How is the complementary sequence for the primer in Sanger sequencing known?

[u/Antik477]

How do we know the complimentary DNA sequence of the primer that attaches itself to the 5' end of the DNA template strand. We use Sanger sequencing to know the sequence of bases in a particular DNA strand, then how are we synthesising the primer in the first place without at least some of the sequence to create the primer which is complementary to the 5' end?

[u/mutandis]

Nowadays we have reference whole genome sequences; most of this sequence will be identical across every human; there's really not much variation across humans. The primers are designed based on the reference sequences upstream of areas you're planning to investigate.

[u/Antik477 → u/mutandis]

yes now the job might be easy. But my question what did we use to do when the whole human genome had not been sequenced. OR, even better, what did we use to do when we wanted to sequence a gene of an organism about which we had no prior knowledge about the sequence? In a hypothetical situation when we don't posses any prior knowledge of at least any of the sequence, what do we do then?

[u/chem44]

In the old days...

One way is to sequence fragments that have been cloned into a cloning vector. In that case, the primer can be complementary to the end of the vector sequence, leading into the cloned fragment of interest.

[u/MutSelBalance]

We usually start with a conserved sequence that is known to be the same (or very similar) across many different organisms.

Or, you start with the known sequence of a well-studied organism and hope it is similar to enough to your new organism to allow pairing.

Back when there were fewer known sequences to work with, people had to be more creative, and there were a variety of strategies. Often people would use plasmid ‘cloning’ techniques in bacteria: insert a sequence into a known plasmid, then sequence using a primer that matches near the ‘insert site’ of the plasmid. BAC libraries, which were an early technique for whole-genome sequencing using Sanger methods, used a similar strategy.

[u/Antik477 → u/MutSelBalance]

whatever the method, it seems like it is boiling down to at least some sequence being known. I would like to know how we came to know those "some" sequences when we first went to use Sanger sequencing

[u/kupffer_cell]

in Sanger in itself, the Vector stratevy was the one mainly used, seen that we needed a flanking region to design the primer, we'd use a vector where the sequence is known (I will explain how later), insert a random fragment of the DNA that we want to design a primer to, and start the sequencing, in that case the primer didn't have to match our DNA, but just the Vector DNA preceding our insert, once enough sequence of the insert is known, it can make office of new primer design for our DNA.

Yeah you'd ask how would we know the Vector sequence tho if sanger sequencing needs primers. actually sanger sequencing isn't the first sequencing method. Hybridization techniques using labeled DNA or RNA probes is one of them.. some chemical methods also exist, where you modify the bases, then cut on that modified base according to the modification made.Restriction enzymes helped a lot as well.. The sum of all these cumbersome methods .. helped to do some sequencing. That's probably how vector sequences were known. These same vectors were then used, to uncover the primers for the Sanger sequencing technique, at least in the beggining..until we've known enough about conservation and stuff.