I have been reading about next-generation sequencing technologies that can sequence long reads. Even though the origin of my question is sequencing technologies, the question I am asking is about the basics of DNA biochemistry.

One of the issues in manipulating DNA samples seems to be the ability to circularize long linear DNA molecules into circular DNA molecules. What are the technical challenges of circularising DNA molecules longer than 50Kbp?

  • $\begingroup$ sorry i mistook 50kb as 50bp.. If you are referring to Moleculo then it still has to undergo the HiSeq type of sequencing. I guess splitting the genome just makes the assembly easier.. Can you indicate the references which quote the 50kbp limit.. Ilumina says it is 10kbp.. $\endgroup$ – WYSIWYG Mar 14 '14 at 16:51
  • $\begingroup$ I am not sure if it is a limit of input or ease of protocol/assembly.. Because it is not impossible to clone 50-100kb in plasmids.. Is circularization an essential step? Illumina doesn't explain the process in detail. $\endgroup$ – WYSIWYG Mar 14 '14 at 17:00
  • $\begingroup$ From Illumina LRS FAQ: "Long reads are generated by creating one or several libraries from an individual sample, depending on each sample type. To create a library, genomic DNA is first fragmented to approximately 10 kilobases (Kb). Next, these fragments are clonally amplified, sheared, and labeled with a unique index. The fragments are then sequenced using Illumina technology. The short sequence reads originating from each molecule are assembled separately into synthetic long reads using proprietary algorithms, resulting in a full sequence of each long fragment." $\endgroup$ – WYSIWYG Mar 14 '14 at 17:08
  • $\begingroup$ @WYSIWYG, I clarified my question. Although I want to know about DNA circularization because of my interest in next-generation sequencing, my question is not about next-gen. $\endgroup$ – 719016 Mar 15 '14 at 11:31

For ligation of a linear molecule to occur, the two ends must come together at the active site of the DNA ligase. In a simple molecular cloning experiment the aim is usually to avoid recircularisation of a cut plasmid vector, and instead to get a new fragment of DNA inserted into the vector. However because the ends of the linearised circular plasmid are tethered together (by being ends of the same molecule) they are more likely to encounter each other than they are to encounter the end of an incoming fragment. This is usually overcome, in part, by increasing the relative concentration of the target fragment.

If you are aiming to recircularise a long fragment then the opposite effect will come into play: the ends of individual long molecules will be much more independent and so the end of another molecule will be likely to compete successfully for ligation. In theory this effect could be overcome by reducing the DNA concentration, but I assume that this is impractical for other reasons.

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  • $\begingroup$ thanks for your answer, which is very clear and something that didn't occur to me before. I have a follow-up question in a new post. $\endgroup$ – 719016 Mar 15 '14 at 11:33
  • $\begingroup$ biology.stackexchange.com/q/15737/658 $\endgroup$ – 719016 Mar 15 '14 at 11:57

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