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.

Learn to be a Pythonista!

Python Playground

Learn to be a Pythonista! Try Python

Python Playground is a collection of fun programming projects that will inspire you to new heights. You'll manipulate images, build simulations, and interact with hardware using Arduino & Raspberry Pi. With each project, you'll get familiarized with leveraging external libraries for specialized tasks, breaking problems into smaller, solvable pieces, and translating algorithms into code.

$ Check price
29.9529.95Amazon 4 logo(14+ reviews)

More Python resources

Take your Linux skills to the next level!

Linux for Beginners

Take your Linux skills to the next level! Try Linux & UNIX

Linux for Beginners doesn't make any assumptions about your background or knowledge of Linux. You need no prior knowledge to benefit from this book. You will be guided step by step using a logical and systematic approach. As new concepts, commands, or jargon are encountered they are explained in plain language, making it easy for anyone to understand.

$ Check price
24.9924.99Amazon 4.5 logo(101+ reviews)

More Linux & UNIX resources

Ad