I understand that in Sanger sequencing we can clone our fragments with the help of e.g. bacteria to make multiple copies of our fragments for further analysis.

I also understand cloning can be a bottleneck in Sanger sequencing - and partly prompted the development of NGS methods.

But doesn't PCR do just that, make multiple copies?

What am I missing?

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    $\begingroup$ Possible duplicate of Sequencing from PCR $\endgroup$ – March Ho Dec 8 '15 at 13:37
  • $\begingroup$ @MarchHo: but... that question is not about Sanger :-| $\endgroup$ – Tobia Tesan Dec 8 '15 at 14:17
  • $\begingroup$ In some cases, you do not know the sequence you are looking for--functional cloning, genome projects, cloning DNA/RNA binding to proteins you are interested in etc. $\endgroup$ – 243 Dec 8 '15 at 14:46
  • $\begingroup$ In addition, PCR can amplify any sequences having primer sequences at the both ends. Sometimes, you are not sure if you get a right sequence from crude DNA mixtures. $\endgroup$ – 243 Dec 8 '15 at 14:49
  • $\begingroup$ This is impossible to answer without knowing the context of what you are trying to sequence. But as someone who has sequenced literally thousands of exons from genomic DNA, cloning is obviously not required for every single Sanger project. I just used PCR to amplify the regions I cared about,. $\endgroup$ – swbarnes2 Dec 8 '15 at 19:16

You can use PCR products in Sanger sequencing; it is very common. Using PCR products instead of cloned genes does raise a set of problems that are less of a concern than with cloned sequences, such as the presence of incomplete or incorrect PCR products, but there are standard and simple solutions for most of these concerns.

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    $\begingroup$ And other minor issues like eventual need for cloning, possibility of using standardized sequencing primers post cloning (like T7) etc. $\endgroup$ – WYSIWYG Nov 26 '15 at 6:45

The error rate of PCR is still very high in comparison to natural bacteria DNA replication of its plasmids.

Natural bacteria DNA replication has an error rate of approximately 1 in 10 billion, and the best PCR polymerase commercially available (Q5 from NEB) has an error rate of approximately 1 in 1 million.

Therefore, cloning fragments into bacteria and sequencing the cloned plasmids ensures that you have the correct sequence for further downstream experiments, since the error rate of natural plasmid replication is far lower. With PCR, you are likely to have some errors introduced due to the higher error rates. This becomes more and more likely with increasing numbers of PCR cycles as well as increasing PCR amplicon lengths.

The fact that the PCR amplicon sequences correctly does not mean that DNA cloned from the PCR amplicon is correct. Therefore, if the PCR is intended for downstream experiments, it is best to sequence the cloned plasmid instead of the amplicon.

In addition, as WYSIWYG has pointed out in the comment, having the PCR amplicon cloned into a plasmid would also allow for the plasmid's regions flanking the cloning site to be used for sequencing. This allows standard sequencing primers on the plasmid to be used, instead of having to design new sequencing primers for each PCR amplicon.

Furthermore, since Sanger sequencing requires the primers to bind ~50-100bp upstream of the region to be sequenced, cloning the amplicon into a plasmid would also allow short amplicons to be sequenced in a single read, instead of requiring a forward and reverse read for sequencing a PCR amplicon.

  • $\begingroup$ This is not correct; or at least, it's only partly correct. It's true that PCR has a much higher error rate than bacterial replication. But that is not a major concern when sequencing pooled DNA from a PCR reaction, because while each amplified strand may have errors, the average of the amplification is still the original sequence, because only a tiny fraction of the strands have a specific error in a specific place. However, if you first amplify the DNA with PCR, and then clone it and sequence it, you have the worst of both worlds, because you may well have errors in the piece you cloned. $\endgroup$ – iayork Nov 25 '15 at 15:13
  • $\begingroup$ @iayork While it is true that directly sequencing a PCR product is doable and is frequently done, the vast majority of cloning is done to transfer a piece of DNA into a vector for expression of some kind. If you sequence the PCR product before cloning it, you will need to sequence it again afterwards, since there is risk of the individual cloned fragment being incorrect. It makes more sense to sequence a known single clone instead. From what I know, PCR product sequencing is usually done either to check that the PCR has worked, or to sequence short stretches of DNA in long vectors. $\endgroup$ – March Ho Dec 8 '15 at 9:26
  • $\begingroup$ I think you have a rather limited view of sequencing; I have been using PCR for sequencing purposes since 1992, and I am far from alone. In any case, the original post wasn't asking how to clone, they were asking whether there was a technical reason for cloning with regard to Sanger sequencing. There isn't one; sequencing from PCR gives highly accurate sequence, and your explanation of error rates is misleading. $\endgroup$ – iayork Dec 8 '15 at 13:03
  • $\begingroup$ @iayork I added a number of technical reasons for cloning. I would consider these to be good reasons to sequence a cloned plasmid instead of the PCR amplicon directly. $\endgroup$ – March Ho Dec 8 '15 at 13:14
  • $\begingroup$ You have still not corrected your misleading comment about PCR error rates, though. $\endgroup$ – iayork Dec 8 '15 at 13:18

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