04. Primer walking

The Sanger method is fast, reliable and accurate, but is limited to its short reads of around 500 nucleotides per run. In order to extend the amount of reads, we can use a technique called primer walking.

In Sanger sequencing, we attached a primer about 10-20 base pairs below the start of the target sequence. Since our strand terminates at around 500 nucleotides, any sequences longer cannot be read.

Procedure

To get around this, we add a second primer that is around 10-20 base pairs upstream of the termination of our first sequence. We can then sequence the next ~500 base pairs, and repeat this process until the entire cloned DNA is sequenced.

In this diagram, we can see that we added four primers - P1, P2, P3, and P4.

Primer Walking Diagram

Upstream vs. downstream

Upstream simply means up the path that transcription acts on. So in our diagram above, upstream would be to the left. Downstream means down the stream as transcription occurs, so in our diagram this would be to our right.

Careful Measures

To avoid any ambiguity, both strands of DNA are sequenced to double-check our work. Additionally, the reaction vessel is kept at stringent annealing conditions to avoid any spurious binding of nonidentical sequences. Furthermore, primers are ensured to be at least 24 nucleotides long to avoid having them bind to the same region.

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