I went through some literature to understand the basic concepts behind the Sanger - Chain Termination Method of sequencing. I have understood the concepts, but I have a doubt. The Sanger sequencing method (invented in 1977) uses PCR, developed in 1985. Am I missing something?
- Did the original sanger protocol use some other method?
PCR has been a way to amplify DNA, there might be a hard way to obtain enough DNA for Sanger sequencing before PCR, should be!
Sanger sequencing differs from PCR in that only a single primer is used in the reaction. Typically, for a given PCR fragment, two Sanger sequencing reactions are set up, one for sequencing the forward strand, the other one for sequencing the reverse strand. ... Primer length should be in the range of 18 to 22 bases. Anurup Mohanty
You do not need pcr to amplify a sequence. If a sequence is cloned into lambda or other viral sequences then infecting bacteria and growing the bacteria and viruses provides the amplification that you need for sequencing. Virus purification was around a long time before pcr. I believe that he sequenced PHIX174 phage lambda dna as a template
Sanger sequencing did exist long before PCR, and I am one of those who did it. It did require more input template DNA that was relatively pure, something that is not an issue if you amplify. Initially the input DNA was single stranded (cloned in M13 for example) because that worked much better, it was only a bit later that double stranded templates were routinely used. Also the signals were notably lower because you only had one round and no amplification. But it did work.
And the original Sanger had 4 reactions - 1 for each of the nucleotide bases. You had to run them out on super thin, super huge acrylamide gels, expose the radiographs, and do the base calling by hand. Glad I missed having to do that particular protocol!
It was worse than that Katie A Burnette .each set of 4 reactions had to be loaded 3 times at 0, 20 and 40 minutes on the gel to bring the long, medium and short sequences into the best separation that the gel was capable of in order to get 400 readable bases
Paul Rutland we ran 1m long gels so only did two loadings. But when the gel and reactions were good could get 700nts
Katie A Burnette you mean you missed all the fun?? And then there was Maxim-Gilbert
Michael J. Benedik I only had 35x43 gels and one of the most memorable experiences from those days was attending a meeting in Oxford and a researcher showed 50 sheets of perfect sequencing autoradiograms created by the simple addition of biotin to one pcr primer and getting good quality ssDNA every time from that weekend probably gave us 50% more readable sequence from every reaction
Paul Rutland and Michael J. Benedik I was finishing up my undergrad when the human genome was sequenced - very exciting time to be in science!
My biochem instructors were dismayed that it was suddenly so cheap and easy to get something Sanger sequenced. They asked "how would you verify that your plasmid has the correct insert?" and we'd answer "just sequence it!". They wanted us to say "draw a plasmid map, set up restriction digests, gel electrophoresis, estimate the sizes, blah, blah blah". Their counter-argument of "but sequencing is expensive!" didn't hold up either when the price was less than $5 per sample. Same with "we don't have that option!" when the department bought a Sanger machine. Back then, the hardest part was getting your data since 400 kb was a HUGE file at the time and you needed to buy the analysis software.
Lots of great free tools came out then too like primer3, BLAST was online and not a set of CDs, Arabidopsis T-DNA and TILLING lines were available.
I think a comparable shift in technology happened with CRISPR. Targeted mutagenesis in living organisms other than yeast used to be impractical to impossible. Exciting times!
Michael J. Benedik, Paul Rutland and Katie A Burnette
Thanks for sharing these stories. It's really inspiring to see (as an undergraduate) how far technology has brought us.
I have a follow-up question: Based on what I read here(https://www.sigmaaldrich.com/IN/en/technical-documents/protocol/genomics/sequencing/sanger-sequencing)
The technique used by Sanger employed polymerases in-vitro to amplify DNA. (just like PCR), but this technique was invented in 1977, while PCR came in 1988.
To make it more clear: How did Sanger use PCR back in 1977?
Would you mind telling me if I have a misconception?
The article that you posted the link to is not actually describing the original Sanger sequencing but rather is describing the current methods we use for Sanger sequencing, which is improved and modified from the original.
In the original Sanger you are making one complementary strand to the template, so there is no amplification but there is an approx “doubling” in the amount of DNA.
So it has similarities to PCR but in PCR you have multiple rounds of copying that give you exponential amplification. In the original Sanger you only have one round of copying but in current versions of Sanger you have multiple rounds but not exponential amplification (only one primer)
- Badges
- Science method