1]
Primer walking: a DNA sequencing method
Primer walking is a method to determine the sequence of DNA up to the 1.3–7.0 kb range whereas
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.[5]
Primer walking versus shotgun sequencing
Primer walking is an example of
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.[7]
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.[8]
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.[9]
Process
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").[10] Primer walking was also the basis for the development of shotgun sequencing, which uses random primers instead of specifically chosen ones.
See also
- Chromosome jumping
- Chromosome landing
- Shotgun sequencing – an alternative method, using random, rather than consecutive, sub-strands.
References
- ^ Company, Azenta Life Sciences. "Primer Walking FAQ". web.genewiz.com. Retrieved 2021-11-20.
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- ISBN 978-0-19-963718-8.
- ISBN 9780128022214.
- PMID 10784293.
- ^ "ScienceDirect.com | Science, health and medical journals, full text articles and books". www.sciencedirect.com. Retrieved 2021-11-20.
- ^ "genetics - Why do we use DNA sequencing methods such as shotgun?". Biology Stack Exchange. Retrieved 2021-11-20.
- ^ "genetics - Why do we use DNA sequencing methods such as shotgun?". Biology Stack Exchange. Retrieved 2021-11-20.
- ^ "genetics - Why do we use DNA sequencing methods such as shotgun?". Biology Stack Exchange. Retrieved 2021-11-20.
- PMID 376402.