biology a level - sanger sequencing

Hi this sounds like a stupid question but for Sanger sequencing i know the DNA needs to be single stranded and textbooks give differing information, should i say add the DNA and heat up to 90 degrees so that the double strands separate or should i say to put a single stranded DNA into the mixture originally from the beginning? Anyone know if this matters which one i put.
Also when i talk about the chain terminating nucleotides is it ok to say terminating bases as the nucleotides are specific to the bases they are cutting or should i still say chain terminating nucleotides and specify in my answer that they are specific to diff bases. I do OCR A btw not sure if that makes a difference

Reply 1

Nitrotoluene

Original post by planteater27

Hello planteater27!
That's a great question — no worries.😀

For Sanger sequencing it is best to have singlestranded DNA. The special nucleotides that halt the DNA chain prematurely do so by latching onto the growing strand and putting it to an early end. So if you start with double-stranded DNA, those nucleotides have to find a complementary base in the template to attach to, which makes them less useful.
You need to add the DNA first and then heat it to 90°C in order to separate the double strands. Afterwards, the DNA polymerase is able to start synthesizing a new strand from the single-stranded template.
And they call them terminating bases rather than chain-terminating nucleotides, which is perfectly fine in the context of Sanger sequencing. Nucleotides simply refer to the basic components of DNA, which include the bases (A, C, G, and T).
Each nucleotide can potentially base pair with specific bases on the growing strand, so calling them terminating bases complementary to other bases is correct.

To sum it up:

Start with single-stranded DNA and heat it to 90°C to break the double strands.